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Molecular characterization of the interaction between cucumber necrosis virus and zoospores of the fungal… Robbins, Marjorie Ann 2000

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Molecular Characterization of the Interaction Between Cucumber Necrosis Virus and Zoospores of the Fungal Vector Olpidium bornovanus By  MARJORIE ANN ROBBINS  B.Sc. (Honours Biology), Dalhousie University, 1991 M.Sc, McGill University, 1994  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY In T H E F A C U L T Y OF GRADUATE STUDIES (Plant Science)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA April 2000 © Marjorie Ann Robbins, 2000  in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his or  her  representatives.  It  is  understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  [fvCjuVVuiAJiJ  Abstract  In this thesis, the m o l e c u l a r b i o l o g y o f the i n t e r a c t i o n b e t w e e n Cucumber necrosis tombusvirus ( C N V )  a n d z o o s p o r e s o f its f u n g a l vector, Olpidium bornovanus, was  e x a m i n e d . S e r i a l p a s s a g e i n the a b s e n c e o f O. bornovanus p r o d u c e d a p o o l o f  CNV  v a r i a n t s d e f i c i e n t i n f u n g u s t r a n s m i s s i o n . V a r i a n t s o f C N V h a d m u t a t i o n s and/or deletions in genomic R N A  that a f f e c t e d v a r i o u s stages o f the v i r u s m u l t i p l i c a t i o n - c y c l e ,  t h e r e b y d e c r e a s i n g t r a n s m i s s i b i l i t y . O n e t r a n s m i s s i o n mutant, L L 5 , was s e l e c t e d f o r f u r t h e r c h a r a c t e r i z a t i o n s i n c e this m u t a n t r e t a i n e d stable, i n t a c t p a r t i c l e s a n d was as i n f e c t i o u s as w i l d - t y p e C N V .  S e q u e n c i n g o f the L L 5 c o a t p r o t e i n ( C P ) a n d  subsequent  s i t e - d i r e c t e d m u t a g e n e s i s o f the C N V C P i n d i c a t e d that a s i n g l e n u c l e o t i d e s u b s t i t u t i o n r e s u l t i n g i n a G l u to L y s s u b s t i t u t i o n i n the C N V C P s h e l l d o m a i n was r e s p o n s i b l e f o r the r e d u c e d t r a n s m i s s i o n o f L L 5 . A n in vitro b i n d i n g assay was d e v e l o p e d to d e t e r m i n e i f L L 5 was d e f i c i e n t at the l e v e l o f attachment to f u n g a l z o o s p o r e s . L L 5 b o u n d z o o s p o r e s at a p p r o x i m a t e l y 5 0 % the l e v e l o f w i l d - t y p e C N V i n d i c a t i n g that the r e d u c e d a b i l i t y to b i n d z o o s p o r e s was at least p a r t i a l l y r e s p o n s i b l e f o r the r e d u c e d t r a n s m i s s i o n . F u r t h e r e x a m i n a t i o n o f the b i n d i n g o f s e v e r a l O. Z w m o v a n w s - t r a n s m i t t e d v i r u s e s to O. bornovanus z o o s p o r e s i n d i c a t e d that the a b i l i t y to b i n d z o o s p o r e s in vitro c o r r e l a t e s w i t h  the k n o w n b i o l o g i c a l s p e c i f i c i t y o f t r a n s m i s s i o n . A d d i t i o n a l l y , i t was f o u n d that b i n d i n g o f C N V to O. bornovanus was saturable, w i t h a m a x i m u m o f 1.75 X 1 0 v i r u s p a r t i c l e s 4  b o u n d p e r z o o s p o r e . It was a l s o d e t e r m i n e d that C N V c a n c o m p e t i t i v e l y i n h i b i t b i n d i n g o f another O. bo rnovanus-trmsmitted v i r u s , Melon necrotic spot carmovirus. T a k e n together, these results suggest that O. bornovanus z o o s p o r e s c o n t a i n s p e c i f i c r e c e p t o r m o l e c u l e ( s ) f o r attachment o f C N V  particles. ii  A m i n o a c i d s e q u e n c e r e l a t i o n s h i p s a m o n g the c o a t p r o t e i n s o f s e v e r a l s m a l l spherical plant R N A  v i r u s e s i n c l u d i n g f o u r k n o w n to b e t r a n s m i t t e d b y O. bornovanus  w e r e e x a m i n e d . Interestingly, the c o a t p r o t e i n s e q u e n c e s o f the f o u r O. bornovanus t r a n s m i t t e d v i r u s e s w e r e c l o s e l y related, d e s p i t e the a f f i n i t i e s o f these v i r u s e s to d i s t i n c t t a x o n o m i c groups. T w o r e g i o n s w i t h i n the o t h e r w i s e n o n - c o n s e r v e d c o a t p r o t e i n p r o t r u d i n g d o m a i n w e r e p a r t i c u l a r l y c o n s e r v e d . T h e s e data f u r t h e r s u g g e s t the i n v o l v e m e n t o f s p e c i f i c c o a t p r o t e i n s e q u e n c e s i n the f u n g u s t r a n s m i s s i o n p r o c e s s .  iii  Table of Contents Abstract  ii  Table o f Contents  iv  List of Tables  •.  v i i  L i s t o f Figures..  viii  List o f Abbreviations  ix  Acknowledgements  xiii  1 C H A P T E R ONE: L I T E R A T U R E R E V I E W 1.1 I n t r o d u c t i o n 1.2 M o d e s o f p l a n t v i r u s t r a n s m i s s i o n 1.2.1 A r t h r o p o d t r a n s m i s s i o n 1.2.1.1 N o n - c i r c u l a t i v e a p h i d t r a n s m i s s i o n 1.2.1.1.1 V i r u s r e t e n t i o n 1.2.1.1.2 C o a t p r o t e i n 1.2.1.1.3 H e l p e r c o m p o n e n t 1.2.1.2 C i r c u l a t i v e a p h i d t r a n s m i s s i o n 1.2.1.2.1 C o a t p r o t e i n 1.2.1.2.2 C h a p e r o n i n s 1.2.1.2.3 R e c e p t o r - m e d i a t e d e n d o c y t o s i s 1.2.1.3 T h r i p s t r a n s m i s s i o n ( c i r c u l a t i v e , r e p l i c a t i v e ) 1.2.2 N e m a t o d e t r a n s m i s s i o n ( n o n - c i r c u l a t i v e ) 1.2.3 F u n g u s t r a n s m i s s i o n 1.2.3.1 Olpidium l i f e - c y c l e 1.2.3.2 In vitro ( n o n - c i r c u l a t i v e ) t r a n s m i s s i o n 1.2.3.2.1 S p e c i f i c i t y o f in vitro f u n g a l t r a n s m i s s i o n 1.2.3.3 In vivo ( c i r c u l a t i v e ) f u n g a l t r a n s m i s s i o n 1.2.3.3.1 In vivo t r a n s m i s s i o n b y Olpidium brassicae 1.2.3.3.2 In vivo t r a n s m i s s i o n b y Polymyxa a n d Spongospora s p p 1.3 C u c u m b e r n e c r o s i s v i r u s 1.3.1 T a x o n o m i c status o f C N V 1.3.2 G e n o m i c o r g a n i z a t i o n : 1.3.3 C N V d e f e c t i v e i n t e r f e r i n g R N A s 1.3.4 C o a t p r o t e i n m u t a n t s 1.3.5 P a r t i c l e s t r u c t u r e 1.3.6 T h e r o l e o f the C N V c o a t p r o t e i n i n f u n g u s t r a n s m i s s i o n 1.4 B r i e f o v e r v i e w o f v i r u s - r e c e p t o r i n t e r a c t i o n s 1.5 S u m m a r y a n d T h e s i s O b j e c t i v e s  1 1 3 7 8 8 9 11 14 15 17 17 20 21 25 29 32 33 34 35 36 38 38 39 40 41 42 43 44 46  2 C H A P T E R TWO: M A T E R I A L S A N D M E T H O D S  49  iv  2.1 M a i n t e n a n c e o f v i r u s c u l t u r e s 2.2 P u r i f i c a t i o n o f v i r u s 2.2.1 C s C l p u r i f i c a t i o n 2.2.2 D i f f e r e n t i a l c e n t r i f u g a t i o n 2.2.3 M i n i p r e p p u r i f i c a t i o n 2.3 M a i n t e n a n c e o f f u n g a l c u l t u r e s 2.4 P r o d u c t i o n o f a n t i b o d i e s 2.4.1 P o l y c l o n a l a n t i b o d y p r o d u c t i o n 2.4.2 M o n o c l o n a l a n t i b o d y p r o d u c t i o n 2.4.3 P u r i f i c a t i o n o f I g G a n d p r o d u c t i o n o f a l k a l i n e p h o s p h a t a s e c o n j u g a t e 2.5 E n z y m e - l i n k e d I m m u n o a s s a y ( E L I S A ) 2.6 I m m u n o b l o t t i n g 2.7 I s o l a t i o n o f t r a n s m i s s i o n - d e f i c i e n t C N V v a r i a n t s 2.8 F u n g u s t r a n s m i s s i o n assay 2.9 R T - P C R a n d c l o n i n g o f the L L 5 c o a t p r o t e i n 2.10 In vitro t r a n s c r i p t i o n a n d i n o c u l a t i o n o f p l a n t s 2.11 In vitro m u t a g e n e s i s 2.12 B i n d i n g A s s a y s 2.13 I n t r o d u c t i o n o f a f r a m e s h i f t m u t a t i o n i n t o t h e C L S V CP O R F 2.14 A n a l y s i s o f J R 3 A X h o 2.15 L e c t i n l a b e l l i n g o f f u n g a l z o o s p o r e s 3 C H A P T E R THREE: IDENTIFICATIONA N D M O L E C U L A R C H A R A C T E R I Z A T I O N OF N A T U R A L C N V V A R I A N T S W I T H  49 49 49 50 51 51 • 53 53 53 54 55 56 57 57 58 58 59 59 60 61 61  REDUCED  TRANSMISSmnJTY  62  3.1 I n t r o d u c t i o n 62 3.2 R e s u l t s 64 3.2.1 M e c h a n i c a l l y p a s s a g e d CNV c o n t a i n s v a r i a n t s d e f i c i e n t i n f u n g u s transmission 64 3.2.2 A G l u t o L y s m u t a t i o n i n t h e L L 5 c o a t p r o t e i n S d o m a i n i s r e s p o n s i b l e f o r reduced fungal transmission 66 3.2.3 T h e G l u t o L y s s u b s t i t u t i o n i n the L L 5 s h e l l d o m a i n i s d i r e c t l y r e s p o n s i b l e f o r the l o s s o f t r a n s m i s s i b i l i t y 69 3.2.3.1 M 5 a n d M 5 / L L 5 S p a r t i c l e s c o n t a i n i n t a c t RNA b u t h a v e a l t e r e d e l e c t r o p h o r e t i c m o b i l i t i e s o n agarose g e l s 70 3.2.3.2 M 5 / L L 5 a n d M 5 / L L 5 s p a r t i c l e s a r e stable a n d as i n f e c t i o u s as W T 72 3.2.3.3 M 5 / L L 5 a n d M 5 / L L 5 a c c u m u l a t e t o W T l e v e l s i n i n f e c t e d p l a n t s 73 3.2.4 M 5 / L L 5 a n d M 5 / L L 5 s p a r t i c l e s b i n d z o o s p o r e s less e f f i c i e n t l y than W T virus 75 3.3 D i s c u s s i o n 76 S  4 C H A P T E R FOUR: C O M P A R A T I V E S E Q U E N C E A N A L Y S I S OF T H E C O A T P R O T E I N S OF FOUR OLPIDIUM BORNOVANUS T R A N S M I T T E D VIRUSES 8 1 4.1 I n t r o d u c t i o n 4.1.1 P r o p e r t i e s o f O. bornovanus t r a n s m i t t e d v i r u s e s u s e d i n this s t u d y 4.1.1.1 Cucumber leaf spot virus  81 82 82  v  4.1.1.2 Melon necrotic spot virus 4.1.1.3 Red clover necrotic mosaic virus 4.1.1.4 Cucumber necrosis virus  4.2 R e s u l t s 4.2.1 C o m p a r a t i v e s e q u e n c e a n a l y s i s o f the C N V , M N S V , a n d R C N M V c o a t proteins 4.2.2 T h e C L S V c o a t p r o t e i n i s n o t r e q u i r e d f o r c e l l - t o - c e l l m o v e m e n t b u t i s r e q u i r e d f o r s y s t e m i c m o v e m e n t i n N. benthamiana 4.3 D i s c u s s i o n 4.3.1 C o m p a r a t i v e s e q u e n c e a n a l y s i s 4.3.2 R o l e o f the C L S V C P i n v i r u s m o v e m e n t 5 C H A P T E R FIVE: E V I D E N C E T H A T F U N G A L Z O O S P O R E S C O N T A I N SPECIFIC RECEPTORS F O R TRANSMISSION O F C N V  83 83 84  84 84 90 95 95 96 98  5.1 I n t r o d u c t i o n 98 5.2 R e s u l t s 100 5.2.1 D e v e l o p m e n t o f a n in vitro assay f o r b i n d i n g o f C N V t o O. bornovanus zoospores 100 5.2.2 B i n d i n g o f v i r u s t o z o o s p o r e s is s p e c i f i c 103 5.2.3 B i n d i n g o f v i r u s t o z o o s p o r e s is saturable 104 5.2.4 C N V c a n c o m p e t e w i t h M N S V a n d T N V b u t n o t w i t h C L S V f o r b i n d i n g sites 107 5.3 D i s c u s s i o n 109 6 C H A P T E R SIX: U S E O F F I T C - L E C T I N S T O I D E N T I F Y S U G A R S O N T H E S U R F A C E O F O. BORNOVANUS A N D O. BRASSICAE Z O O S P O R E S  6.1 I n t r o d u c t i o n 6.2 R e s u l t s 6.2.1 C o n c a n a v a l i n A - F I T C b i n d s t o Olpidium z o o s p o r e s 6.2.2 Triticum vulgaris l e c t i n - F I T C d o e s n o t b i n d t o s e c o n d a r y z o o s p o r e s o f Olpidium b u t d o e s b i n d t o t o p r i m a r y z o o s p o r e s o f Olpidium 6.2.3 Bauhinia purpurea l e c t i n - F I T C b i n d s t o O. brassicae b u t n o t O. bornovanus z o o s p o r e s  113  113 115 115 117 124  6.2.4 Erythrina corallodendron l e c t i n - F I T C d o e s n o t b i n d Olpidium z o o s p o r e s . 1 2 5 6.2.5 Tetragonolobuspurpureas l e c t i n - F I T C b i n d s t o Olpidium zoospores........ 1 2 5  6.2.6 D i s c u s s i o n  126  7 CHAPTER SEVEN: GENERAL DISCUSSION  130  References  137  vi  List of Tables T a b l e 1.1 P r i n c i p a l v e c t o r s o f p l a n t v i r u s g e n e r a a n d their m e c h a n i s m o f t r a n s m i s s i o n . 4 T a b l e 1.2 K n o w n a n d p u t a t i v e f u n g a l v e c t o r s f o r v i r u s e s a n d v i r u s - l i k e agents  26  T a b l e 1.3 S o m e k n o w n r e c e p t o r s f o r a n i m a l v i r u s e s  45  T a b l e 3.1 P h e n o t y p e s o f C N V t r a n s m i s s i o n m u t a n t s  66  T a b l e 3.2 B i n d i n g o f C N V t o O. bornovanus z o o s p o r e s in vitro  76  T a b l e 4.1 V i r u s e s u s e d i n the C P s e q u e n c e a l i g n m e n t s a n d d e n d o g r a m  85  T a b l e 6.1 L e c t i n s u s e d i n b i n d i n g assays w i t h Olpidium z o o s p o r e s  115  T a b l e 6.2 B i n d i n g o f F T T C - l e c t i n c o n j u g a t e s t o Olpidium z o o s p o r e s in vitro  124  vii  List of Figures F i g u r e 1.1 L i f e c y c l e o f Olpidium brassicae  27  F i g u r e 1.2 G e n o m i c o r g a n i z a t i o n o f C N V  39  F i g u r e 1.3 S t r u c t u r e o f the T B S V p a r t i c l e  42  F i g u r e 3.1 L o c a t i o n o f m u t a t i o n s i n the L L 5 C P g e n e  67  F i g u r e 3.2 S u m m a r y o f f u n g u s t r a n s m i s s i o n assays  68  F i g u r e 3.3 A g a r o s e g e l e l e c t r o p h o r e s i s o f m u t a n t a n d W T v i r u s  70  F i g u r e 3.4 A g a r o s e g e l e l e c t r o p h o r e s i s o f R N A  extracted from mutant and  WT  virions F i g u r e 3.5 A c c u m u l a t i o n o f v i r a l R N A  71 or virus particles i n infected leaves  73  F i g u r e 3.6 S u m m a r y o f in vitro b i n d i n g assays  74  F i g u r e 4.1 D e n d o g r a m d e p i c t i n g r e l a t i o n s h i p s a m o n g the C P s o f s e v e r a l i c o s a h e d r a l viruses  86  F i g u r e 4.2 A l i g n m e n t o f the C P s o f s e v e r a l s m a l l s p h e r i c a l v i r u s e s  90  F i g u r e 4.3 A l i g n m e n t o f the C P a m i n o a c i d s e q u e n c e s o f f o u r O. bornovanustransmitted viruses F i g u r e 4.4 S y m p t o m s o n N. benthamiana i n o c u l a t e d w i t h t r a n s c r i p t s o f J R 3 o r JR3AXho F i g u r e 4.5 S y m p t o m s o n N. benthamiana o f l e a v e s i n o c u l a t e d w i t h t r a n s c r i p t s o f J R 3 A X h o or JR3 (wild-type C L S V ) F i g u r e 4.6 A g a r o s e g e l o f total R N A  90 92 93  e x t r a c t e d f r o m s i n g l e l e a v e s o f N. benthamiana. 94  F i g u r e 5.1 E f f e c t o f t i m e o n b i n d i n g o f C N V to O. bornovanus z o o s p o r e s  101  F i g u r e 5.2 E f f e c t o f p H o n b i n d i n g o f C N V to O. bornovanus z o o s p o r e s  102  F i g u r e 5.3 S p e c i f i c i t y o f v i r u s b i n d i n g to Olpidium spp  105  F i g u r e 5.4 S a t u r a t i o n b i n d i n g o f v i r u s to Olpidium s p p  106  F i g u r e 5.5 C o m p e t i t i o n b i n d i n g e x p e r i m e n t s  -.  108  F i g u r e 6.1 B i n d i n g o f C o n A - F I T C to O. bornovanus z o o s p o r e s  118  F i g u r e 6.2 B i n d i n g o f C o n A - F I T C to O. brassicae z o o s p o r e s  119  F i g u r e 6.3 B i n d i n g o f W G A - F I T C to O. bornovanus a n d O. brassicae r e s t i n g spores 120 F i g u r e 6.4 B i n d i n g o f B P A - F I T C to O. brassicae z o o s p o r e s  121  F i g u r e 6.5 E C A - F I T C d o e s n o t b i n d to O. bornovanus o r O. brassicae z o o s p o r e s 1 2 2 F i g u r e 6.6 B i n d i n g o f T P A - F I T C to O. bornovanus a n d O. brassicae z o o s p o r e s  viii  123  List of Abbreviations  3' 5'  three p r i m e five prime arm a d e n o s i n e i n t h e c o n t e x t o f n u c l e o t i d e s e q u e n c e ; a l a n i n e i n the c o n t e x t of amino acid sequence amino acid antibody alanine alkaline phosphatase arginine  a  A aa Ab Ala ALP Arg ArMV  Arabis mosaic vims  Asn Asp ATP bp  asparagine aspartic acid adenosine-5-triphosphate base pairs Bauhinia purpurea a g g l u t i n i n  BPA BNYVV  Beet necrotic yellow vein virus  BRL BSA  Bethesda Research Laboratories bovine serum albumin  BYDV  Barley yellow dwarf virus  C  c y t i d i n e i n the c o n t e x t o f n u c l e o t i d e s e q u e n c e ; c y s t e i n e i n the c o n t e x t of a protein sequence  Ca  ++  calcium  CaMV  Cauliflower mosaic virus  cDNA  complementary  CLRV  cm  cucumber leaf roll virus centimetre  CMV CNV  Cucumber mosaic virus Cucumber necrosis virus  ConA  concanavalin A  CP CsCl C-terminal  cv. Cys D DI DIC DNA DTT E ECA EDTA  DNA  coat protein cesium chloride carboxyl-terminal cultivar cysteine aspartic acid defective interfering differential interference contrast deoxyribonucleic acid dithiothreitol glutamic acid Erythrinia corallodendron a g g l u t i n i n  ethylenediaminetetraacetic acid ix  F  e n z y m e - l i n k e d i m m u n o s o r b e n t assay phenylalanine  FMDV  Foot and mouth disease virus  Fig. FITC g G  His  Figure fluorescein isothiocyanate gram(s); g e n o m i c i n the c o n t e x t o f v i r a l R N A g u a n o s i n e i n the c o n t e x t o f a n u c l e o t i d e s e q u e n c e ; g l y c i n e i n t h e context o f a protein sequence gene activator protein glutamic acid glycine glycoprotein hinge histidine highly aphid transmissible helper component protein histidine  HIV  Human immunodeficiency virus  Hr  hour  HRV  Human rhinovirus  I ICAM He K  MAb  isoleucine intercellular adhesion molecule isoleucine l y s i n e i n the c o n t e x t o f a m i n o a c i d s e q u e n c e ; t h o u s a n d i n t h e c o n t e x t of size kilobase kilodalton(s) litre(s) leucine l o w density lipoprotein lysine micro milli m o l a r i n the c o n t e x t o f c o n c e n t r a t i o n ; m e t h i o n i n e i n the c o n t e x t o f protein sequence monoclonal antibody  MDMV  Maize dwarf mosaic virus  min MP  minute(s) movement protein  ELISA  GAP Glu Gly GP h H HAT HC-Pro  kb kDa 1 L LDL Lys |i m M  mRNA  N NAT ng NMR nt  messenger R N A  asparagine not aphid transmissible nanogram nuclear magnetic resonance nucleotide  x  N-terminal  oligo(s)  ORF P  PAGE PBS PCR PEBV  PEG Phe  arnino-terrninal oligonucleotide(s)  open reading frame p r o l i n e i n the c o n t e x t o f p r o t e i n sequence; p r o t r u d i n g d o m a i n i n the context of C N V C P polyacrylamide gel electrophoresis phosphate buffered saline polymerase chain reaction Pea early browning virus  Pro  polyethylene glycol phenylalanine full length infectious c D N A clone of C N V proline  PVY  Potato virus Y  Q R  glutamine arginine ribonucleic acid(s)  pK2/M5  RNA(s) RRSV  RTD  RT-PCR  S SDS sec Ser T TAE TBE  Raspberry ringspot virus  readthrough domain reverse-transcription P C R s e r i n e i n the c o n t e x t o f p r o t e i n sequence; s h e l l d o m a i n i n the c o n t e x t of C N V C P s o d i u m d o d e c y l sufate seconds serine t h y m i d i n e i n the c o n t e x t o f a n u c l e o t i d e sequence; t h r e o n i n e i n the context of protein sequence T r i s acetate E D T A Tris borate E D T A  TBRV  Tomato blackring virus  TBS  Tris buffered saline t h e c h e r r y s t r a i n o f t o m a t o b u s h y stunt v i r u s  TBSV-Ch TEV  Tobacco etch virus  Thr  threonine  TMV TNV  Tobacco mosaic virus Tobacco necrosis virus  TPA  Tetragonobolus purpureas a g g l u t i n i n  Tris  Trp  Tris-hydroxymethyl amino methane Tryptophan  TRV TSWV TuMV TVMV  Tobacco rattle virus Tomato spotted wilt virus Turnip mosaic virus Tobacco vein mottling virus  Tyr  Tyrosine  xi  u V Val VAP W WGA WT Y ZYMV  uridine i n the context o f nucleotide sequence; units w h e n referring to amount o f enzyme volts i n the context o f electrophoresis; valine i n the context o f protein sequence valine viral attachment protein tryptophan wheat germ agglutinin wild-type tyrosine  Zucchini yellow mosaic virus  xii  Acknowledgements I w o u l d l i k e to t h a n k D r . D ' A n n R o c h o n f o r h e r g e n e r o u s g u i d a n c e c o n c e r n i n g all t h e o r e t i c a l a n d t e c h n i c a l a s p e c t s o f this p r o j e c t a n d a l s o f o r h e r i n c r e d i b l e p a t i e n c e as m y i n t e l l e c t u a l p a r e n t d u r i n g t h o s e t u m u l t u o u s a d o l e s c e n t years. I w o u l d a l s o l i k e to t h a n k the N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l a n d t h e U n i v e r s i t y o f B r i t i s h C o m u m b i a f o r f i n a n c i a l assistance. M a n y t h a n k s to m y s u p e r v i s o r y c o m m i t t e e , D r . C a r o l i n e Astell, Dr. J i m Kronstad, and Dr. B r i a n Ellis f o r their helpful input o n the project. I e s p e c i a l l y w o u l d l i k e to t h a n k D r . B r i a n E l l i s f o r the g e n e r o u s u s e o f l a b o r a t o r y s p a c e a n d e q u i p m e n t d u r i n g p a r t o f this study. I w o u l d a l s o l i k e to t h a n k t h e P a c i f i c A g r i c u l t u r e R e s e a r c h C e n t r e f o r the u s e o f their facilities a n d e x p e r t i s e , e s p e c i a l l y M i c h a e l W e i s f o r p h o t o g r a p h y , c o m p u t e r , a n d m i c r o s c o p y a d v i c e . M a n y t h a n k s to the R o c h o n L a b ; D r . T i m Sit w h o t a u g h t m e h o w to c l o n e a n d h o w to c o o l a c o c a - c o l a to p e r f e c t n e a r - f r e e z i n g t e m p e r a t u r e s , D r . R e n e e F i n n e n , w h o s h o w e d m e h o w to b e n i c e to m y R N A , D r . J u l i e J o h n s t o n , f o r m a n y late n i g h t e x p e r i m e n t s a n d l a u g h t e r a n d D r . H o w a r d D a m u d e f o r m a n y f u n a d v e n t u r e s . I w o u l d l i k e to t h a n k R o n R e a d e f o r all h i s technical help and for being my friend i n Summerland.  I w o u l d a l s o l i k e to t h a n k D r .  Jane Miller, K i m M a c D o n a l d , Shannon W a r d , Steve Orbin, Dr. Joan M a c P h e r s o n , Dr. M a y n a r d and M i c h e l l e M i l k s , Jane Theilmann, Berend-Jan B o s c h and K i s h o r e K a k a n i for their f r i e n d s h i p i n S u m m e r l a n d .  I w o u l d l i k e to t h a n k D r . J u l i e J o h n s t o n , E l i a n e  Ubalijoro, Shannon M c L e a n , Dr. Claire Huguenot, Dr. K a r m a Carrier and my f a m i l y for their e m o t i o n a l s u p p o r t w h i l e I w a s i n S u m m e r l a n d .  I w o u l d e s p e c i a l l y l i k e to t h a n k m y  partner, A n d r e w P a r k e r , who, a s i d e f r o m b e i n g m y b e s t f r i e n d , has b e e n the m a j o r i n s t i g a t o r a n d s u p p o r t e r o f m y s c i e n t i f i c c a r e e r t h u s far. xiii  1 CHAPTER ONE: LITERATURE REVIEW  1.1 Introduction  Transmission o f most plant viruses is a highly specific process i n w h i c h particular i n v e r t e b r a t e s a n d f u n g i act as v e c t o r s ( M a t t h e w s , 1991). V e c t o r s o f p l a n t v i r u s e s a i d i n b r e a c h i n g the p l a n t c e l l w a l l entry b a r r i e r a n d a l s o c o n t r i b u t e t o t h e s p e c i f i c i t y o f t r a n s m i s s i o n . I n a n i m a l v i r u s - h o s t c e l l i n t e r a c t i o n s , attachment t o s p e c i f i c r e c e p t o r s o n the p l a s m a m e m b r a n e o f s u s c e p t i b l e c e l l s is a n e c e s s a r y step i n t h e i n f e c t i o n p r o c e s s a n d is a m a j o r d e t e r m i n a n t o f v i r u s - t i s s u e t r o p i s m ( T a r d i e u , 1982; M a r s h a n d H e l e n i u s , 1989). T h e r e h a v e b e e n n o reports o f r e c e p t o r - m e d i a t e d r e c o g n i t i o n o f p l a n t c e l l s b y viruses. T h e specificity o f natural plant virus infection is probably controlled v i a receptors, b u t m o r e l i k e l y b y r e c e p t o r s a s s o c i a t e d w i t h the v e c t o r rather than the p l a n t c e l l membrane. A l t h o u g h there i s c o n s i d e r a b l e k n o w l e d g e c o n c e r n i n g the b i o l o g y o f p l a n t v i r u s v e c t o r i n t e r a c t i o n s , l i t t l e is k n o w n about s u c h i n t e r a c t i o n s at the m o l e c u l a r l e v e l . A n u n d e r s t a n d i n g o f the s p e c i f i c i n t e r a c t i o n s that o c c u r b e t w e e n v i r u s p a r t i c l e s a n d t h e p u t a t i v e r e c e p t o r s o f the v e c t o r s w h i c h t r a n s m i t t h e m s h o u l d p r o v i d e  fundamental  k n o w l e d g e c o n c e r n i n g v i r u s - v e c t o r relations, and, m o r e o v e r , m a y l e a d t o t h e d e v e l o p m e n t o f n e w strategies f o r v i r u s d i s e a s e c o n t r o l . Interactions b e t w e e n Cucumber necrosis virus ( C N V ) a n d the f u n g u s Olpidium bornovanus are b e i n g s t u d i e d t o d e t e r m i n e h o w p l a n t v i r u s e s a n d t h e i r v e c t o r s i n t e r a c t at  1  the m o l e c u l a r l e v e l . C N V is n a t u r a l l y t r a n s m i t t e d to c u c u m b e r t h r o u g h the s o i l v i a z o o s p o r e s o f O. bornovanus ( C a m p b e l l , 1996; C a m p b e l l a n d S i m , 1994; D i a s , 1970a). P r e v i o u s w o r k i n this l a b o r a t o r y has s h o w n that the C N V c o a t p r o t e i n d e t e r m i n e s the s p e c i f i c i t y o f f u n g u s t r a n s m i s s i o n ( M c L e a n et al, 1994). D e l i n e a t i o n o f r e g i o n s o f c o a t p r o t e i n s i n v o l v e d i n v e c t o r - t r a n s m i s s i o n has b e e n f a c i l i t a t e d b y the p r e s e n c e o f n a t u r a l v i r a l v a r i a n t s w h i c h h a v e l o s t t h e i r a b i l i t y to b e t r a n s m i t t e d v i a l o n g t e r m m e c h a n i c a l p a s s a g e (see C a m p b e l l , 1996; G r a y , 1996). P r i o r to the w o r k d e s c r i b e d i n t h i s thesis, n o v a r i a n t s o f C N V h a d p r e v i o u s l y b e e n i d e n t i f i e d w h i c h h a d l o s t t h e i r a b i l i t y to be f u n g u s - t r a n s m i t t e d . F u r t h e r m o r e , n o s p e c i f i c r e g i o n s o f the C N V c o a t p r o t e i n (i.e. p o t e n t i a l r e c e p t o r b i n d i n g sites) h a d y e t b e e n d e l i n e a t e d i n the specificity of fungus transmission. Z o o s p o r e s o f O. bornovanus h a v e b e e n s h o w n b y D i a s ( 1 9 7 0 a ) to t r a n s m i t  CNV  t o c u c u m b e r roots. T r a n s m i s s i o n o f v i r u s e s b y O. bornovanus i s s p e c i f i c , w i t h p a r t i c u l a r strains o f the f u n g u s f r o m m e l o n , s q u a s h o r c u c u m b e r b e i n g a b l e to t r a n s m i t o n l y s p e c i f i c v i r u s e s ( C a m p b e l l et al, 1995). T h e b a s i s f o r the s p e c i f i c i t y o f i n t e r a c t i o n i s u n k n o w n , b u t I h y p o t h e s i z e that s p e c i f i c r e c e p t o r m o l e c u l e s o n the s u r f a c e o f f u n g a l z o o s p o r e s interact w i t h s p e c i f i c sites o n the v i r u s to e n a b l e t r a n s m i s s i o n . T h i s literature r e v i e w is o r g a n i z e d i n t o three s e c t i o n s . T h e first s e c t i o n d e a l s w i t h p l a n t v i r u s t r a n s m i s s i o n . G e n e r a l aspects o f n e m a t o d e , insect, a n d f u n g a l t r a n s m i s s i o n w i l l b e r e v i e w e d f o l l o w e d b y a r e v i e w o f Olpidium t r a n s m i s s i o n w i t h p a r t i c u l a r e m p h a s i s o n w h a t is k n o w n a b o u t t r a n s m i s s i o n o f C N V b y O. bornovanus a n d T N V b y O. brassicae. In the s e c o n d s e c t i o n , the b i o l o g i c a l a n d m o l e c u l a r p r o p e r t i e s o f C N V  2  as w e l l  as its c a p s i d a r c h i t e c t u r e w i l l b e d e s c r i b e d . In the t h i r d s e c t i o n , g e n e r a l i n f o r m a t i o n a b o u t receptor-mediated animal virus cell recognition and entry will be provided.  1.2 Modes of plant virus transmission V i r u s e s are o b l i g a t e parasites a n d m o v e m e n t f r o m o n e s u s c e p t i b l e h o s t to a n o t h e r is n e c e s s a r y f o r v i r u s p e r p e t u a t i o n i n nature ( M a t t h e w s , 1991). P l a n t v i r u s e s are u n a b l e to penetrate the i n t a c t p l a n t c u t i c l e . T o o v e r c o m e this barrier, v i r u s e s e i t h e r a v o i d the n e e d to penetrate the c e l l w a l l b y n o n s p e c i f i c m e a n s s u c h as m e c h a n i c a l  transmission,  seed transmission, vegetative propagation, grafting, or dodder, or viruses f o r m specific a s s o c i a t i o n s w i t h i n v e r t e b r a t e o r f u n g a l v e c t o r s w h i c h penetrate p l a n t c u t i c l e s to e n a b l e their entry ( M a t t h e w s , 1991; T a b l e 1.1). F o r an i n v e r t e b r a t e o r f u n g u s to f u n c t i o n as a vector, v i r u s p a r t i c l e s f r o m an i n f e c t e d p l a n t m u s t a s s o c i a t e w i t h the v e c t o r a n d b e r e t a i n e d i n an i n f e c t i o u s f o r m l o n g e n o u g h to s u b s e q u e n t l y d i s s o c i a t e a n d b e i n o c u l a t e d i n t o a n e w host plant. P l a n t v i r u s e s , l i k e a n i m a l v i r u s e s , h a v e e v o l v e d m e c h a n i s m s to use e x i s t i n g structures a n d p a t h w a y s a s s o c i a t e d w i t h t h e i r v e c t o r s (or hosts i n the c a s e o f a n i m a l v i r u s e s ) i n o r d e r to p e r f o r m the three p h a s e s o f the t r a n s m i s s i o n p r o c e s s : a c q u i s i t i o n , r e t e n t i o n , a n d i n o c u l a t i o n ( G r a y , 1996; P i r o n e a n d B l a n c , 1996).  3  Table  1.1 P r i n c i p a l v e c t o r s o f p l a n t v i r u s g e n e r a a n d their m e c h a n i s m o f t r a n s m i s s i o n 1,2  Vector  Virus  Transmission Mechanism  Aphids  Caulimovirus Faba virus Potyvirus Carlavirus Cucumovirus Alfamovirus Potexvirus Closterovirus Sequiviurs Trichovirus Waikavirus Luteovirus Nanavirus Umbravirus Enamovirus Machlomovirus Tospovirus Machlomovirus Bromo virus Carmovirus Comovirus Sobemovirus Tymovirus Badnavirus Closterovirus Trichovirus Badnavirus Waikavirus Monogeminivirus  Non-circulative  Thrips Beetles  Mealybugs  Leafhoppers  Circulative  Non-circulative Circulative Non-circulative Circulative  Non-circulative  Non-circulative Circulative  Marafivirus  Phytoreovirus Hybrigeminivirus Circulative Closterovirus Non-circulative Bigemini virus Circulative Potexvirus Mites Non-circulative Trichovirus Rymovirus Nepovirus Nematodes Non-circulative Tobravirus Fungi Necrovirus Non-circulative (In vitro) a Tombusvirus Aureusvirus Dianthovirus Varicosavirus Circulative (In vivo) Bymovirus Furovirus This table was abstracted and modified from Gray and Rochon (1999). It should be noted that in some cases, not all members of a genus are transmitted by the indicated vector. Treehoppers Whiteflies  2  4  Several m e c h a n i s m s o f plant virus transmission have been proposed based on i n f o r m a t i o n f r o m studies o v e r the last 50 years ( G r a y a n d R o c h o n , 1999; C a m p b e l l ,  1996;  G r a y , 1996; P i r o n e a n d B l a n c , 1996). T h e s e m o d e l s o f t r a n s m i s s i o n r a n g e f r o m t h o s e b a s e d o n b i o l o g i c a l studies to m o r e recent o n e s u t i l i z i n g r e s u l t s o b t a i n e d f r o m m o l e c u l a r studies. U l t i m a t e l y , a k n o w l e d g e o f the p r e c i s e structures o f v i r u s a n d v e c t o r w h i c h m e d i a t e the s p e c i f i c i t y o f t r a n s m i s s i o n at the m o l e c u l a r l e v e l w i l l a i d i n c l a r i f i c a t i o n o f these m o d e l s . Early examinations of aphid- and leafhopper-transmission of plant viruses p r o d u c e d c r i t e r i a f o r v i r u s c l a s s i f i c a t i o n b a s e d o n the l e n g t h o f t i m e that i n f e c t i v e v i r u s r e s i d e d i n the v e c t o r b e f o r e l o s i n g its a b i l i t y to b e t r a n s m i t t e d to a h e a l t h y p l a n t ( G r a y a n d R o c h o n , 1999). N o n - p e r s i s t e n t v i r u s e s are r e t a i n e d i n t h e i r v e c t o r f o r s e v e r a l hours, s e m i - p e r s i s t e n t v i r u s e s f o r d a y s a n d s o m e t i m e s w e e k s , a n d p e r s i s t e n t v i r u s e s f o r the l i f e o f the v e c t o r ( G r a y a n d R o c h o n , 1999). N o n - p e r s i s t e n t a n d s e m i - p e r s i s t e n t v i r u s e s are acquired and inoculated within seconds or minutes while persistent viruses require hours to days. T h e use o f the a b o v e c r i t e r i a to c a t e g o r i z e v i r u s - v e c t o r a s s o c i a t i o n s b e c a m e p r o b l e m a t i c w i t h the d i s c o v e r y o f a d d i t i o n a l a r t h r o p o d , n e m a t o d e a n d f u n g a l v e c t o r s ( G r a y a n d R o c h o n , 1999). Recently, plant viruses with k n o w n vectors have been divided into two categories b a s e d o n their m o d e o f t r a n s m i s s i o n : c i r c u l a t i v e o r n o n - c i r c u l a t i v e ( G r a y , 1 9 9 6 , T a b l e 1.1). C i r c u l a t i v e v i r u s e s are a c t i v e l y t r a n s p o r t e d a c r o s s v e c t o r m e m b r a n e s a n d s u r v i v e i n s i d e the vector. T h e s e v i r u s e s c a n f u r t h e r b e c h a r a c t e r i z e d i n t o p r o p a g a t i v e v i r u s e s , w h i c h r e p l i c a t e i n their a r t h r o p o d vector, a n d n o n - p r o p o g a t i v e v i r u s e s , w h i c h d o n o t ( G r a y a n d R o c h o n , 1999). N o n - c i r c u l a t i v e v i r u s e s are a s s o c i a t e d e x t e r n a l l y a n d d o n o t  5  c r o s s m e m b r a n e b a r r i e r s ( P i r o n e a n d B l a n c , 1996). T h e m a j o r i t y o f p l a n t v i r u s e s are n o t i n t e r n a l i z e d b y t h e i r v e c t o r s a n d t h e r e f o r e are c l a s s i f i e d as n o n - c i r c u l a t i v e . T h e c l a s s i f i c a t i o n o f v i r u s e s as e i t h e r c i r c u l a t i v e o r n o n - c i r c u l a t i v e c a n b e u s e d f o r a l l p l a n t v i r u s e s that r e q u i r e a s p e c i f i c i n v e r t e b r a t e v e c t o r f o r s u r v i v a l i n nature ( G r a y a n d R o c h o n , 1999). H o w e v e r , the debate c o n t i n u e s as to w h e t h e r the t e r m s c i r c u l a t i v e a n d nonc i r c u l a t i v e c a n b e u s e d to d e s c r i b e v i r u s e s w i t h f u n g a l v e c t o r s ( G r a y a n d R o c h o n , 1999; C a m p b e l l , 1996; C a m p b e l l , 1993). It has b e e n a r g u e d that the i n t e r a c t i o n b e t w e e n v i r u s e s a n d t h e i r f u n g a l v e c t o r s c a n n o t be c h a r a c t e r i z e d as c i r c u l a t i v e / n o n - c i r c u l a t i v e b e c a u s e f u n g i h a v e n o c i r c u l a t o r y s y s t e m ( C a m p b e l l , 1993). T h e t e r m s in vitro a n d in vivo a c q u i s i t i o n are u s e d b y those i n the f i e l d to d e s c r i b e the i n t e r a c t i o n b e t w e e n a v i r u s  a n d its f u n g a l v e c t o r ( C a m p b e l l , 1996). In in vitro a c q u i s i t i o n , v i r u s c o m e s i n c o n t a c t w i t h its f u n g a l v e c t o r o u t s i d e the c e l l s o f l i v i n g p l a n t s a n d c a n s u r v i v e i n the s o i l i n d e p e n d e n t o f v e c t o r o r plant. In in vivo a c q u i s i t i o n , v i r u s is a c q u i r e d w h i l e the f u n g u s is w i t h i n p l a n t r o o t c e l l s a n d s u r v i v e s o n l y w i t h i n r e s t i n g spores ( C a m p b e l l , 1993). T h e t e r m p e r s i s t e n t ( c i r c u l a t i v e ) has b e e n u s e d to r e f e r to v i r u s e s t r a n s m i t t e d b y f u n g i i n the in vitro m a n n e r w i t h n o n - p e r s i s t e n t ( n o n - c i r c u l a t i v e ) f o r v i r u s e s a c q u i r e d in vivo ( T e a k l e ,  1983). T h i s a n a l o g y has b e e n u s e d b y others w i t h the c a u t i o n that the c o m p a r i s o n i s n o t e n t i r e l y v a l i d d u e to the d i f f e r e n c e s i n b i o l o g y b e t w e e n f u n g i a n d i n v e r t e b r a t e s ( A d a m s , 1991). T h e r e a p p e a r to b e e n o u g h c o m m o n a l i t i e s b e t w e e n t r a n s m i s s i o n m e c h a n i s m s i n v o l v i n g i n v e r t e b r a t e s a n d f u n g i to w a r r a n t the use o f c i r c u l a t i v e / n o n - c i r c u l a t i v e to d e s c r i b e the i n t e r a c t i o n s o f a l l v i r u s e s w i t h t h e i r v e c t o r s as p r o p o s e d b y G r a y a n d R o c h o n (1999). H o w e v e r , the t e r m s in vitro a n d in vivo are n e c e s s a r y i n the d i s c u s s i o n o f f u n g u s  6  t r a n s m i s s i o n as these t e r m s g i v e a d d i t i o n a l a n d n e c e s s a r y i n f o r m a t i o n c o n c e r n i n g the f u n g a l l i f e - c y c l e a n d m o d e o f t r a n s m i s s i o n ( C a m p b e l l , 1996). T h e r e f o r e , b o t h sets o f terms, c i r c u l a t i v e / n o n c i r c u l a t i v e a n d in vivo/in vitro, w i l l b e u s e d i n d i s c u s s i o n s o f f u n g u s t r a n s m i s s i o n i n this thesis. T h e f o l l o w i n g s e c t i o n s d e s c r i b e the b i o l o g y o f s p e c i f i c i n t e r a c t i o n s b e t w e e n i n v e r t e b r a t e a n d f u n g a l v e c t o r s w i t h the v i r u s e s t h e y transmit, m o d e l s o f t r a n s m i s s i o n a r i s i n g f r o m this i n f o r m a t i o n , a n d w h a t is k n o w n at the m o l e c u l a r l e v e l o f these r e l a t i o n s . T h e i n t e r a c t i o n s o f i n v e r t e b r a t e v e c t o r s s u c h as a p h i d s , w h i t e f l i e s , a n d n e m a t o d e s w i t h the v i r u s e s t h e y t r a n s m i t w i l l be e x a m i n e d i n d e t a i l s i n c e the m a j o r i t y o f r e s e a r c h i n t o p l a n t v i r u s t r a n s m i s s i o n has d e a l t w i t h these s y s t e m s . P a r a l l e l s c a n b e d r a w n b e t w e e n p l a n t v i r u s t r a n s m i s s i o n b y i n v e r t e b r a t e a n d f u n g a l v e c t o r s w i t h i n the b r o a d e r m o d e l s o f vector transmission. •1.2.1 A r t h r o p o d t r a n s m i s s i o n A t least 7 0 % o f p l a n t v i r u s e s are k n o w n to be t r a n s m i t t e d b y a r t h r o p o d s ( v a n d e n H e u v e l et al., 1999). K u n k e l ( 1 9 2 2 ) was the first to r e p o r t t r a n s m i s s i o n o f a v i r u s b y a n insect; i n this c a s e the t r a n s m i s s i o n o f y e l l o w stripe d i s e a s e b y a p l a n t h o p p e r .  Two  c l a s s e s o f the A r t h r o p o d a c o n t a i n m e m b e r s w i t h v i r u s v e c t o r s , the I n s e c t a a n d A r a c h n i d a ( M a t t h e w s , 1991). T h e m a j o r c l a s s i m p o r t a n t f o r p l a n t v i r u s t r a n s m i s s i o n i s the Insecta, c o n t a i n i n g the o r d e r H o m o p t e r a w h i c h f e e d b y s u c k i n g o n g r e e n p l a n t s a n d i n c l u d e s a p h i d s , w h i t e f l i e s , h o p p e r s , a n d m e a l y b u g s ( M a t t h e w s , 1991). In the f o l l o w i n g s e c t i o n , the m e c h a n i s m s b y w h i c h i n s e c t s i n the H o m o p t e r a t r a n s m i t p l a n t v i r u s e s w i l l b e d e s c r i b e d .  7  1.2.1.1 N o n - c i r c u l a t i v e a p h i d t r a n s m i s s i o n T h e m a j o r i t y o f the 2 5 0 k n o w n a p h i d - t r a n s m i t t e d v i r u s e s a r e t r a n s m i t t e d i n a n o n c i r c u l a t i v e m a n n e r ( M a t t h e w s , 1991). A s p r e v i o u s l y d i s c u s s e d , n o n - c i r c u l a t i v e v i r u s e s d o n o t c r o s s m e m b r a n e b a r r i e r s i n t h e i r v e c t o r a n d s o are d e s c r i b e d as a s s o c i a t i n g e x t e r n a l l y ( P i r o n e a n d B l a n c , 1996). I n t h e c a s e o f a p h i d s , n o n - c i r c u l a t i v e t r a n s m i s s i o n refers t o v i r u s e s w h i c h are r e t a i n e d i n t h e a p h i d stylets ( m o u t h p a r t s ) after f e e d i n g p r o b e s o n i n f e c t e d plants. V i r u s c a n a l s o b e f o u n d i n t h e a p h i d f o o d c a n a l a n d f o r e g u t b u t d o e s n o t c r o s s m e m b r a n e b a r r i e r s i n t h e a p h i d a n d t h e r e f o r e d o e s n o t enter the a p h i d h e m o c o e l ( c i r c u l a t o r y system). T h e r e t e n t i o n o f v i r i o n s i n t h e a p h i d stylet i s a s p e c i f i c p r o c e s s ( d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n ) i m p l y i n g that s p e c i f i c m o l e c u l e s i n t h e stylets as w e l l as s p e c i f i c r e g i o n s o f the v i r u s p a r t i c l e are i n v o l v e d i n t r a n s m i s s i o n . 1.2.1.1.1 V i r u s r e t e n t i o n A f t e r l a n d i n g o n l e a v e s , a p h i d s m a k e b r i e f p r o b e s to test t h e s u i t a b i l i t y o f the f o o d s o u r c e ( M a t t h e w s , 1991). A p h i d - t r a n s m i s s i o n h a s b e e n s h o w n t o o c c u r d u r i n g b r i e f i n t r a c e l l u l a r f e e d i n g p r o b e s o f 5-10 s e c o n d s o n p l a n t e p i d e r m a l o r m e s o p h y l l c e l l s ( P o w e l l , 1991; P o l l a r d , 1973). S a p s a m p l i n g o n v i r u s - i n f e c t e d p l a n t s c o n t a m i n a t e s t h e a p h i d stylet tips, f o o d c a n a l , a n d f o r e g u t w i t h v i r u s ( M a t t h e w s , 1991). A q u e s t i o n d e b a t e d i n r e c e n t years c o n c e r n s t h e site o f v i r u s i n o c u l u m r e t e n t i o n i n t h e a p h i d . Is t h e stylet, f o o d c a n a l , and/or f o r e g u t t h e p r i m a r y r e s e r v e o f i n f e c t i o u s v i r i o n s f o r n o n circulative transmission b y aphids? H o g g a n ( 1 9 3 3 ) w a s t h e first t o suggest that v i r i o n s i n t h e stylets p l a y a s i g n i f i c a n t r o l e i n n o n - c i r c u l a t i v e t r a n s m i s s i o n . L a t e r , i t w a s d e m o n s t r a t e d that the a b i l i t y o f a p h i d s  8  to t r a n s m i t P V Y i s r e d u c e d b y c h e m i c a l a n d p h y s i c a l treatments o f a p h i d stylet tips ( B r a d l e y , 1964). M e m b r a n e f e e d i n g e x p e r i m e n t s w i t h d y e s s h o w e d that a p h i d s h a v e the a b i l i t y to transfer v i r u s f r o m i n f e c t e d to h e a l t h y p l a n t s v i a a r e g u r g i t a t i o n m e c h a n i s m ( H a r r i s a n d B a t h , 1973). B a s e d o n t h i s w o r k , a n ' i n g e s t i o n - e g e s t i o n ' m o d e l o f n o n - c i r c u l a t i v e a p h i d t r a n s m i s s i o n was s u g g e s t e d w h e r e v i r u s a c q u i s i t i o n o c c u r s d u r i n g i n g e s t i o n o f p l a n t c e l l contents f o l l o w e d b y i n o c u l a t i o n d u r i n g r e g u r g i t a t i o n o f p r e v i o u s l y i n g e s t e d sap f r o m the a l i m e n t a r y c a n a l a n d f o r e g u t ( H a r r i s , 1977). T h i s h y p o t h e s i s i s w i d e l y a c c e p t e d a n d g i v e s the i m a g e o f a p h i d s as ' f l y i n g s y r i n g e s ' a c t i v e l y c o n t r i b u t i n g to a c q u i s i t i o n a n d r e l e a s e o f virus. T h e a p h i d - l o c a t i o n o f v i r i o n s i n v o l v e d i n t r a n s m i s s i o n was s t u d i e d b y W a n g et  al.  (1996). O f 523 a p h i d s f e d I - l a b e l l e d T E V - H A T (a h i g h l y t r a n s m i s s i b l e i s o l a t e o f 125  Tobacco etch virus), 4 8 % r e t a i n e d l a b e l i n the stylets v e r s u s 0 . 7 7 % f e d T E V - N A T (not  t r a n s m i s s i b l e ) . It was c o n c l u d e d that v i r i o n s r e t a i n e d w i t h i n the f o o d c a n a l (distal t h i r d ) o f the stylets w e r e t h o s e w h i c h w e r e p r i m a r i l y i n v o l v e d i n p o t y v i r u s t r a n s m i s s i o n ( W a n g etal., 1996).  1.2.1.1.2 C o a t p r o t e i n T h e r e i s a h i g h d e g r e e o f s p e c i f i c i t y i n the i n t e r a c t i o n b e t w e e n n o n c i r c u l a t i v e v i r u s e s a n d t h e i r a p h i d v e c t o r s ( K e n n e d y et al., 1962; M a t t h e w s , 1991). S t r a i n s o f the s a m e v i r u s m a y d i f f e r i n e f f i c i e n c y o f t r a n s m i s s i o n b y a p a r t i c u l a r a p h i d s p e c i e s . In a d d i t i o n , a p h i d s p e c i e s v a r y w i d e l y i n the n u m b e r o f d i f f e r e n t v i r u s e s t h e y transmit. T h i s s p e c i f i c i t y is best e x p l a i n e d b y s p e c i f i c a d s o r p t i o n b e t w e e n v i r u s e s a n d r e c e p t o r o r r e c e p t o r - l i k e sites i n the a p h i d f o o d c a n a l ( W a t s o n a n d P l u m b , 1972). 9  T h e m a j o r i t y o f w o r k thus f a r has f o c u s e d o n the v i r u s p a r t i c l e , n a m e l y the v i r a l c o a t p r o t e i n ( C P ) , a n d its r o l e i n the s p e c i f i c i t y o f t r a n s m i s s i o n . N o n - t r a n s m i s s i b l e v i r u s v a r i a n t s h a v e b e e n d e s c r i b e d f o r s e v e r a l v i r u s - v e c t o r c o m b i n a t i o n s after serial p a s s a g i n g i n the a b s e n c e o f the vector, i.e. d i r e c t l y f r o m p l a n t to p l a n t v i a m e c h a n i c a l i n o c u l a t i o n ( G r a y , 1996). F o r s o m e o f these v a r i a n t s i t was f o u n d that the C P o f the n o n t r a n s m i s s i b l e v a r i a n t s was d i f f e r e n t f r o m that o f the t r a n s m i s s i b l e v i r u s . C o m p a r i s o n s o f C P a m i n o acid sequences between transmissible and nont r a n s m i s s i b l e p o t y v i r u s e s s h o w e d c h a n g e s i n an A s p - A l a - G l y ( D A G ) triplet as a c o n s i s t e n t feature o f n o n - t r a n s m i s s i b l e p o t y v i r u s i s o l a t e s ( H a r r i s o n a n d R o b i n s o n , 1988). It was h y p o t h e s i z e d that the p o t y v i r u s D A G  s e q u e n c e was i n v o l v e d i n b i n d i n g o f v i r i o n s  to a p h i d m o u t h p a r t s ( H a r r i s o n a n d R o b i n s o n , 1988). In this m o d e l , v i r i o n r e l e a s e f o r i n o c u l a t i o n o c c u r r e d b y c l e a v a g e o f the N - t e r m i n u s o f the c o a t p r o t e i n b y t r y p s i n - l i k e e n z y m e s i n the s a l i v a o f the a p h i d . T h e N - t e r m i n u s o f the C P o f p o t y v i r u s e s i s e x p o s e d o n the p a r t i c l e s u r f a c e ( A l l i s o n et al., 1985; S h u k l a et ah, 1988). E n z y m a t i c r e m o v a l o f the C P N - t e r m i n a l d o m a i n has b e e n s h o w n to r e s u l t i n the l o s s o f a p h i d t r a n s m i s s i b i l i t y ( S a l o m o n , 1989). D i r e c t e v i d e n c e that the D A G  sequence is important for non-circulative aphid  t r a n s m i s s i o n o f p o t y v i r u s e s was p r o v i d e d i n e x p e r i m e n t s w h e r e c D N A o f the C P g e n e f r o m a n o n - t r a n s m i s s i b l e i s o l a t e o f Tobacco vein mottling virus ( T V M V ) ( w i t h a m u t a t i o n i n the D A G  m o t i f ) was c l o n e d i n p l a c e o f the C P g e n e o f a t r a n s m i s s i b l e T V M V  i s o l a t e c o n t a i n i n g the D A G  m o t i f ( A t r e y a et al, 1990). T h e t r a n s c r i b e d R N A  i n o c u l a t e d o n t o t o b a c c o p l a n t s a n d a p h i d s w e r e u n a b l e to t r a n s m i t the r e s u l t a n t m u t a n t ( A t r e y a et al, 1990).  10  was DAG  F u r t h e r s t u d i e s s h o w e d that d e l e t i o n o f the D A G triplet f r o m a p o t y v i r a l C P r e s u l t e d i n the l o s s o f a p h i d t r a n s m i s s i b i l i t y ( A t r e y a et ah, 1991). D e l e t i o n o f e a c h i n d i v i d u a l a m i n o a c i d o f D A G h a d a s i m i l a r effect. L a t e r , s e v e r a l s u b s t i t u t i o n s w e r e i n t r o d u c e d b y s i t e - d i r e c t e d m u t a g e n e s i s i n a n d a r o u n d the D A G m o t i f ( e x p a n d e d to D A G X b a s e d o n the e f f e c t o f c h a n g i n g r e s i d u e s i n c l o s e p r o x i m i t y to D A G ) to d e t e r m i n e a m i n o a c i d s n e c e s s a r y a n d s u f f i c i e n t f o r t r a n s m i s s i o n ( A t r e y a et al., 1995). I n the first p o s i t i o n A s p o r A s n are r e q u i r e d , i n the s e c o n d p o s i t i o n o n l y A l a g i v e s t r a n s m i s s i o n , a n d i n the t h i r d p o s i t i o n G l y appears c r i t i c a l f o r t r a n s m i s s i o n . S t u d i e s h a v e s h o w n that the C P i s s u f f i c i e n t f o r the t r a n s m i s s i o n o f c u c u m o v i r u s e s [the type m e m b e r Cucumber mosaic virus ( C M V ) b e i n g the m o s t s t u d i e d ( C h e n a n d F r a n c k i , 1990)]. P r o p e r t i e s o f the C M V C P a f f e c t v a r i a b i l i t y i n t r a n s m i s s i o n e f f i c i e n c y o f the v i r u s ( G e r a et al., 1979; P e r r y et al., 1998). T h i s n a t u r a l l y o c c u r i n g v a r i a b i l i t y m a y b e s e l e c t e d f o r b y r e p e a t e d m e c h a n i c a l p a s s a g e o f the v i r u s i n the a b s e n c e o f the a p h i d vector. M u t a g e n e s i s s t u d i e s h a v e i n d i c a t e d that s i n g l e a m i n o a c i d c h a n g e s i n t w o r e g i o n s o f the C M V C P c a n a b o l i s h its a b i l i t y to b e a p h i d - t r a n s m i t t e d ( P e r r y et al., 1994). S u c h c h a n g e s m a y d i r e c t l y o r i n d i r e c t l y a f f e c t the a b i l i t y o f C M V to i n t e r a c t w i t h its v e c t o r . N o l i n e a r a m i n o a c i d s e q u e n c e m o t i f s s u c h as the D A G X o f p o t y v i r u s e s h a v e b e e n i d e n t i f i e d f o r C M V . R e p e a t e d m e c h a n i c a l p a s s a g e p r o d u c e d a m u t a n t that i s o n l y t r a n s m i s s i b l e b y o n e o f the t w o a p h i d s p e c i e s n o r m a l l y t r a n s m i t t i n g C M V ( N g a n d Perry, 1999). 1.2.1.1.3 H e l p e r c o m p o n e n t A p h i d transmission of potyviruses requires a functional virally-encoded nonstructural h e l p e r c o m p o n e n t ( H C - P r o ) p r o t e i n ( G o v i e r a n d K a s s a n i s , 1974; T h o r n b u r y et 11  al., 1985; r e v i e w e d b y P i r o n e a n d B l a n c , 1996). H C - P r o a c t i v i t y i s e x c h a n g e a b l e a m o n g p o t y v i r u s s p e c i e s w i t h d i f f e r i n g degrees o f s p e c i f i c i t y ( P i r o n e , 1981; S a k o a n d Ogata, 1981). O n e h y p o t h e s i s f o r the f u n c t i o n o f the p o t y v i r a l H C - P r o i n n o n - c i r c u l a t i v e a p h i d t r a n s m i s s i o n i s that i t m e d i a t e s the b i n d i n g o f v i r u s to sites w i t h i n the a p h i d a l i m e n t a r y c a n a l ( B e r g e r a n d P i r o n e , 1986). V i r u s - l i k e p a r t i c l e s h a v e b e e n o b s e r v e d i n a m a t r i x a s s o c i a t e d w i t h the stylets a n d f o r e g u t o f a p h i d s f e d o n a m i x t u r e o f v i r u s a n d H C - P r o . N o b o u n d particles were observed w h e n aphids were fed o n virus alone or w h e n viral o r H C - P r o m u t a n t s i n c o m p e t e n t f o r a p h i d t r a n s m i s s i o n w e r e u s e d ( A m m a r et al, 1994). T h e a c t i o n o f H C - P r o c o u l d b e direct, b y l i n k i n g v i r u s v i a the C P D A G X s e q u e n c e to the a p h i d ( b r i d g e h y p o t h e s i s ) , o r i n d i r e c t , b y m o d i f y i n g c o m p o u n d s i n the a p h i d to a l l o w v i r u s b i n d i n g ( P i r o n e , 1991). T o test w h e t h e r the N - t e r m i n u s o f a p o t y v i r u s C P , w h i c h c o n t a i n s the D A G X m o t i f described above, w a s i n v o l v e d i n virus retention i n aphids, recombinant protein c o n t a i n i n g the a m i n o - t e r m i n a l r e g i o n o f Maize dwarf mosaic potyvirus ( M D M V ) w a s f e d to a p h i d s b e f o r e f e e d i n g t h e m M D M V v i r i o n s a n d H C - P r o ( S a l o m o n a n d B e r n a r d i , 1995). A p h i d t r a n s m i s s i o n o f M D M V w a s s i g n i f i c a n t l y r e d u c e d i n these e x p e r i m e n t s . T h e s e d a t a s u g g e s t e d that the r e c o m b i n a n t p r o t e i n c o m p e t e d w i t h v i r u s - b i n d i n g sites i n the a p h i d , p r e v e n t i n g s u b s e q u e n t v i r u s b i n d i n g . S a l o m o n a n d B e r n a r d i ( 1 9 9 5 ) h y p o t h e s i z e d that the a m i n o - t e r m i n a l r e g i o n o f the C P , rather t h a n H C - P r o , attaches to sites a l o n g the a p h i d f o o d c a n a l a n d that H C - P r o m e d i a t e s a c o n f o r m a t i o n a l c h a n g e i n the c o a t p r o t e i n a l l o w i n g the b i n d i n g o f v i r u s to a p h i d .  12  C u r r e n t e v i d e n c e s u p p o r t s t h e h y p o t h e s i s that H C - P r o acts as a 'bridge' b e t w e e n virus particle a n d aphid instead o f mediating a conformational change i n virus C P . In this m o d e l , H C - P r o b i n d s t o b o t h v i r u s p a r t i c l e a n d t h e stylet f o o d c a n a l , a l l o w i n g v i r i o n s t o b e r e t a i n e d i n t h e stylets w h e r e they are a v a i l a b l e to b e i n o c u l a t e d i n t o n e w hosts ( P i r o n e a n d B l a n c , 1996; W a n g et al., 1996; B l a n c et al., 1997). B l a n c et al. ( 1 9 9 7 ) s h o w e d a d i r e c t in vitro a s s o c i a t i o n b e t w e e n a p o t y v i r a l H C - P r o a n d t h e C P o f t r a n s m i s s i b l e strains, where non-functional C P s (mutated i n D A G ) failed to bind HC-Pro. T h e p o s s i b l e r o l e o f H C - P r o i n v e c t o r s p e c i f i c i t y w a s first s u g g e s t e d b y t h e w o r k o f S a k o et al. (1984). L a t e r , W a n g et al. ( 1 9 9 8 ) p r o v i d e d d i r e c t e v i d e n c e that t h e specificity a n d efficiency o f potyvirus transmission is regulated b y HC-Pro. Punctures o f the e p i d e r m a l p l a s m a l e m m a b y t h e m a x i l l a r y stylet t i p s c a n b e r e c o r d e d as d r o p s i n e l e c t r i c a l p o t e n t i a l ( T j a l l i n g i i , 1985). E l e c t r i c a l l y r e c o r d e d i n t r a c e l l u l a r stylet activities a p p e a r e d t o b e q u a l i t a t i v e l y s i m i l a r f o r a l l three a p h i d s p e c i e s e x a m i n e d , i n d i c a t i n g that aphid behaviour was not responsible for the differences i n virus transmissibility between a p h i d s p e c i e s ( W a n g et al, 1998). I n e x p e r i m e n t s t o test t h e h y p o t h e s i s that t h e s o u r c e o f H C - P r o w a s r e s p o n s i b l e f o r t h e d i f f e r e n t i a l a b i l i t y o f L. erysimi t o t r a n s m i t T E V a n d T u M V a n d f o r t h e l e s s e f f i c i e n t t r a n s m i s s i o n o f T u M V b y A. gossypii, a p h i d s w e r e a l l o w e d t o a c q u i r e h e t e r o l o g o u s a n d h o m o l o g o u s v i r i o n - H C - P r o c o m b i n a t i o n s ( W a n g et al, 1998). L. erysimi w a s u n a b l e t o t r a n s m i t T E V f r o m i n f e c t e d p l a n t s o r m i x t u r e s w i t h  P V Y H C - P r o b u t c o u l d t r a n s m i t T E V at a l e v e l c o m p a r a b l e t o Af. persicae w h e n t h e v i r u s w a s a c q u i r e d i n t h e p r e s e n c e o f the T u M V H C - P r o , i n d i c a t i n g that H C - P r o c a n r e g u l a t e t r a n s m i s s i o n s p e c i f i c i t y ( W a n g etal, 1998). A c o r r e l a t i o n w a s a l s o s h o w n f o r r e t e n t i o n  13  o f v i r u s i n the a p h i d stylets w i t h t r a n s m i s s i o n , f u r t h e r s t r e n g t h e n i n g the i d e a that d i f f e r e n c e s i n r e t e n t i o n e x p l a i n d i f f e r e n c e s i n t r a n s m i s s i o n ( W a n g et al, 1998). C o m p a r i s o n s o f H C - P r o sequences f r o m several transmissible and nont r a n s m i s s i b l e p o t y v i r u s e s h a v e i d e n t i f i e d t w o h i g h l y c o n s e r v e d d o m a i n s , the l y s i n e i s o l e u c i n e - t h r e o n i n e - c y s t e i n e ( K T T C ) d o m a i n ( T h o r n b u r y et al, 1990) a n d the p r o l i n e t h r e o n i n e - l y s i n e ( P T K ) d o m a i n ( G r a n i e r et al, 1993). A c h a n g e f r o m K to E i n the K T T C m o t i f o f the H C - P r o o f T V M V r e s u l t e d i n l o s s o f H C - P r o a c t i v i t y ( A t r e y a et al, 1992). A s u b s t i t u t i o n o f the T t o A i n the P T K m o t i f o f Z Y M V H C - P r o v i r t u a l l y e l i m i n a t e d H C P r o a c t i v i t y ( H u e t et al, 1994). It was h y p o t h e s i z e d that the K T T C m o t i f is i n v o l v e d i n b i n d i n g o f H C - P r o t o the a p h i d m o u t h p a r t s w h i l e the P T K m o t i f m a y b i n d to the v i r i o n s ( P e n g etal, 1998). Transmission of caulimoviruses also requires a nonstructural virally-encoded h e l p e r ( B l a n c et al, 1993). S t u d i e s h a v e s h o w n that the c a r b o x y l - t e r m i n u s o f the Cauliflower mosaic virus ( C a M V ) h e l p e r f a c t o r (or a p h i d t r a n s m i s s i o n f a c t o r ) b i n d s c o a t  p r o t e i n in vitro ( B l a n c et al, 1993; S c h m i d t et al, 1994). T h e s e s t u d i e s are c o n s i s t e n t w i t h the h y p o t h e s i s that the C a M V h e l p e r f a c t o r m e d i a t e s b i n d i n g o f C P to i n s e c t mouthparts. 1.2.1.2 C i r c u l a t i v e a p h i d t r a n s m i s s i o n C i r c u l a t i v e p l a n t v i r u s e s m u s t c r o s s m e m b r a n e b a r r i e r s i n the v e c t o r to b e t r a n s m i t t e d to n e w h o s t plants. L u t e o v i r u s e s are c i r c u l a t i v e , n o n - p r o p a g a t i v e p l a n t v i r u s e s w h i c h are t r a n s m i t t e d b y a p h i d s ( G r a y et al, 1991; P o w e r etal, 1991). V i r i o n s are i s o m e t r i c a n d c o n t a i n a s i n g l e - s t r a n d e d v i r a l g e n o m i c R N A .  Luteoviruses have a high  l e v e l o f v e c t o r s p e c i f i c i t y , w i t h greater t h a n 2 0 s p e c i e s o f a p h i d s r e p o r t e d as v e c t o r s , b u t 14  o n l y o n e o r a f e w s p e c i e s o f a p h i d h a v i n g the a b i l i t y to t r a n s m i t a n y o n e l u t e o v i r u s ( J e d l i n s k i , 1981; G i l d o w a n d G r a y , 1993). 1.2.1.2.1 C o a t p r o t e i n V i r u s t r a n s c a p s i d a t i o n s t u d i e s s u g g e s t e d that v e c t o r s p e c i f i c i t y o f the M A V  and  R P V i s o l a t e s o f Barley yellow dwarf luteovirus ( B Y D V ) was a f u n c t i o n o f the v i r u s c a p s i d ( R o c h o w , 1970). In these e x p e r i m e n t s s o m e M A V  nucleic acid became coated  w i t h R P V c o a t p r o t e i n d u r i n g c o - i n f e c t i o n o f a s i n g l e plant. M A V t r a n s m i t t e d b y R. padi, w h i c h n o r m a l l y o n l y t r a n s m i t t e d R P V .  MAV  c o u l d then be r e p l i c a t i o n was  d e t e c t e d i n the n e w h o s t p l a n t b u t t h i s v i r u s c o u l d not b e t r a n s m i t t e d b y R. padi. T h e s e results s u g g e s t e d that an i n t e r a c t i o n b e t w e e n the v i r u s c a p s i d a n d s p e c i f i c sites w i t h i n the a p h i d are the m a j o r d e t e r m i n a n t s f o r B Y D V - v e c t o r s p e c i f i c i t y ( R o c h o w , 1970). T h e luteovirus particle is c o m p o s e d o f two capsid proteins (Jolly and M a y o , 1994). F r o m n u c l e o t i d e s e q u e n c e a n a l y s i s , o p e n r e a d i n g f r a m e 3 ( O R F 3), e n c o d i n g the C P , i s f o l l o w e d i n - f r a m e b y a l a r g e r 3'-terminal O R F 5. O R F 5 is e x p r e s s e d b y r e a d t h r o u g h o f the t e r m i n a t i o n c o d o n o f O R F 3, a p r o c e s s w h i c h r e s u l t s i n the f o r m a t i o n o f C P s u b u n i t s w i t h a d d i t i o n a l s e q u e n c e at the C - t e r m i n u s t e r m e d the r e a d t h r o u g h d o m a i n ( R T D ) ( J o l l y a n d M a y o , 1994). V i r u s p a r t i c l e s o f s e v e r a l l u t e o v i r u s e s w e r e f o u n d to c o n t a i n s o m e C P  molecules  w i t h R T D a t t a c h e d ( J o l l y a n d M a y o , 1994). T h e R T D is e x p o s e d o n the s u r f a c e o f the l u t e o v i r u s p a r t i c l e i n a C - t e r m i n a l l y t r u n c a t e d f o r m ( B r a u l t et al., 1995) a n d i s p r o p o s e d to be i n v o l v e d i n a p h i d t r a n s m i s s i o n ( V i n c e n t et al., 1990). T r u n c a t e d R T D p r o t e i n has b e e n d e t e c t e d i n p u r i f i e d v i r u s a n d c r u d e extracts o f p l a n t s a n d p r o t o p l a s t s i n f e c t e d w i t h s e v e r a l l u t e o v i r u s e s ( B a h n e r et al., 1990; M a r t i n et al., 1990; F i l i c h k i n et al., 1994; W a n g 15  et al,  1995), s u g g e s t i n g that R T D c l e a v a g e is due to a c o n s e r v e d p r o c e s s i n g e v e n t a n d  not to r a n d o m d e g r a d a t i o n . In s e q u e n c e c o m p a r i s o n s b e t w e e n a h i g h l y t r a n s m i s s i b l e a n d a p o o r l y t r a n s m i s s i b l e l u t e o v i r u s , i t was f o u n d that the m a j o r i t y o f d i f f e r e n c e s o c c u r r e d i n the Ct e r m i n a l h a l f o f the R T D ( J o l l y a n d M a y o , 1994). A m i n o a c i d s e q u e n c e a l i g n m e n t s b e t w e e n s i x l u t e o v i r u s C P s s h o w e d a h i g h e r d e g r e e o f h o m o l o g y i n the N - t e r m i n u s o f the r e a d t h r o u g h d o m a i n ( R T D ) than i n the C - t e r m i n u s ( G u i l l e y et al., 1994). T h e s e r e s u l t s raise the h y p o t h e s i s that the N - t e r m i n u s o f the R T D i s i n v o l v e d i n n o n - s p e c i f i c aspects o f t r a n s m i s s i o n w h i l e the C - t e r m i n u s c o n t a i n s the d e t e r m i n a n t s f o r v e c t o r s p e c i f i c i t y . A p h i d t r a n s m i s s i o n o f a l u t e o v i r u s was r e d u c e d b y d e l e t i n g the C - t e r m i n a l h a l f o f its R T D , i n d i c a t i n g that the R T D p l a y s a r o l e i n t r a n s m i s s i o n ( B r a u l t et al., 1995). It was s h o w n that R T D - d e f i c i e n t v i r u s i s n o t a p h i d t r a n s m i s s i b l e ( C h a y et al., 1996). In b o t h studies, R T D m u t a n t s s h o w e d r e d u c e d a c c u m u l a t i o n i n p l a n t c e l l s so i t i s p o s s i b l e that the l a c k o f t r a n s m i s s i o n o f at least s o m e o f the m u t a n t s was d u e to l o w v i r u s titer rather than to a s p e c i f i c d e f e c t i n t r a n s m i s s i o n . D o m a i n s i n the l u t e o v i r u s C P are s u f f i c i e n t f o r a c q u i s i t i o n o f v i r u s t h r o u g h the h i n d g u t e p i t h e l i u m s i n c e v i r u s m u t a n t s w i t h o u t a R T D c a n b e a c q u i r e d i n this m a n n e r ( C h a y et al., 1996). T h e f u n c t i o n s o f d i f f e r e n t r e g i o n s o f the R T D w e r e e x a m i n e d b y p r o d u c i n g short i n - f r a m e d e l e t i o n s a l o n g the R T D ( B r u y e r e et al,  1997). A p r e l i m i n a r y  m a p o f f u n c t i o n s o f d i f f e r e n t parts o f the R T D was p r o d u c e d but the a s s o c i a t i o n o f m u l t i p l e a n d p o t e n t i a l l y i n t e r a c t i n g f u n c t i o n s o f the R T D m a k e h i g h - r e s o l u t i o n a n a l y s i s o f this p r o t e i n d i f f i c u l t ( B r u y e r e et al,  1997).  16  1.2.1.2.2 C h a p e r o n i n s C h a p e r o n i n s h a v e b e e n p r o p o s e d to p l a y a r o l e i n a p h i d t r a n s m i s s i o n o f l u t e o v i r u s e s , p o l e r o v i r u s e s , e n a m o v i r u s e s ( v a n d e n H e u v e l et al, 1994, 1997; F i l i c h k i n et al., 1997) a n d m o r e r e c e n t l y i n w h i t e f l y t r a n s m i s s i o n o f t o m a t o y e l l o w l e a f c u r l b e g o m o v i r u s ( M o r i n et al., 1999). C h a p e r o n i n s b i n d to a n d s t a b i l i z e n e w l y t r a n s l a t e d o r t r a n s l o c a t e d a g g r e g a t i o n - p r o n e p r o t e i n s a n d a i d i n their f o l d i n g a n d a s s e m b l y v i a a n A T P d e p e n d a n t p r o c e s s ( H a i t i , 1996). T h e l u t e o v i r u s R T D d e t e r m i n e s its i n t e r a c t i o n w i t h a G r o E L h o m o l o g u e ( s y m b i o n i n ) , a c h a p e r o n i n s e c r e t e d i n t o the a p h i d h e m o c o e l b y the e n d o s y m b i o t i c b a c t e r i u m , Buchnera ( v a n d e n H e u v e l et al, 1997). V i r i o n s w i t h o u t a R T D are s i g n i f i c a n t l y l e s s p e r s i s t e n t after i n j e c t i o n i n t o the a p h i d h e m o l y m p h t h a n w i l d type v i r u s ( v a n d e n H e u v e l et al, 1997). T r e a t m e n t o f a p h i d l a r v a e w i t h a n t i b i o t i c s s i g n i f i c a n t l y r e d u c e s Buchnera G r o E L l e v e l s . T r a n s m i s s i o n i s i n h i b i t e d d u e to a p o s t u l a t e d l o s s o f v i r a l c a p s i d i n t e g r i t y i n the a p h i d h e m o l y m p h i n the a b s e n c e o f G r o E L ( v a n d e n H e u v e l et al, 1994). B a s e d o n these studies, the l u t e o v i r u s - G r o E L i n t e r a c t i o n i s p o s t u l a t e d to b e a n e c e s s a r y c o m p o n e n t o f v i r a l stability i n the a p h i d h e m o l y m p h , r e d u c i n g p r o t e o l y t i c b r e a k d o w n a n d t h e r e b y a l l o w i n g v i r u s m o v e m e n t t h r o u g h the a p h i d and subsequent transmission. 1.2.1.2.3 R e c e p t o r - m e d i a t e d e n d o c y t o s i s T h e m o v e m e n t o f l u t e o v i r u s e s t h r o u g h the a p h i d has b e e n e l u c i d a t e d u s i n g u l t r a s t r u c t u a l s t u d i e s o f s e c t i o n s o f a p h i d s i n j e c t e d w i t h B Y D V ( G i l d o w , 1987; G i l d o w , 1993; G i l d o w a n d G r a y , 1993). V i r u s i s a c q u i r e d b y the a p h i d t h r o u g h i n g e s t i o n o f i n f e c t e d p h l o e m c o n t e n t s d u r i n g f e e d i n g ( G i l d o w , 1987). V i r u s i s t h e n t r a n s p o r t e d t h r o u g h the h i n d g u t e p i t h e l i u m a n d r e l e a s e d i n t o the h e m o c o e l ( G i l d o w , 1993). T h e r e is 17  l i t t l e s e l e c t i v i t y at t h e h i n d g u t m e m b r a n e barrier, w i t h t h e m a j o r i t y o f l u t e o v i r u s e s h a v i n g the ability to pass through the hindgut e p i t h e l i u m o f all k n o w n aphid vectors w h i l e other v i r u s e s cannot. T h e h i g h l e v e l o f s e l e c t i v i t y a m o n g l u t e o v i r u s e s i s b e l i e v e d t o o c c u r at the n e x t stage, w h e n v i r u s i n t h e h e m o c o e l m u s t p a s s t h r o u g h t h e b a s a l l a m i n a a n d b a s a l p l a s m a l e m m a i n o r d e r t o enter t h e s a l i v a r y g l a n d a n d b e t r a n s m i t t e d d u r i n g s u b s e q u e n t f e e d i n g p r o b e s ( G i l d o w a n d R o c h o w , 1980; G i l d o w a n d G r a y , 1993). A f t e r e n t e r i n g t h e s a l i v a r y g l a n d , v i r u s is t r a n s p o r t e d i n c o a t e d v e s i c l e s a n d r e l e a s e d i n t o t h e s a l i v a r y c a n a l w h e r e i t f l o w s w i t h s a l i v a t h r o u g h the s a l i v a r y d u c t a n d i n t o n e w h o s t p l a n t s d u r i n g f e e d i n g ( P o n s e n , 1977). M o v e m e n t o f B Y D V into the aphid accessory salivary gland was studied to d e t e r m i n e i f this i s a site o f s p e c i f i c i t y a n d a l s o t o i d e n t i f y p o s s i b l e m e c h a n i s m s o f v i r u s m o v e m e n t t h r o u g h t h e a p h i d l e a d i n g t o t r a n s m i s s i o n ( G i l d o w a n d G r a y , 1993). V i r i o n s w e r e o b s e r v e d a t t a c h e d to t h e s a l i v a r y b a s a l p l a s m a l e m m a a n d i n c o a t e d p i t s i n t h e process o f being transported into the cytoplasm b y endocytosis. There was specificity i n this p r o c e s s i n that t h e M A V i s o l a t e o f B Y D V c o u l d o n l y penetrate t h e a c c e s s o r y s a l i v a r y g l a n d b a s a l l a m i n a o f s p e c i f i c a p h i d s p e c i e s . T h r e e types o f v i r u s - b a s a l l a m i n a interactions were described b y G i l d o w a n d G r a y (1993); nonpenetrating-nontransmitted, p e n e t r a t i n g - n o n t r a n s m i t t e d , a n d p e n e t r a t i n g - t r a n s m i t t e d . I n t h e t w o types o f nontransmitted luteoviruses, virus c o u l d not be transmitted either because it c o u l d not cross the basal l a m i n a (nonpenetrating) o r because it c o u l d cross b u t was then prevented f r o m e n t e r i n g t h e s a l i v a r y g l a n d at t h e c e l l m e m b r a n e ( p e n e t r a t i n g ) ( G i l d o w a n d G r a y , 1993).  18  A m o d e l f o r the m o v e m e n t o f v i r i o n s t h r o u g h the a p h i d a c c e s s o r y s a l i v a r y g l a n d has b e e n p r o p o s e d ( G i l d o w a n d G r a y , 1993). T r a n s m i s s i b l e l u t e o v i r u s e s are s p e c i f i c a l l y r e c o g n i z e d at the b a s a l p l a s m a l e m m a , w h e r e they attach to the m e m b r a n e a n d enter the c e l l b y e n d o c y t o s i s . V i r i o n s a c c u m u l a t e i n t u b u l a r v e s i c l e s a d j a c e n t to the a p i c a l p l a s m a l e m m a . C o a t e d v e s i c l e s c o n t a i n i n g i n d i v i d u a l v i r i o n s b u d o f f the t u b u l a r v e s i c l e s a n d t r a n s p o r t the v i r i o n s to the a p i c a l p l a s m a l e m m a w h e r e they are r e l e a s e d b y e n d o c y t o s i s i n t o the s a l i v a r y c a n a l . T h e v i r i o n s are t h e n a b l e to m o v e w i t h s a l i v a r y s e c r e t i o n s o u t o f the a p h i d a n d t h r o u g h the stylets i n t o n e w p l a n t hosts d u r i n g f e e d i n g . T h e r o l e o f the a p h i d s a l i v a r y g l a n d i n t r a n s m i s s i o n s p e c i f i c i t y was f u r t h e r e x a m i n e d ( P e i f f e r et al,  1997). T h e a b i l i t y o f R P V a n d P A V i s o l a t e s o f B Y D V to  a s s o c i a t e w i t h the b a s a l l a m i n a o f v e c t o r a n d n o n - v e c t o r a p h i d s p e c i e s was e x a m i n e d a n d c o m p a r e d to the b i n d i n g o f v i r u s e s to the i s o l a t e d b a s a l l a m i n a e in vitro. It was c o n f i r m e d that the b a s a l l a m i n a s e r v e s as a s e l e c t i o n step i n c i r c u l a t i v e a p h i d t r a n s m i s s i o n ( P e i f f e r et al,  1997). T h e r e are t w o stages o f r e g u l a t i o n at the l e v e l o f the  b a s a l l a m i n a ; w h e t h e r o r n o t v i r i o n s c a n attach to the b a s a l l a m i n a a n d w h e t h e r c h a n g e s i n the p e r m e a b i l i t y o f this s t r u c t u r e o c c u r to a l l o w v i r i o n s to c r o s s t h i s barrier. T h e a c c e s s o r y s a l i v a r y g l a n d p l a s m a l e m m a is a s e c o n d d i s t i n c t s e l e c t i o n b a r r i e r to l u t e o v i r u s t r a n s m i s s i o n ( P e i f f e r et al,  1997).  M o v e m e n t o f B Y D V s t h r o u g h the h i n d g u t e p i t h e l i u m was e x a m i n e d u s i n g u l t r a s t r u c t u r a l m e t h o d s a n d i t was h y p o t h e s i z e d that r e c e p t o r - m e d i a t e d e n d o c y t o s i s r e g u l a t e d l u t e o v i r u s a c q u i s i t i o n b y a p h i d s ( G i l d o w , 1993). F r o m t h i s m o d e l i t was p r e d i c t e d that r e c e p t o r - m e d i a t e d a t t a c h m e n t o f v i r i o n s to m e m b r a n e s c o n t r o l s a c q u i s i t i o n and vector specificity. Transmission efficiency may be partially regulated by virus 19  a f f i n i t y f o r p u t a t i v e r e c e p t o r s o n the a c c e s s o r y s a l i v a r y g l a n d b a s a l l a m i n a ( P e i f f e r et al, 1997) . N o c a n d i d a t e r e c e p t o r s h a v e yet b e e n i d e n t i f i e d f r o m v e c t o r aphids. 1.2.1.3 T h r i p s t r a n s m i s s i o n ( c i r c u l a t i v e , r e p l i c a t i v e ) Tomato spotted wilt tospovirus ( T S W V ) i s i n the f a m i l y Bunyaviridae a n d i s  t r a n s m i t t e d i n a c i r c u l a t i v e , p r o p a g a t i v e f a s h i o n b y s e v e r a l s p e c i e s o f thrips, the m o s t a b u n d a n t o f w h i c h is Frankliniella occidentalis ( T h y s a n o p t e r a : T h r i p i d a e ) ( B a n d l a et al., 1998) . T S W V i s a c q u i r e d b y l a r v a e o f F. occidentalis v i a entry to m i d g u t e p i t h e l i a l c e l l s ( U l l m a n et al., 1995) a n d c a n n o t b e a c q u i r e d b y adults d u e to a m i d g u t b a r r i e r ( U l l m a n et al, 1992). T S W V is membrane-bound encoding t w o m e m b r a n e glycoproteins (GPs) ( G P 1 a n d G P 2 ) o n its v i r a l R N A ( S h o p e , 1985). U l t r a s t r u c t u r a l e v i d e n c e i m p l i c a t e s the t w o T S W V e n v e l o p e G P s as v i r a l attachment p r o t e i n s ( U l l m a n et al., 1995). I m m u n o l a b e l e d T S W V G P s w e r e o b s e r v e d b o u n d to the b r u s h b o r d e r p l a s m a l e m m a o f serial s e c t i o n s f r o m l a r v a l m i d g u t s ( U l l m a n et al, 1992). A m o r p h o u s m a s s e s o f T S W V n u c l e o c a p s i d p r o t e i n w e r e o b s e r v e d i n the c y t o p l a s m o f these c e l l s as w e r e T S W V - e n c o d e d p r o t e i n s w i t h i n i n c l u s i o n s , v a c u o l e s , a n d v e s i c l e s ( U l l m a n et al, 1992). It w a s p r o p o s e d b a s e d o n these f i n d i n g s that v i r a l G P s i n t e r a c t w i t h a p u t a t i v e r e c e p t o r i n the p l a s m a m e m b r a n e o f larval thrips' m i d g u t epithelial cells, initiating virus fusion and release o f viral c o m p o n e n t s i n t o the c e l l c y t o p l a s m ( U l l m a n et al, 1992; U l l m a n et al, 1995). R e p e a t e d m e c h a n i c a l p a s s a g e o f T S W V to p l a n t s results i n e n v e l o p e - d e f i c i e n t m u t a n t s w h i c h are n o l o n g e r t r a n s m i s s i b l e a n d w h i c h l a c k G P - e n c o d i n g s e q u e n c e s (de R e s e n d e etal, 1991). G P 2 c o n t a i n s a n a r g i n i n e - g l y c i n e - a s p a r t a t e ( R G D ) m o t i f c h a r a c t e r i s t i c o f c e l l - a d h e s i o n m o l e c u l e s ( K o r m e l i n k et al, 1992). B a s e d o n this, B a n d l a 20  et al. ( 1 9 9 8 ) h y p o t h e s i z e d that the T S W V G P s are v i r a l attachment p r o t e i n s i n t e r a c t i n g w i t h p u t a t i v e c e l l u l a r r e c e p t o r s i n t h r i p s m i d g u t c e l l s to m e d i a t e v i r u s a c q u i s i t i o n . G e l o v e r l a y assays a n d i m m u n o l a b e l i n g  w e r e u s e d to assess the r o l e o f the T S W V  G P s as v i r a l attachment p r o t e i n s a n d to attempt to i d e n t i f y c o r r e s p o n d i n g p u t a t i v e c e l l u l a r r e c e p t o r s f r o m t h r i p s ( B a n d l a et al., 1998). T S W V G P s b o u n d s e l e c t i v e l y to a s i n g l e 5 0 k D a b a n d o f separated  F. occidentalis  p r o t e i n s b u t d i d not b i n d to s i m i l a r o v e r l a y s u s i n g  separated p r o t e i n s f r o m n o n - v e c t o r i n s e c t s o r another s p e c i e s f r o m the f a m i l y  Thripidae  ( B a n d l a et al., 1998). T h e i n t e n s i t y o f the 5 0 k D a b a n d w a s c o n s i s t e n t l y greater i n l a r v a e than i n adults w h i c h is c o n s i s t e n t w i t h the f a c t that l a r v a e t r a n s m i t T S W V b u t adults don't. A n t i - i d i o t y p i c a n t i b o d i e s to the G P 1 a n d G P 2 b o u n d s p e c i f i c a l l y to the 5 0 k D a p r o t e i n , f u r t h e r i n d i c a t i n g the s p e c i f i c i t y o f the i n t e r a c t i o n ( B a n d l a et ah, 1998). T h e m i d g u t p l a s m a l e m m a w a s s p e c i f i c a l l y l a b e l e d b y f l u o r e s c e n t a n t i - i d i o t y p i c a n t i b o d i e s to G P 1 a n d G P 2 w i t h n o f l u o r e s c e n c e i n the m i d g u t s o f c o n t r o l i n s e c t s , f u r t h e r s u p p o r t i n g the h y p o t h e s i s that the 5 0 k D a p r o t e i n i s a c e l l u l a r r e c e p t o r i n the t h r i p s m i d g u t m e d i a t i n g the a c q u i s i t i o n o f T S W V ( B a n d l a et al., 1998). F u r t h e r w o r k i s n e c e s s a r y to d e t e r m i n e i f b i n d i n g o f G P 1 a n d G P 2 to the p u t a t i v e 5 0 k D a r e c e p t o r o c c u r s  in vivo  a n d i f i t is  s p e c i f i c . T h e latter c a n b e d o n e b y d e m o n s t r a t i n g saturable b i n d i n g a n d c o m p e t i t i o n f o r binding.  1.2.2 Nematode transmission (non-circulative)  L o n g i d o r i d a n d trichodorid nematodes transmit nepo- and tobraviruses, respectively in a non-circulative manner, acquiring virus from feeding o n infected plant  21  roots ( B r o w n et al, 1995). S e v e n n e m a t o d e s p e c i e s w i t h i n the g e n u s Paratrichodorus a n d f o u r w i t h i n Trichodorus are k n o w n to b e v e c t o r s o f t o b r a v i r u s e s ( B r o w n et al., 1995). Specificity exists between nematode species and their associated viruses where particular t o b r a v i r u s i s o l a t e s are o n l y t r a n s m i t t e d b y c e r t a i n n e m a t o d e s p e c i e s a n d o n l y p a r t i c u l a r n e m a t o d e s p e c i e s m a y t r a n s m i t c e r t a i n v i r u s e s . T h e P p K 2 0 i s o l a t e o f Tobacco rattle tobravirus ( T R V ) is t r a n s m i t t e d b y P. pachydermus b u t n o t b y T. primitivus, a n d P. pachydermus t r a n s m i t s s o m e T R V  i s o l a t e s ( P p K 2 0 , P p B l , P p W l ) b u t n o t others ( T v C 4 7 ,  T p E l ) ( B r o w n et al, 1989; P l o e g et al, 1991, 1992). T h i s s p e c i f i c i t y i s d e t e r m i n e d b y the a b i l i t y o f n e m a t o d e s to r e t a i n v i r u s p a r t i c l e s at s p e c i f i c sites w i t h i n t h e i r f e e d i n g apparatus. T r a n s m i s s i b l e v i r u s was f o u n d i n s t a r v e d Longidorus f o r u p to 12 w e e k s , i n Xiphinema f o r a year, a n d f o r m o r e t h a n a y e a r f o r Trichodorus, i n d i c a t i n g that the v i r u s  m a y p e r s i s t f r o m o n e g r o w i n g s e a s o n to a n o t h e r w i t h i n t h e i r n e m a t o d e v e c t o r s ( M a t t h e w s , 1991; v a n H o o f , 1970). D i s e a s e c o n t r o l is c u r r e n t l y a c h i e v e d b y a p p l y i n g s o i l sterilant c h e m i c a l s to e l i m i n a t e n e m a t o d e p o p u l a t i o n s a n d b y the a p p l i c a t i o n o f o x i m a c a r b a m a t e s w h i c h t e m p o r a r i l y i m m o b i l i s e the n e m a t o d e s , thus p r e v e n t i n g t h e m f r o m t r a n s m i t t i n g the v i r u s ( V i s s e r et al, 1999). T h e m e c h a n i s m o f v i r u s r e t e n t i o n a l o n g the n e m a t o d e f o o d c a n a l is u n k n o w n . T h e m o l e c u l a r b i o l o g y o f v i r u s - n e m a t o d e i n t e r a c t i o n s i s l i t t l e u n d e r s t o o d d u e to a l a c k o f an in vitro s y s t e m f o r n e m a t o d e f e e d i n g s u c h as e x i s t s f o r a p h i d studies. In f e e d i n g , n e m a t o d e s i n j e c t t h e i r h o l l o w n e e d l e - l i k e f e e d i n g stylet i n t o r o o t c e l l s , i n j e c t s a l i v a c a u s i n g l i q u i f i c a t i o n o f the c y t o p l a s m , a n d f i n a l l y w i t h d r a w p l a n t c e l l c o n t e n t s b y a s u c k i n g m e c h a n i s m ( M a t t h e w s , 1991). It is k n o w n that a c q u i r e d v i r u s p a r t i c l e s b i n d to  22  s p e c i f i c r e g i o n s o f the stylet sheath, p h a r y n x , o r e s o p h a g u s d u r i n g the f e e d i n g p r o c e s s ( G r a y , 1996). T h e t o b r a v i r u s g e n o m e c o n s i s t s o f t w o p o s i t i v e - s e n s e R N A s that are s e p a r a t e l y e n c a p s i d a t e d i n t o r o d - s h a p e d particles. R N A - 1 i s l o n g e r t h a n R N A - 2 a n d e n c o d e s the g e n e s f o r r e p l i c a t i o n a n d m o v e m e n t o f the v i r u s a n d a s m a l l p r o t e i n o f u n k n o w n f u n c t i o n w h i c h i s h i g h l y c o n s e r v e d a m o n g t o b r a v i r u s e s ( H a r r i s o n a n d R o b i n s o n , 1988). T h e smaller R N A - 2 is highly variable a m o n g different isolates o f T R V i n both nucleotide s e q u e n c e a n d g e n o m e length. R N A - 2 e n c o d e s the v i r a l C P as w e l l as o n e o r m o r e n o n s t r u c t u r a l p r o t e i n s . It has b e e n s h o w n that s o m e o f these p r o t e i n s are i n v o l v e d i n t r a n s m i s s i o n ( M a c F a r l a n e et al., 1996; H e r n a n d e z et al., 1997). T R V p a r t i c l e s h a v e b e e n a s s o c i a t e d w i t h the c u t i c u l a r l i n i n g o f the e s o p h a g u s o f P. pachydermus and T. similis n e m a t o d e s , t e n d i n g to lie p a r a l l e l to the l o n g a x i s o f the f o o d c a n a l ( T a y l o r a n d R o b e r t s o n , 1970; B r o w n et al, 1996). T h e r e t e n t i o n o f v i r u s i n s i d e the n e m a t o d e f o o d c a n a l l i k e l y r e q u i r e s s p e c i f i c structures o f the v i r u s p a r t i c l e a n d the n e m a t o d e e s o p h a g e a l cuticle. T h e sites o f v i r u s r e t e n t i o n o n the w a l l o f the f o o d c a n a l i n X. diversicaudatum a n d P. pachydermus c o n t a i n c a r b o h y d r a t e s ( B r o w n et al., 1995). A d d i t i o n a l l y , c a r b o h y d r a t e - s t a i n i n g m a t e r i a l has b e e n o b s e r v e d s u r r o u n d i n g p a r t i c l e s o f Arabis mosaic nepovirus ( A r M V ) r e t a i n e d w i t h i n the f e e d i n g apparatus o f X. diversicaudatum  ( R o b e r t s o n a n d H e n d r y , 1986). B a s e d o n e v i d e n c e o f c a r b o h y d r a t e s i n the v i c i n i t y o f v i r u s i n the n e m a t o d e f o o d c a n a l , B r o w n et a/.(1995) s u g g e s t e d that the v i r u s c o a t p r o t e i n may have lectin-like properties, with carbohydrates being i n v o l v e d i n both retention a n d d i s s o c i a t i o n o f v i r u s particles.  23  T o b r a v i r u s p a r t i c l e s a n d n e p o v i r u s e s are u n r e l a t e d e x c e p t f o r the f a c t that t h e y are nematode-transmitted.  V i r a l coat p r o t e i n i s l i k e l y i n v o l v e d i n the r e c o g n i t i o n p r o c e s s  b e t w e e n n e m a t o d e v e c t o r a n d these v i r u s e s . T h u s f a r i t has b e e n s h o w n that the d e t e r m i n a n t s f o r t r a n s m i s s i o n o f Raspberry ringspot nepovirus ( R R S V ) , Tomato black ring nepovirus ( T B R V ) a n d Tobacco ringspot nepovirus ( T R V ) are o n R N A 2 , w h i c h i s  the s e g m e n t o f the v i r u s g e n o m e c o d i n g f o r the c o a t p r o t e i n ( B r o w n et al., 1995). T h e c o a t p r o t e i n i s a l m o s t c e r t a i n l y i n v o l v e d i n t r a n s m i s s i o n b u t w h e t h e r o r not o t h e r p r o t e i n s e n c o d e d o n R N A 2 are a l s o i n v o l v e d is not k n o w n . T h e structure o f t o b r a v i r u s p a r t i c l e s i n d i c a t e s that t o b r a v i r u s C P s u b u n i t s s t a c k i n a tight s p i r a l to f o r m a h o l l o w r o d - s h a p e d structure. S e q u e n c e a n d s t r u c t u r a l c o m p a r i s o n s b e t w e e n the C P s o f T R V a n d the r o d - s h a p e d Tobacco mosaic tobamovirus ( T M V ) i n d i c a t e that the N - a n d C - t e r m i n a l r e g i o n s o f T R V are l o c a t e d o n the o u t s i d e o f the v i r u s p a r t i c l e ( G o u l d e n et al., 1992). N M R  m e a s u r e m e n t s suggest that the r e l a t i v e l y l o n g  protruding C-terminal amino acid sequences of tobravirus C P s form mobile arms w h i c h are g o o d c a n d i d a t e s f o r i n v o l v e m e n t i n the r e t e n t i o n o f t o b r a v i r u s e s b y t h e i r a s s o c i a t e d v e c t o r n e m a t o d e s ( M a y o et ah,  1995).  T h e f l e x i b l e d o m a i n o f the C P ( O R F 1 o f R N A - 2 ) i s d i f f e r e n t i n s i z e i n a l l three nematode-transmissible  t o b r a v i r u s e s s e q u e n c e d to date [ T R V i s o l a t e T p O l , 17 aa; P E B V  (Pea early browning virus) i s o l a t e T p A 5 6 , 2 9 aa, T R V  i s o l a t e P p K 2 0 , 22 aa]  ( M a c F a r l a n e et al, 1999). It was p r e v i o u s l y s h o w n that r e m o v i n g the C - t e r m i n a l 15 a a o f the C P o f P E B V a b o l i s h e d n e m a t o d e t r a n s m i s s i o n ( M a c F a r l a n e et al., 1996). R e s i d u e s at the e x t r e m e C - t e r m i n u s o f the f l e x i b l e c o a t p r o t e i n d o m a i n w o u l d b e m o s t i m p o r t a n t i n  24  s p e c i f i c r e c o g n i t i o n o f v e c t o r s u r f a c e s o r t r a n s m i s s i o n h e l p e r p r o t e i n , a n d i n that r e g i o n a l l three v i r u s s e q u e n c e s are d i f f e r e n t ( M a c F a r l a n e et al, 1999). B o t h the 9- a n d 2 9 - k D a n o n - s t r u c t u r a l p r o t e i n s o f the t w o v i r u s e s t r a n s m i t t e d b y T. primitivus ( T R V T p O l a n d P E B V T p A 5 6 ) are m o r e s i m i l a r to e a c h o t h e r t h a n t h e y are  to the 2 9 . 4 - k D a p r o t e i n o f T R V P p K 2 0 w h i c h is t r a n s m i t t e d b y P. pachydermus ( M a c F a r l a n e et al, 1999). T h e s e data suggest that n o n s t r u c t u r a l p r o t e i n s h a v e a r o l e i n n e m a t o d e t r a n s m i s s i o n . It has b e e n s u g g e s t e d that the n o n s t r u c t u r a l p r o t e i n s m a y s p e c i f i c a l l y l i n k v i r u s p a r t i c l e s to r e c e p t o r sites i n the n e m a t o d e e s o p h a g u s ( H e r n a n d e z et al, 1997). T h e s e n o n s t r u c t u r a l p r o t e i n s m a y f u n c t i o n as h e l p e r c o m p o n e n t s a n a l o g o u s to the h e l p e r c o m p o n e n t s o f p o t y v i r u s e s o r the a p h i d t r a n s m i s s i o n f a c t o r o f c a u l i m o v i r u s e s (see a b o v e ; P i r o n e a n d B l a n c , 1996). D e t a i l e d m u t a g e n e s i s studies o f the n o n s t r u c t u r a l a n d structural p r o t e i n s are n e c e s s a r y to better u n d e r s t a n d the p r o c e s s o f n e m a t o d e t r a n s m i s s i o n o f t o b r a v i r u s e s , w i t h p a r t i c u l a r e m p h a s i s o n r e s i d u e s p r e d i c t e d to b e s u r f a c e l o c a t e d ( M a c F a r l a n e et al, 1999). 1.2.3 F u n g u s t r a n s m i s s i o n F u n g i w h i c h t r a n s m i t v i r u s e s are z o o s p o r i c , o b l i g a t e parasites o f p l a n t r o o t s ( A d a m s , 1991). T w o d i s t i n c t types o f f u n g i b e l o n g i n g to d i f f e r e n t c l a s s e s o f the subdivision Mastigomycotina  are k n o w n to t r a n s m i t p l a n t v i r u s e s ( T a b l e 1.2; G r a y a n d  R o c h o n , 1999). P l a s m o d i o p h o r o m y c e t e v e c t o r s (Polymyxa a n d Spongospora spp.) h a v e b i f l a g e l l a t e z o o s p o r e s w h i l e the C h y t r i d i o m y c e t e v e c t o r s (Olpidium spp.) h a v e a s i n g l e f l a g e l l u m ( A d a m s , 1991).  25  T a b l e 1.2 K n o w n a n d p u t a t i v e f u n g a l v e c t o r s f o r v i r u s e s a n d v i r u s - l i k e a g e n t s . 1  Virus (Family)  Acronym  Fungal Vector  Acquisition Mode  Tombusviruses (Tombusviridae) Cucumber necrosis virus  CNV  Olpidium bornovanus  in vitro  CLSV  O. bornovanus  in vitro  CSBV MNSV SqNV  0. bornovanus 0. bornovanus 0. bornovanus  in vitro in vitro in vitro  RCNMV  0. bornovanus  in vitro  ChNV LNV TNV  0. brassicae  in vitro in vitro in vitro  FLNA LBVV TStV  o.brassicae o.brassicae 0. brassicae  in vivo in vivo in vivo  BaMMV BaYMV OMV RNMV WSSMV WYMV  Polymyxa graminis P. graminis P. graminis P. graminis P. graminis P. graminis  in vivo in vivo in vivo in vivo in vivo in vivo  BNYYV BSBV IPCV PCV PMTV  P. betae P. betae P. graminis P. graminis Spongospora  in vivo in vivo in vivo in vivo in vivo  OGSV RSNV SBWMV  f.sp. subterranea P. graminis P. graminis P. graminis  in vivo in vivo in vivo  Aureusvirus (Tombusviridae) Cucumber leaf spot virus Carmoviruses (Tombusviridae) Cucumber soil-borne virus Melon necrotic spot virus Squash necrosis virus Dianthoviruses (Tombusviridae) Red clover necrotic mosaic Necroviruses (Tombusviridae) Chenopodium necrosis virus Lisianthus necrosis Tobacco necrosis virus Varicosviruses Freesia leaf necrosis agent Lettuce big vein Tobacco stunt virus Bymoviruses (Potyviridae) Barley mild mosaic virus Barley yellow mosaic virus Oat mosaic virus Rice necrosis mosaic virus Wheat spindle streak mosaic virus Wheat yellow mosaic virus Furoviruses Beet necrotic yellow vein Beet soil-borne virus Indian peanut clump virus Peanut clump virus Potato mop top virus 3  5  Oat golden stripe virus Rice stripe necrosis virus Soil-borne wheat mosaic Uncharacterized viruses Lettuce ring necrosis agent Pepper yellow vein agent Watercress yellow spot virus Watercress chlorotic leaf spot  2  4  o. o.  brassicae brassicae  LRNA PYVA WYSV  O. brassicae O. brassicae S. subterranea f.sp. nasturtii S. subterranea f.sp. nasturtii  WCLA  Table is from D. Rochon (unpublished). A family name is provided if assigned. S q N V is a tentative carmovirus. W S S M V and W Y M V are possibly strains of each other. Beet necrotic yellow vein is a tentative species of the furovirus genus.  3  4  5  26  in vivo in vivo not known not known  #  Q  zoospore discharge  in root cell  F i g u r e 1.1 L i f e c y c l e o f Olpidium brassicae ( a d a p t e d f r o m T e m m i n k , 1971). T h e q u e s t i o n m a r k i n d i c a t e s that it i s n o t c e r t a i n h o w r e s t i n g s p o r a n g i a are f o r m e d i n r o o t cells o f O. brassicae.  B o t h t y p e s o f f u n g i share c o m m o n f e a t u r e s i n their l i f e c y c l e s ( A d a m s , 1991; F i g u r e 1.1). T h i c k - w a l l e d r e s t i n g s p o r e s s u r v i v e i n the s o i l f o r m a n y y e a r s after d e c a y o f the i n f e c t e d plant. W i t h p r o p e r e n v i r o n m e n t a l c o n d i t i o n s , r e s t i n g s p o r e s (cell w a l l , n o flagella) will germinate, b e c o m i n g primary zoospores (flagellated, no cell wall) w h i c h s w i m to h o s t r o o t s a n d e n c y s t o n r o o t e p i d e r m a l cells. E n c y s t m e n t i n v o l v e s w i t h d r a w a l  27  o f the f l a g e l l a , a d h e s i o n to the h o s t w a l l a n d s e c r e t i o n o f a c y s t w a l l s e p a r a t i n g the f u n g a l c y t o p l a s m f r o m the p l a n t c e l l c y t o p l a s m . T h e z o o s p o r e p r o t o p l a s t i s t h e n i n j e c t e d i n t o the h o s t c e l l w h e r e the t h a l l u s d i v i d e s i n t o a m u l t i n u c l e a t e P l a s m o d i u m a n d t h e n d e v e l o p s into a zoosporangium.  S e c o n d a r y z o o s p o r e s are r e l e a s e d f r o m the z o o s p o r a n g i u m i n t o  s o i l w h e n the p r e s e n c e o f w a t e r c a u s e s the d i s s o l u t i o n o f a g e l a t i n o u s p l u g i n t h i s s a c - l i k e structure ( A d a m s , 1991). T h e r e are t w o m a i n strategies f o r t r a n s m i s s i o n o f f u n g a l l y t r a n s m i t t e d v i r u s e s . In o n e strategy, v i r u s i s a d s o r b e d to the z o o s p o r e s u r f a c e b u t is not f o u n d i n r e s t i n g s p o r e s (in vitro t r a n s m i s s i o n ) . In the other, v i r u s enters the t h a l l u s i n the h o s t a n d i s l o c a t e d  w i t h i n r e s t i n g s p o r e s (in vivo t r a n s m i s s i o n ) ( C a m p b e l l , 1996). V i r u s e s t r a n s m i t t e d i n t h i s m a n n e r h a v e b e e n t e r m e d n o n p e r s i s t e n t a n d persistent, r e s p e c t i v e l y ( A d a m s , 1991). T h e s e t r a n s m i s s i o n strategies are a n a l o g o u s to n o n c i r c u l a t i v e a n d c i r c u l a t i v e v i r u s t r a n s m i s s i o n b y i n v e r t e b r a t e v e c t o r s ( G r a y , 1996; see above). A n a l o g i e s between m e c h a n i s m s o f transmission by invertebrate vectors and those u s e d b y f u n g i w e r e c h a l l e n g e d b a s e d o n the f a c t that f u n g i h a v e n o c i r c u l a t o r y s y s t e m ( C a m p b e l l , 1993). T h e u s e o f the terms 'persistent' a n d 'non-persistent' are not a p p r o p r i a t e f o r f u n g a l v e c t o r s b e c a u s e they are b a s e d o n v i r u s s u r v i v a l i n dormant ( f u n g a l ) r e s t i n g s p o r e s i n c o n t r a s t to r e t e n t i o n o f v i r u s b y feeding ( i n v e r t e b r a t e ) v e c t o r s ( C a m p b e l l , 1996). D e s p i t e the o b v i o u s b i o l o g i c a l d i f f e r e n c e s b e t w e e n f u n g i a n d invertebrates, the o v e r a l l s i m i l a r i t i e s b e t w e e n t r a n s m i s s i o n o f f u n g i a n d i n v e r t e b r a t e s i n d i c a t e s that s u c h c o m p a r i s o n s m i g h t g i v e f u r t h e r i n s i g h t i n t o g e n e r a l t r a n s m i s s i o n m e c h a n i s m s ( A d a m s , 1991).  28  T h e t e r m s in vitro a n d in vivo d i s t i n g u i s h b e t w e e n f u n g a l v e c t o r s b a s e d o n w h e t h e r v i r u s e s are e x t e r n a l l y o r i n t e r n a l l y borne, r e s p e c t i v e l y ( C a m p b e l l a n d F r y , 1966). T h e s e t e r m s g i v e i m p o r t a n t b i o l o g i c a l i n f o r m a t i o n about the m o d e o f f u n g a l t r a n s m i s s i o n a n d a l l u d e to the d i f f e r e n c e s i n b i o l o g y b e t w e e n these t w o m o d e s . T h e r e f o r e , I w i l l use the t e r m s in vitro a n d in vivo to d e s c r i b e f u n g u s - t r a n s m i s s i o n m e c h a n i s m s i n t h i s r e v i e w but w i l l a l s o p e r i o d i c a l l y use the t e r m s n o n - c i r c u l a t i v e a n d c i r c u l a t i v e , r e s p e c t i v e l y , to m a i n t a i n c o n t i n u i t y w i t h the s e c t i o n s o n i n v e r t e b r a t e vectors. 1.2.3.1 Olpidium l i f e - c y c l e O. brassicae was d e m o n s t r a t e d  to b e the n a t u r a l v e c t o r o f Tobacco necrosis virus  ( T N V ) ( T e a k l e , 1960) a n d m u c h o f o u r k n o w l e d g e o f the b i o l o g y o f Olpidium spp. c o m e s f r o m studies o f O. brassicae ( T e m m i n k a n d C a m p b e l l , 1968, 1969a, 1 9 6 9 b ; T e m m i n k , 1971; F i g u r e 1.1). T h e c e l l s o f l e t t u c e O. brassicae z o o s p o r e s are 2 x 3 urn, o v o i d , a n d s u r r o u n d e d b y a c o n t i n u o u s p l a s m a l e m m a ( T e m m i n k a n d C a m p b e l l , 1969a). T h e a x o n e m e i s 0.2 to 0.25 J i m w i d e a n d s u r r o u n d e d b y a m e m b r a n o u s sheath c o n t i n u o u s w i t h the p l a s m a l e m m a o f the z o o s p o r e b o d y ( T e m m i n k a n d C a m p b e l l , 1969a). T h e c e l l s o f O. bornovanus i n f e c t i n g c u c u m b e r are l a r g e r than that o f O. brassicae, b e i n g 6-7 |im. T h e l e t t u c e i s o l a t e o f O. brassicae e n c y s t s o n a n d penetrates r o o t h a i r s a n d e p i d e r m a l c e l l s i n the z o n e o f e l o n g a t i o n o f the roots ( T e m m i n k a n d C a m p b e l l , 1 9 6 9 b ) . I n f e c t i o n o f the r o o t has n o o b s e r v a b l e p a t h o g e n i c e f f e c t o n host p l a n t s b u t s u c h p l a n t s m a y b e m o r e s u s c e p t i b l e to other p a t h o g e n i c o r g a n i s m s a n d m a y i n c u r a s m a l l d e c r e a s e i n o v e r a l l g r o w t h ( G r o g a n a n d C a m p b e l l , 1966). A t the t i m e o f i n f e c t i o n o f n e w r o o t e p i t h e l i a l c e l l s , Olpidium z o o s p o r e s h a v e r e t r a c t e d t h e i r a x o n e m e s . T w o d i f f e r e n t m o d e s  29  o f a x o n e m a l retraction, " w r a p - a r o u n d ' a n d 'reeling-in', h a v e b e e n s u g g e s t e d f o r Olpidium ( T e m m i n k , 1971). In the 'wrap-around' m o d e , the a x o n e m e i s c o i l e d a r o u n d the b o d y o f the z o o s p o r e s o that the a x o n e m a l s h e a t h a n d p l a s m a l e m m a o f the z o o s p o r e b o d y are f u s e d d u r i n g a x o n e m a l uptake. In the 'reeling-in' m o d e , the a x o n e m e i s p u l l e d i n s i d e ('reeled in') the z o o s p o r e b o d y . T e m m i n k a n d C a m p b e l l ( 1 9 6 9 b ) s u g g e s t that the 'reeling-in' m o d e o f a x o n e m a l r e t r a c t i o n o c c u r s i n Olpidium z o o s p o r e s . T h e m o d e o f r e t r a c t i o n is i m p o r t a n t i n t r a n s m i s s i o n b e c a u s e it is h y p o t h e s i z e d that v i r u s p a r t i c l e s b o u n d to the a x o n e m e e n t e r the z o o s p o r e c y t o p l a s m f o l l o w i n g 'reeling-in' o f the f l a g e l l u m . T h i s t h e n a l l o w s v i r u s to e n t e r p l a n t s w h e n the z o o s p o r e c y t o p l a s m is i n j e c t e d i n t o the r e c i p i e n t c e l l . A f t e r a x o n e m a l w i t h d r a w a l a c y s t w a l l i s s e c r e t e d a r o u n d the z o o s p o r e b o d y . In p h o t o s o f c r o s s - s e c t i o n s a n d l o n g i t u d i n a l s e c t i o n s o f e n c y s t e d O. brassicae z o o s p o r e s , a x o n e m a l f i b r i l s w e r e o b s e r v e d c o i l e d w i t h i n the cyst, p r o v i d i n g e v i d e n c e f o r t h e ' r e e l i n g i n ' h y p o t h e s i s ( T e m m i n k , 1971). In s o m e c y s t s b u n d l e s o f m e m b r a n e s c o u l d b e o b s e r v e d i n c l o s e p r o x i m i t y to the p l a s m a l e m m a . T h e p r e s e n c e o f this w h o r l o f m e m b r a n e s s u g g e s t s that the s h e a t h is p u l l e d i n a n d b r e a k s d u r i n g a x o n e m a l w i t h d r a w a l ( T e m m i n k a n d C a m p b e l l , 1 9 6 9 b ; T e m m i n k , 1971). T h e 'reeling-in' m o d e o f a x o n e m a l r e t r a c t i o n w o u l d i n d i c a t e that v i r u s a d s o r b e d to the a x o n e m a l s h e a t h is p u l l e d i n t o the c y s t c y t o p l a s m , w h e r e it is a v a i l a b l e to e n t e r the r o o t c e l l c y t o p l a s m d u r i n g i n f e c t i o n ( T e m m i n k a n d C a m p b e l l , 1969b). T h i s h y p o t h e s i s w a s s u p p o r t e d w i t h p h o t o g r a p h s o f v i r u s - l i k e p a r t i c l e s c l o s e to w h o r l s o f m e m b r a n e s i n the c y s t s ( T e m m i n k , 1971). U p o n e n c y s t m e n t , the f u n g a l p r o t o p l a s m enters the r o o t c e l l ( S a h t i y a n c i , 1962). A h o l e a p p e a r s i n the c y s t w a l l a n d t h e n the c y s t c y t o p l a s m , s u r r o u n d e d b y a n e w l y f o r m e d  30  p l a s m a l e m m a , m o v e s i n t o t h e r o o t c e l l t h r o u g h a h o l e i n its c e l l w a l l (Terrvrnink, 1971, T e m m i n k a n d C a m p b e l l , 1 9 6 9 b ) . T h e c y s t p l a s m a l e m m a r e m a i n s i n t h e e m p t y cyst. T h e c y s t p r o t o p l a s t t h e n m o v e s t h r o u g h a h o l e i n the r o o t c e l l p l a s m a l e m m a i n t o t h e r o o t c e l l cytoplasm, b e c o m i n g a thallus contained within the thallus p l a s m a l e m m a ( T e m m i n k , 1971). T h e t h a l l u s g r o w s w i t h i n t h e r o o t c e l l f o r 2 4 h o u r s a n d t h e n b e c o m e s m u l t i n u c l e a t e a n d b e g i n s to f o r m a c e l l w a l l ( T e m m i n k , 1971). A f t e r 7 2 h o u r s t h e t h a l l u s forms cleavage vesicles w h i c h then fuse to b e c o m e zoospores. A varying n u m b e r o f z o o s p o r e s are c o n t a i n e d w i t h i n a m a t u r e z o o s p o r a n g i u m , w h i c h r e l e a s e s t h e z o o s p o r e s t h r o u g h its e x i t tube after c o n t a c t w i t h f r e s h w a t e r ( T e m m i n k , 1971). T w o i m p o r t a n t p o i n t s r e l e v a n t t o in vitro t r a n s m i s s i o n arise f r o m k n o w l e d g e o f the Olpidium l i f e c y c l e . F i r s t , v i r u s b o u n d t o t h e z o o s p o r e a x o n e m e m a y enter t h e z o o s p o r e t h r o u g h t h e 'reeling-in' m o d e o f r e t r a c t i o n w h i l e v i r u s b o u n d t o t h e p l a s m a l e m m a w i l l b e left b e h i n d i n the c y s t after t r a n s f e r o f c y s t c y t o p l a s m to t h e r o o t c e l l ( T e m m i n k , 1971). S e c o n d , t h e f u n g a l t h a l l u s i s s e p a r a t e d f r o m t h e r o o t c e l l c y t o p l a s m b y a s i n g l e m e m b r a n e d u r i n g t h e first 2 4 h o u r s after i n f e c t i o n . V i r u s m u s t enter t h e r o o t c e l l c y t o p l a s m b e f o r e t h e f o r m a t i o n o f a c e l l w a l l b y t h e t h a l l u s a p p r o x i m a t e l y 2 4 h o u r s p o s t - i n f e c t i o n ( T e m m i n k , 1971). O n e hypothesis for the m e c h a n i s m o f transfer o f virus f r o m the cyst cytoplasm ( w h i c h b e c o m e s t h e t h a l l u s ) to t h e r o o t c y t o p l a s m is b y v a c u o l e s ( T e m m i n k , 1971). V a c u o l e - l i k e structures w e r e n o t e d near t h e t h a l l i 4-24 h o u r s after i n o c u l a t i o n , b e f o r e t h e f o r m a t i o n o f the t h a l l u s w a l l . T h e s e v a c u o l e - l i k e structures s o m e t i m e s c o n t a i n e d v i r u s l i k e p a r t i c l e s a n d s o m e t i m e s these v a c u o l e s h a d r u p t u r e d a n d r e l e a s e d v i r u s - l i k e p a r t i c l e s . T h i s release m e c h a n i s m w a s t h o u g h t t o r e s e m b l e e x c r e t i o n o r r e g u r g i t a t i o n b y e x o c y t o s i s  31  as d e s c r i b e d b y de D u v e a n d W a t t i a u x ( 1 9 6 6 ) . L a b e l l i n g w i t h a n t i b o d i e s and/or g o l d p a r t i c l e s m a y h e l p to d e t e r m i n e the i d e n t i t y o f v i r u s - l i k e p a r t i c l e s i n the v a c u o l e s . E i g h t e e n h o u r s after i n f e c t i o n o f roots w i t h O. brassicae a n d T N V ,  irregular  clusters or c r y s t a l - l i k e arrays o f v i r u s p a r t i c l e s w e r e present i n n e c r o t i c c e l l s ( T e m m i n k , 1971). N e c r o s i s s e e m e d to o c c u r o n l y i n the f u n g u s - i n f e c t e d cells. N o g r o w i n g t h a l l i w e r e present i n the n e c r o t i c cells, so that i f v i r u s h a d e n t e r e d the c e l l a n d b e g u n to replicate, g r o w t h o f t h a l l i a n d s u b s e q u e n t p r o d u c t i o n o f z o o s p o r e s was p r e c l u d e d . In a c e l l c o n t a i n i n g g r o w i n g thalli, n o v i r u s p a r t i c l e s w e r e o b s e r v e d ( T e m m i n k , 1971). L i t t l e cell-to-cell spread o f T N V  o c c u r r e d i n i n f e c t e d lettuce roots, w i t h o b s e r v a b l e n e c r o t i c  l e s i o n s o n the roots but n o s y s t e m i c n e c r o s i s o f the entire plant. 1.2.3.2 In vitro ( n o n - c i r c u l a t i v e )  transmission  T w o species o f Olpidium (O. brassicae a n d O. bornovanus) h a v e b e e n s h o w n to transmit s e v e r a l s o i l - b o r n e p l a n t v i r u s e s i n the in vitro ( n o n - c i r c u l a t i v e ) m a n n e r ( T a b l e 1.2; G r a y a n d R o c h o n , 1999). In this m o d e o f t r a n s m i s s i o n , v i r u s e s are not f o u n d w i t h i n r e s t i n g s p o r e s a n d a c q u i s i t i o n o f v i r u s o c c u r s o u t s i d e o f l i v i n g c e l l s ( C a m p b e l l , 1996). V i r u s s u r v i v a l f r o m s e a s o n to s e a s o n d e p e n d s o n the stability o f v i r i o n s i n the s o i l a n d d e c a y i n g p l a n t debris, i n d e p e n d e n t o f f u n g a l a n d p l a n t s u r v i v a l ( C a m p b e l l , 1996). S e v e r a l d i s t i n c t p r o c e s s e s are r e q u i r e d f o r in vitro a c q u i s i t i o n o f v i r u s b y z o o s p o r e s : v i r u s a t t a c h m e n t to z o o s p o r e s , f u n g u s i n f e c t i o n o f the host, v i r u s release i n t o the host, a n d m u l t i p l i c a t i o n o f the v i r u s i n the host ( T e m m i n k et al, 1970). Z o o s p o r e s a n d v i r u s p a r t i c l e s are r e l e a s e d i n t o the s o i l i n d e p e n d e n t l y f r o m i n f e c t e d roots. V i r u s p a r t i c l e s t h e n a d h e r e to the p l a s m a l e m m a a n d a x o n e m a l s h e a t h o f the z o o s p o r e s ( T e m m i n k , 1971; S t o b b s et al, 1982). T h e a x o n e m e a l o n g w i t h b o u n d v i r u s is t h e n 32  w i t h d r a w n i n t o the z o o s p o r e c y t o p l a s m a n d v i r u s is then t h o u g h t to b e c a r r i e d i n t o r o o t c e l l s o f the h o s t p l a n t d u r i n g d i s c h a r g e o f the z o o s p o r e p r o t o p l a s m ( T e m m i n k , 1971; S t o b b s et al., 1982). V i r u s i s not f o u n d i n r e s t i n g s p o r e s as w i t h in vivo t r a n s m i s s i o n (Campbell,  1996).  1.2.3.2.1 S p e c i f i c i t y o f in vitro f u n g a l t r a n s m i s s i o n I n v e s t i g a t i o n s i n t o the m o d e o f T N V t r a n s m i s s i o n b y O. brassicae w e r e u n d e r t a k e n w i t h p a r t i c u l a r e m p h a s i s o n e x p l a i n i n g the o b s e r v e d v e c t o r s p e c i f i c i t y ( T e m m i n k et al, 1970). T h e l e t t u c e a n d t o m a t o i s o l a t e s o f O. brassicae t r a n s m i t  TNV  e f f i c i e n t l y w h i l e the m u s t a r d i s o l a t e d o e s not, e v e n t h o u g h the m u s t a r d i s o l a t e i s a b l e to i n f e c t p l a n t s ( T e m m i n k et ah, 1970). O. bornovanus ( f o r m e r l y O. cucurbitacearum) t r a n s m i t s C N V b u t n o t T N V to s e v e r a l h o s t p l a n t s ( D i a s , 1970a). D i f f e r e n c e s i n a d s o r p t i o n o f v i r u s to f u n g a l z o o s p o r e s w e r e t h o u g h t to c a u s e the d i f f e r e n t i a l t r a n s m i s s i o n , o r s p e c i f i c i t y , o b s e r v e d i n in vitro t r a n s m i s s i o n ( T e m m i n k et al., 1970). T o d e t e r m i n e i f this was the case, d i f f e r e n t isolates o f O. brassicae a n d O. bornovanus w e r e m i x e d w i t h T N V  o r C N V a n d t h e n n e g a t i v e l y stained, s e c t i o n e d , a n d  o b s e r v e d b y e l e c t r o n m i c r o s c o p y f o r v i r u s p a r t i c l e a d s o r p t i o n ( T e m m i n k et al., 1970). T N V b o u n d to l e t t u c e b u t n o t to m u s t a r d O. brassicae. C N V d i d n o t b i n d to e i t h e r i s o l a t e o f O. brassicae. In contrast, C N V b o u n d to O. bornovanus w h i l e T N V d i d n o t ( T e m m i n k et al., 1970). T a k e n together, these r e s u l t s i n d i c a t e d that the s p e c i f i c i t y o f in vitro v i r u s  t r a n s m i s s i o n b y Olpidium i s at the l e v e l o f z o o s p o r e b i n d i n g . A c o m p r e h e n s i v e a n a l y s i s o f the i n t e r a c t i o n s b e t w e e n s i x v i r u s e s k n o w n to b e a c q u i r e d in vitro a n d t r a n s m i t t e d b y at l e a s t o n e i s o l a t e o f Olpidium spp. f u r t h e r e x a m i n e d the s p e c i f i c i t y o f i n t e r a c t i o n b e t w e e n these v i r u s e s a n d t h e i r f u n g a l v e c t o r s ( C a m p b e l l 33  et  al., 1995). Cucumber soil borne carmovirus ( C S B V ) , Melon necrotic spot carmovirus ( M N S V ) , Squash necrosis carmovirus ( S q N V ) , Cucumber leaf spot aureusvirus ( C L S V ) ,  C N V a n d T N V w e r e e x a m i n e d f o r t r a n s m i s s i b i l i t y b y s e v e r a l s i n g l e s p o r a n g i a l isolates o f 0.  bornovanus a n d 0. brassicae o b t a i n e d f r o m s e v e r a l d i f f e r e n t g e o g r a p h i c r e g i o n s . F i v e  c u c u m b e r , five m e l o n , a n d t w o s q u a s h isolates o f O. bornovanus a n d three isolates o f O. brassicae w e r e e x a m i n e d f o r their a b i l i t y to t r a n s m i t these s i x v i r u s e s in vitro ( C a m p b e l l et al., 1995). A l l isolates o f O. bornovanus t r a n s m i t t e d C N V b u t there w a s a h i g h  v a r i a b i l i t y i n the l e v e l s o f t r a n s m i s s i o n as s h o w n b y a b s o r b a n c e v a l u e s f r o m a d o u b l e a n t i b o d y s a n d w i c h E L I S A o f extracts o f i n f e c t e d r o o t s i n o c u l a t e d w i t h in vitro m i x t u r e s o f v i r u s a n d z o o s p o r e s ( C a m p b e l l et al., 1995). A l l isolates o f O. bornovanus t r a n s m i t t e d M N S V . A l l f i v e c u c u m b e r , o n l y t w o o f the m e l o n a n d n o n e o f the s q u a s h isolates o f O. bornovanus t r a n s m i t t e d C L S V . A l l c u c u m b e r a n d s q u a s h b u t o n l y t w o o f the m e l o n isolates o f O. bornovanus t r a n s m i t t e d S q N V . T w o s q u a s h isolates o f O. bornovanus w e r e the o n l y v e c t o r s o f C S B V . N o n e o f the 12 isolates o f O. bornovanus  t r a n s m i t t e d T N V . T h r e e c u c u r b i t isolates o f O.brassicae w e r e tested f o r t r a n s m i s s i o n o f a l l s i x v i r u s e s a n d o n l y T N V w a s t r a n s m i t t e d , w i t h v a r i a b l e e f f i c i e n c y ( C a m p b e l l et ai, 1995). T h e h i g h s p e c i f i c i t y o f the b i o l o g i c a l i n t e r a c t i o n b e t w e e n Olpidium a n d the v i r u s e s it t r a n s m i t s is s t r o n g l y s u g g e s t i v e o f a s p e c i f i c m o l e c u l a r i n t e r a c t i o n b e t w e e n v i r u s p a r t i c l e s a n d Olpidium z o o s p o r e s . 1.2.3.3 In vivo ( c i r c u l a t i v e ) f u n g a l t r a n s m i s s i o n F u n g a l t r a n s m i s s i o n i s c h a r a c t e r i z e d as in vivo ( c i r c u l a t i v e ) i f i n f e c t i v e v i r u s c a n b e i d e n t i f i e d f r o m r e s t i n g s p o r e s o f f u n g u s after a i r - d r y i n g o f r o o t s o r l o n g - t e r m s t o r a g e o f i n f e s t e d s o i l o r after a c i d ( 5 N H C 1 ) o r t r i s o d i u m p h o s p h a t e ( 2 0 % ) treatments, w h i c h 34  k i l l e x t e r n a l l y - b o r n e (in vitro t r a n s m i t t e d ) v i r u s ( C a m p b e l l , 1985; H i r u k i , 1987).  The  p r o c e s s o f in vivo v i r u s a c q u i s i t i o n a n d t r a n s m i s s i o n is u n k n o w n b u t it m a y b e that i n o c u l a t i o n o c c u r s s o o n after the f u n g u s penetrates the v i r u s - i n f e c t e d h o s t c e l l , b e f o r e the f o r m a t i o n o f a t h a l l u s w a l l ( C a m p b e l l , 1996). V i r u s e s are w i t h i n z o o s p o r e s u p o n r e l e a s e f r o m r o o t c e l l s a n d c a n n o t b e a c q u i r e d b y z o o s p o r e s o u t s i d e o f the p l a n t i n c o n t r a s t to v i r u s e s t r a n s m i t t e d i n the in vitro m a n n e r (see b e l o w ) . V i r u s e s w i t h i n r e s t i n g s p o r e s c a n s u r v i v e f o r l o n g p e r i o d s a n d s o i n f e s t a t i o n s b y in v/vo-transmitted v i r u s e s are v i r t u a l l y p e r m a n e n t , w i t h v i r u s r e s i s t a n c e i n p l a n t s b e i n g the p r i m a r y c o n t r o l m e a s u r e ( C a m p b e l l , 1996). 1.2.3.3.1 In vivo t r a n s m i s s i o n b y Olpidium brassicae A n a s s o c i a t i o n b e t w e e n O. brassicae a n d b i g v e i n d i s e a s e o f l e t t u c e was the first d e s c r i p t i o n o f Olpidium spp. i n v o l v e d i n d i s e a s e p r o d u c t i o n o n p l a n t s ( G r o g a n et al., 1958; F r y , 1958). T h e c a u s a t i v e agent o f the d i s e a s e was d e t e r m i n e d to b e Lettuce bigvein virus ( L B V V ) ( C a m p b e l l et al., 1961; C a m p b e l l a n d G r o g a n , 1963). Tobacco stunt virus ( T S t V ) was a l s o s h o w n to b e t r a n s m i t t e d b y O. brassicae ( H i r u k i , 1970). V i r i o n s o f  L B V V a n d T S t V are r o d - s h a p e d a n d c l o s e l y r e l a t e d s e r o l o g i c a l l y ( C a m p b e l l ,  1996).  T S t V c o n t a i n s t w o d s R N A s a n d it i s l i k e l y that L B V V a l s o c o n t a i n s d s R N A s . T h e r e l a t i o n s h i p s o f T S t V a n d L B V V w i t h O. brassicae are v e r y s i m i l a r ( G r o g a n a n d Campbell,  1966).  O. brassicae-trmsmitted L B V V i s a c q u i r e d as the f u n g u s d e v e l o p s i n v i r u s -  i n f e c t e d h o s t c e l l s ( C a m p b e l l a n d G r o g a n , 1963). A p p r o x i m a t e l y 5 0 % o f s i n g l e s p o r a n g i a l c u l t u r e s c o n t a i n L B V V ( L i n et al, 1970; C a m p b e l l , 1988). T h e r e i s l i m i t e d e v i d e n c e that L B V V d o e s not r e p l i c a t e i n O. brassicae ( C a m p b e l l , 1996). T h e l a c k o f 35  r e p l i c a t i o n o f L B V V i n its v e c t o r w o u l d e x p l a i n w h y c u l t u r e s o f O. brassicae, w h i c h r e a d i l y i n f e c t a n d r e p r o d u c e o n s u g a r b e e t roots, l o s e their a b i l i t y t o t r a n s m i t L B V V after serial p a s s a g e t o v i r u s - r e s i s t a n t s u g a r - b e e t r o o t s ( C a m p b e l l , 1962). L B V V - i n f e c t e d O. brassicae c o n t i n u e d t o b e v i r u l i f e r o u s after serial p a s s a g e t o lettuce roots, i n w h i c h t h e  v i r u s r e p l i c a t e s r e a d i l y ( C a m p b e l l , 1962). I n t h e a b s e n c e o f r e p l i c a t i o n i n the v e c t o r , v i r u s titer falls w i t h e a c h g e n e r a t i o n u n l e s s the f u n g u s i n f e c t s h o s t c e l l s i n w h i c h the v i r u s c a n m u l t i p l y ( C a m p b e l l , 1996). L B V V a n d T S t V s u r v i v e i n r e s t i n g s p o r e s a n d c a n remain i n infested soil indefinitely. 1.2.3.3.2 In vivo t r a n s m i s s i o n b y Polymyxa a n d Spongospora spp. S e v e r a l m e m b e r s o f the f u r o - a n d b y m o v i r u s g r o u p s are f u n g a l l y t r a n s m i t t e d i n the in vivo m a n n e r b y Polymyxa spp. ( T a b l e 1.2; G r a y a n d R o c h o n , 1999). O n e f u r o v i r u s , Potato mop top virus ( P M T V ) a n d t w o u n c h a r a c t e r i z e d v i r u s e s , Watercress yellow spot virus ( W Y S V ) a n d Watercress chlorotic leaf spot agent ( W C L A ) , are t r a n s m i t t e d b y Spongospora s p p ( T a b l e 1.2, G r a y a n d R o c h o n , 1999).  Fungally transmissible furoviruses contain sequence homologies i n a conserved d o m a i n o f a C P r e a d t h r o u g h p r o t e i n . In this r e g a r d , t h e f u r o v i r u s r e a d t h r o u g h d o m a i n m a y h a v e a f u n c t i o n s i m i l a r t o that o f the R T D o f the a p h i d - t r a n s m i t t e d l u t e o v i r u s e s (see s e c t i o n 1.2.1; C a m p b e l l , 1996). B y m o v i r u s e s d o n o t e n c o d e a C P R T b u t d o e n c o d e a p r o t e i n f r o m t h e s e c o n d O R F o f R N A - 2 w h i c h is i n v o l v e d i n t r a n s m i s s i o n ( C a m p b e l l , 1996). F u r o - a n d b y m o v i r u s e s are r o d - s h a p e d b u t t h e y are m o r p h o l o g i c a l l y distinguishable. Furoviruses have two to five separately encapsidated s s R N A s and b y m o v i r u s e s h a v e t w o s s R N A s ( C a m p b e l l , 1996). T h e g e n o m e s o f t h e s e v i r u s e s c a n b e a l t e r e d t h r o u g h serial p a s s a g i n g o r m a i n t e n a n c e o n o l d e r plants. S p o n t a n e o u s d e l e t i o n 36  m u t a t i o n s i n the r e g i o n e n c o d i n g the r e a d t h r o u g h p r o t e i n o f the C P o f Soil borne wheat mosaic virus ( S B W M V ) , B N Y V V , Peanut clump virus ( P C V ) , a n d B a M M V h a v e b e e n  i d e n t i f i e d f r o m serial m e c h a n i c a l p a s s a g e i n the a b s e n c e o f their f u n g a l v e c t o r s ( C h e n et al., 1995; S h i r a k o a n d B r a k k e , 1984; T a m a d a a n d K u s u m e , 1991; M a n o h a r et al.,  1993;  D e s s e n s et al., 1995; J a c o b i et al, 1995). It has b e e n s u g g e s t e d that the d e l e t e d r e g i o n s c o r r e s p o n d to p o r t i o n s o f the C P i n v o l v e d i n f u n g u s t r a n s m i s s i o n . B N Y V V was m e c h a n i c a l l y p a s s a g e d 17 t i m e s o v e r a p e r i o d o f three y e a r s i n o r d e r to p r o d u c e d e l e t i o n m u t a n t s i n R N A - 2 ( T a m a d a a n d K u s u m e , 1991). T h e s e B N Y V V m u t a n t s h a d d e l e t i o n s i n the C - t e r m i n u s o f the R T p r o t e i n a n d w e r e n o t t r a n s m i s s i b l e b y the v e c t o r o f B N Y V V , Polymyxa betae, s u g g e s t i n g that the f u l l - l e n g t h 75 k D a R T p r o t e i n was r e q u i r e d f o r t r a n s m i s s i o n ( T a m a d a a n d K u s u m e , 1991). T h i s was the first d i r e c t p r o o f that a C P R T p r o t e i n p l a y s a r o l e i n v e c t o r t r a n s m i s s i o n o f a p l a n t v i r u s . D e l e t i o n analysis and scanning alanine mutagenesis have identified a K T E R a m i n o acid m o t i f near the C - t e r m i n u s o f the B N Y V V R T p r o t e i n ( T a m a d a et al, 1996). T h e Polymyxa gramzms-transmitted  v i r u s , S B W M V , has a s i m i l a r s e q u e n c e m o t i f ( K T E I R ) l o c a t e d n e a r  the C - t e r m i n u s o f its R T p r o t e i n ( S h i r a k o a n d W i l s o n , 1993). B a s e d o n these s i m i l a r i t i e s , i t has b e e n s u g g e s t e d that these m o t i f s p l a y i m p o r t a n t r o l e s i n f u n g u s t r a n s m i s s i o n . A l i g n m e n t s o f the C P r e a d t h r o u g h d o m a i n o f s e v e r a l f u n g a l l y t r a n s m i t t e d f u r o - a n d b y m o v i r u s e s h a v e i d e n t i f i e d the c o m m o n a m i n o a c i d d y a d s E R a n d Q R ( P e e r e n b o o m et al,  1996). T h e s e m o t i f s are l o c a t e d o n the o u t s i d e o f the p r o t e i n , s u g g e s t i n g that they  m a y facilitate i n t e r a c t i o n s w i t h the f u n g a l vector.  37  1.3 Cucumber necrosis virus C N V w a s i d e n t i f i e d i n the 1950's as the c a u s a t i v e agent o f a s e v e r e d i s e a s e i n g r e e n h o u s e g r o w n c u c u m b e r s i n O n t a r i o , C a n a d a ( M c K e e n , 1959). C N V c a u s e d n e c r o t i c s p o t t i n g a n d m a l f o r m a t i o n o f the f o l i a g e , s t u n t i n g o f g r o w t h a n d r e d u c e d fruit size, w i t h d e a t h o f the p l a n t s i x to e i g h t w e e k s p o s t - i n f e c t i o n i n s y s t e m i c cases. C N V is r e g u l a r l y f o u n d i n f e c t i n g g l a s s h o u s e g r o w n c u c u m b e r s i n O n t a r i o a n d has b e e n i d e n t i f i e d as a c a u s a l agent o f d i s e a s e o f t o m a t o s i n T e x a s a n d A r i z o n a . It is a l s o f o u n d i n f e c t i n g l e t t u c e a s y m p t o m a t i c a l l y i n the S a l i n a s V a l l e y o f C a l i f o r n i a ( D . M . R o c h o n , p e r s o n a l c o m m u n i c a t i o n ) . A l t h o u g h C N V has a l i m i t e d n a t u r a l h o s t range, its e x p e r i m e n t a l h o s t range is wide. C N V can infect plants f r o m several dicotyledonous plant families.  1.3.1 Taxonomic status of  CNV  C N V r e p r e s e n t s o n e o f s e v e r a l s p e c i e s i n the T o m b u s v i r u s g e n u s ( f a m i l y Tombusviridae). T o m b u s v i r u s e s h a v e 3 0 n m s p h e r i c a l p a r t i c l e s c o n t a i n i n g a s i n g l e  m o l e c u l e o f p o s i t i v e s e n s e R N A , c o m p r i s e d o f a p p r o x i m a t e l y 4,700 nt. C N V i s s e r o l o g i c a l l y d i s t i n c t f r o m o t h e r t o m b u s v i r u s e s , l i k e l y d u e to the v a r i a b l e n a t u r e o f the t o m b u s v i r u s C P p r o t r u d i n g d o m a i n . M o s t t o m b u s v i r u s e s are s o i l - t r a n s m i t t e d b u t C N V i s the o n l y t o m b u s v i r u s w h i c h has b e e n s h o w n to h a v e a s p e c i f i c s o i l v e c t o r . T o m b u s v i r u s e s r e p r e s e n t p a r t o f the f a m i l y Tombusviridae. A l t h o u g h C N V i s the o n l y t o m b u s v i r u s f o r w h i c h a s o i l v e c t o r has b e e n u n e q u i v o c a l l y d e m o n s t r a t e d , s e v e r a l m e m b e r s o f the Tombusviridae are k n o w n to b e f u n g a l l y t r a n s m i t t e d ( T a b l e 1.2). It i s p o s s i b l e that a d d i t i o n a l t o m b u s v i r u s e s as w e l l as Tombusviridae m e m b e r s w i l l b e f o u n d to h a v e Olpidium v e c t o r s .  38  ORF  5' —I  TJAG 33 kDa ORF1  |  TH  92 kDa ORF 2  5  I 20 kDa  1  3'  41 kDa ( C P i n - l 2 1 k D a l — ORF 3 ORF 4 sgRNA 1 sgRNA 2  Figure 1.2 G e n o m i c o r g a n i z a t i o n o f C N V .  The five open reading frames ( O R F s ) e n c o d e d b y the C N V g e n o m e are s h o w n . P r o t e i n s p r o d u c e d b y e a c h O R F are i n d i c a t e d . T h e 33 a n d 9 2 k D a p r o t e i n s are t r a n s l a t e d f r o m g e n o m i c l e n g t h R N A a n d the 4 1 k D a a n d 21/20 k D a p r o t e i n s are t r a n s l a t e d f r o m s u b g e n o m i c ( s g ) R N A s 1 a n d 2, r e s p e c t i v e l y . S g R N A 2 is b i c i s t r o n i c w i t h the 2 0 k D a p r o t e i n b e i n g p r o d u c e d b y l e a k y s c a n n i n g ( J o h n s t o n a n d R o c h o n , 1996).  1.3.2 Genomic organization  T h e g e n o m e o f C N V is 4 7 0 1 nt, h a s b e e n f u l l y s e q u e n c e d a n d a f u l l - l e n g t h i n f e c t i o u s c D N A c l o n e has b e e n p r o d u c e d ( R o c h o n a n d T r e m a i n e , 1989; R o c h o n a n d J o h n s t o n , 1991). A t least f i v e p r o t e i n s o f m o l e c u l a r w e i g h t 33, 92, 41, 21, a n d 2 0 k D a are p r e d i c t e d b y the s e q u e n c e o f C N V ( F i g u r e 1.2). B a s e d o n s e q u e n c e c o m p a r i s o n s i t has b e e n s u g g e s t e d that p 9 2 is the r e p l i c a s e ( R o c h o n a n d T r e m a i n e , 1989). A d d i t i o n a l w o r k h a s s h o w n that p 4 1 is the c o a t p r o t e i n ( C P ) , p 2 1 i s a c e l l - t o - c e l l m o v e m e n t p r o t e i n , a n d p 2 0 is i n v o l v e d i n s y m p t o m i n d u c t i o n as w e l l as s u p p r e s s i n g the f o r m a t i o n o f d e f e c t i v e i n t e r f e r i n g R N A s ( J o h n s t o n a n d R o c h o n , 1990; R o c h o n a n d J o h n s t o n , 1991; R o c h o n , 1991). T h e f u n c t i o n o f p 3 3 is n o t k n o w n b u t i t is b e l i e v e d t o b e a n e c e s s a r y c o m p o n e n t o f the v i r a l r e p l i c a s e . T w o 3' c o - t e r m i n a l s u b g e n o m i c R N A s ( s g R N A 1 a n d s g R N A 2 ) are g e n e r a t e d f r o m C N V g e n o m i c R N A d u r i n g i n f e c t i o n ( R o c h o n a n d J o h n s t o n , 1991). S g R N A l  39  serves as template f o r the c o a t p r o t e i n a n d s g R N A 2 is a b i f u n c t i o n a l m R N A  encoding  p21 a n d p20. T r a n s l a t i o n o f p 2 0 f r o m s g R N A 2 o c c u r s v i a a l e a k y s c a n n i n g m e c h a n i s m ( J o h n s t o n a n d R o c h o n , 1990, 1996). 1.3.3 CNV defective interfering RNAs  D e f e c t i v e i n t e r f e r i n g (DI) R N A s are n a t u r a l v i r a l d e l e t i o n m u t a n t s p r o d u c e d w h e n errors are m a d e d u r i n g r e p l i c a t i o n o f the p a r e n t a l v i r u s . D I R N A s are u n a b l e to r e p l i c a t e w i t h o u t the p r o d u c t i o n o f trans-acting factors, s u c h as the v i r a l r e p l i c a s e , b y the p a r e n t a l virus. D I g e n o m e s retain a m i n i m u m o f cis-acting sequences required for replication. C o m p e t i t i o n b e t w e e n the m o r e e f f i c i e n t l y r e p l i c a t i n g D I R N A s a n d that o f the p a r e n t a l v i r u s f o r these f a c t o r s results i n i n t e r f e r e n c e w i t h the n o r m a l r e p l i c a t i o n o f the parental, o r helper, v i r u s (Jones et al., 1990). D I R N A s are t h o u g h t to o c c u r u n i v e r s a l l y i n a n i m a l v i r u s i n f e c t i o n s (Perrault, 1981) a n d h a v e b e e n r e p o r t e d f o r s e v e r a l p o s i t i v e - s t r a n d s s R N A p l a n t v i r u s e s ( r e v i e w e d b y R o u x et al., 1991) i n c l u d i n g C N V  ( R o c h o n , 1991;  J o h n s t o n a n d R o c h o n , 1990; F i n n e n a n d R o c h o n , 1993), T B S V - C h ( H i l l m a n et al, 1987), Cymbidium ringspot tombusvirus ( B u r g y a n et al., 1989), Turnip crinkle carmovirus ( L i et al., 1989), Clover yellow mosaic potexvirus ( W h i t e et al., 1991), Tomato spotted wilt tospovirus ( R e s e n d e et al., 1992) a n d s e v e r a l other p l a n t v i r u s e s .  RNA  extracts o f plants i n f e c t e d w i t h a l a b o r a t o r y c u l t u r e o f C N V  contained high levels of a virus-related R N A  (CNV-Lc)  s p e c i e s o f a p p r o x i m a t e l y 4 0 0 nt ( J o h n s t o n  a n d R o c h o n , 1990). S y m p t o m s o n plants i n o c u l a t e d w i t h C N V - L c w e r e persistent a n d attenuated w h i l e a severe s y s t e m i c n e c r o s i s o f plants i n o c u l a t e d w i t h w i l d - t y p e transcripts o c c u r r e d , i n d i c a t i n g that this 4 0 0 nt v i r u s - l i k e R N A  40  c o u l d be a DI.  CNV  S e q u e n c i n g o f s e v e r a l c l o n e s f r o m the the C N V - L c s m a l l R N A s i n d i c a t e d that these s m a l l R N A s w e r e i n f a c t D I R N A s d e r i v e d f r o m the C N V  g e n o m e ( F i n n e n a n d R o c h o n , 1993).  In a d d i t i o n to the attenuated a n d persistent s y m p t o m s o c c u r i n g i n the p r e s e n c e o f D I R N A s , a reduced y i e l d of virus particles (wild-type C N V ) is observed. The coat protein of C N V al,  is not r e q u i r e d f o r e f f i c i e n t g e n e r a t i o n or a c c u m u l a t i o n o f D I R N A s ( R o c h o n et  1994).  1.3.4 Coat protein mutants  A c o a t p r o t e i n d e l e t i o n m u t a n t w h i c h d o e s not c o n t a i n the s e q u e n c e c o d i n g f o r the p r o t r u d i n g d o m a i n ( P - d o m a i n ; see p a r t i c l e structure b e l o w ) o f its C P ( M c L e a n et al, 1993) was e x a m i n e d to d e t e r m i n e the r o l e o f the P - d o m a i n i n C N V  infection. This  mutant, n a m e d PD(-), r e p l i c a t e d w e l l a n d m o v e d s y s t e m i c a l l y i n the C N V N. clevelandii. N e i t h e r C N V  s y s t e m i c host,  v i r i o n s n o r C P s u b u n i t s c o u l d be d e t e c t e d f r o m sap o f P D ( -  )-infected plants. Interestingly, the m o s t p r o m i n e n t R N A  species i n systemically infected  l e a v e s o f P D ( - ) - i n o c u l a t e d plants was a p p r o x i m a t e l y 3 8 0 0 nt, c o r r e s p o n d i n g to the d e l e t i o n o f a l l b u t 74 nt o f the 1140 nt C P c o d i n g s e q u e n c e ( C P ( - ) ) ( M c L e a n T h e s e results s h o w e d that C N V  RNA  et al, 1993).  c a n r e p l i c a t e a n d m o v e s y s t e m i c a l l y i n p l a n t s i n the  a b s e n c e o f a f u n c t i o n a l C P . A n o t h e r t o m b u s v i r u s , Tomato bushy stunt virus, c h e r r y s t r a i n ( T B S V - C h ) , i s a l s o able to m o v e c e l l - t o - c e l l a n d s y s t e m i c a l l y i n N. clevelandii a n d N. benthamiana i n the a b s e n c e o f its C P ( S c h o l t o f et al, 1993).  In f u r t h e r attempts to p r o d u c e a P - d o m a i n l e s s C N V  particle, several P - d o m a i n  f r a m e s h i f t a n d d e l e t i o n m u t a n t s w e r e p r o d u c e d a n d e x a m i n e d f o r the p r e s e n c e o f v i r u s o r v i r u s - l i k e p a r t i c l e s u s i n g e l e c t r o n m i c r o s c o p y ( S i t et al, 1995). P a r t i c l e s w e r e not  41  o b s e r v e d f o r a n y o f the m u t a n t s , i n c l u d i n g those l a c k i n g o n l y t w e l v e a m i n o a c i d s at the carboxy-terminus  o f the P - d o m a i n . C P s u b u n i t s w e r e i d e n t i f i e d f o r s e v e r a l o f these P-  d o m a i n m u t a n t s i n sap but at l o w e r l e v e l s than w i l d - t y p e C N V , i n d i c a t i n g that the d e l e t e d C P s u b u n i t s m a y be l e s s stable t h a n w i l d - t y p e ( S i t et al., 1995). T h e a b s e n c e o f v i r u s p a r t i c l e s i n p l a n t s i n f e c t e d w i t h P - d o m a i n m u t a n t s d e m o n s t r a t e s that the P - d o m a i n is required for C N V  p a r t i c l e f o r m a t i o n and/or stability.  (c)  F i g u r e 1.3 S t r u c t u r e o f (a) the T B S V p a r t i c l e , (b) c o a t p r o t e i n subunit, a n d (c) l i n e a r o r d e r o f d o m a i n s o f the c o a t protein. T h e n u m b e r s i n (c) c o r r e s p o n d to the n u m b e r o f a m i n o a c i d s i n e a c h d o m a i n (adapted f r o m H o p p e r et al., 1984). S e e text f o r a d d i t i o n a l details.  1.3.5 P a r t i c l e s t r u c t u r e D e t a i l s o f the C N V  p a r t i c l e structure h a v e not b e e n d e t e r m i n e d . H o w e v e r , the  t h r e e - d i m e n s i o n a l structure o f the p a r t i c l e a n d c o n s t i t u e n t c o a t p r o t e i n s u b u n i t s o f the c l o s e l y r e l a t e d type m e m b e r o f the t o m b u s v i r u s g r o u p , T B S V , has b e e n d e t e r m i n e d b y X -  42  r a y c r y s t a l l o g r a p h y to h i g h r e s o l u t i o n ( H a r r i s o n et al,  1978). T h e 180 i d e n t i c a l C P  s u b u n i t s are a r r a n g e d i n T = 3 i c o s a h e d r a l s y m m e t r y i n the v i r a l c a p s i d . E a c h C P s u b u n i t f o l d s i n t o three d o m a i n s : the R N A - b i n d i n g ( R ) , s h e l l (S), a n d p r o t r u d i n g (P) d o m a i n s ( F i g u r e 1.3; H a r r i s o n et al,  1978; H o p p e r et al,  1984). T h e R a n d S d o m a i n s are  c o n n e c t e d b y an a r m (a) a n d the S a n d P d o m a i n s are j o i n e d b y a short h i n g e (h). T B S V C P s u b u n i t s o c c u r i n three d i f f e r e n t e n v i r o n m e n t s i n the c a p s i d architecture, d e s i g n a t e d A , B, a n d C. T h e a r m s o f the C s u b u n i t s i n t e r c a l a t e i n the i n t e r i o r o f the p a r t i c l e to f o r m a c a g e - l i k e structure w h i l e C a  + +  s t a b i l i z e s the i n t e r a c t i o n b e t w e e n s h e l l d o m a i n s o f  s u b u n i t s A - B , B-C, a n d C - A at the p a r t i c l e s u r f a c e ( H a r r i s o n et al, c h e l a t o r s s u c h as E D T A r e m o v e C a  + +  1978). A d d i t i o n o f  f r o m these s u b u n i t contacts, c a u s i n g a s p a r t i c a c i d  r e s i d u e s to r e p e l e a c h o t h e r a n d the p a r t i c l e to s w e l l i n a p H d e p e n d e n t f a s h i o n ( H a r r i s o n etal, 1978).  A l t h o u g h the c r y s t a l structure o f C N V has n o t b e e n d e t e r m i n e d , h o m o l o g y b a s e d structural m o d e l i n g has b e e n u s e d to m o d e l the structures o f the C N V  C P subunit  f o l l o w i n g a l i g n m e n t s w i t h the T B S V subunit. T h e s e s t u d i e s h a v e i n d i c a t e d that the o v e r a l l s e c o n d a r y structure o f C N V d o e s n o t d i f f e r r a d i c a l l y f r o m that o f T B S V (K. K a k a n i a n d D. R o c h o n , p e r s o n a l  1.3.6 The role of the CNV  communication).  coat protein in fungus transmission  T h e C P O R F o f C N V a n d that o f T B S V - C h , a t o m b u s v i r u s w i t h n o k n o w n f u n g a l vector, w e r e e x c h a n g e d i n f u l l - l e n g t h i n f e c t i o u s c D N A c l o n e s o f e a c h v i r u s to d e t e r m i n e i f the C P i s i n v o l v e d i n the s p e c i f i c i t y o f t r a n s m i s s i o n ( M c L e a n et al, 1994). P a r t i c l e s o f c h i m e r i c T B S V - C h c o n t a i n i n g the C N V  C P g e n e w e r e t r a n s m i s s i b l e v i a z o o s p o r e s o f O.  43  bornovanus, w h i l e p a r t i c l e s o f c h i m e r i c C N V  c o n t a i n i n g the T B S V - C h C P g e n e w e r e not  t r a n s m i s s i b l e b y O. bornovanus ( M c L e a n et al., 1994). T h e s e results w e r e the first d i r e c t e v i d e n c e that the C P o f a p l a n t v i r u s i s r e s p o n s i b l e f o r the s p e c i f i c i t y o f its t r a n s m i s s i o n b y a fungus. W e h y p o t h e s i z e that s e q u e n c e s and/or structures o f the C N V C P i n t e r a c t w i t h s p e c i f i c factors(s) i n the z o o s p o r e p l a s m a l e m m a to facilitate attachment a n d thus t r a n s m i s s i o n . T h e s h e l l a n d p r o t r u d i n g d o m a i n s o f the t o m b u s v i r u s p a r t i c l e f o r m d i s t i n c t j e l l y - r o l l b a r r e l s ( H a r r i s o n et al., 1978), c o m m o n s t r u c t u r a l m o t i f s o f m a n y i c o s a h e d r a l v i r u s e s a n d c e r t a i n p r o t e i n s k n o w n to h a v e r e c e p t o r b i n d i n g f u n c t i o n s ( C h e l v a n a y a g a m et ah, 1992; G i b s o n a n d A r g o s , 1990). T h e j e l l y - r o l l b a r r e l s o f C N V m a y b e i n v o l v e d i n the i n t e r a c t i o n o f this v i r u s w i t h p u t a t i v e r e c e p t o r m o l e c u l e s o n the s u r f a c e o f O. bornovanus zoospores.  1.4 Brief overview of virus-receptor interactions V i r u s i n f e c t i o n o f host c e l l s b e g i n s b y attachment o f the v i r u s to the host c e l l s u r f a c e ( f o r r e v i e w s see D a l e s , 1973; L o n b e r g - H o l m a n d P h i l i p s o n , 1974; D i m m o c k , 1982; T a r d i e u et al., 1982; P a u l s o n , 1985; M a r s h a n d H e l e n i u s , 1989). T h e nature, n u m b e r a n d d i s t r i b u t i o n o f the s p e c i f i c c e l l u l a r r e c e p t o r s to w h i c h m o s t v i r u s e s attach p l a y s a r o l e i n d e t e r m i n i n g t h e i r host r a n g e a n d t i s s u e t r o p i s m ( H o l l a n d , 1961;  Lonberg-  H o l m a n d P h i l i p s o n , 1974; C r o w e l l a n d L a n d a u , 1979; P a u l s o n , 1985; M i m s , 1986). A n i m a l v i r u s e s u t i l i z e a w i d e v a r i e t y o f c e l l u l a r s u r f a c e m o l e c u l e s as r e c e p t o r s i n c l u d i n g p r o t e i n s , c a r b o h y d r a t e s a n d g l y c o l i p i d s ( R o s s m a n n , 1994; T a b l e 1.3). A t t a c h m e n t is  44  m e d i a t e d b y the b i n d i n g o f a site o n t h e v i r u s particle, a v i r a l attachment p r o t e i n ( V A P ) , to a m o l e c u l e o n the c e l l s u r f a c e a c t i n g as a v i r u s r e c e p t o r ( L e n t z , 1990). Table  1.3  S o m e k n o w n receptors for animal viruses . 1  Cellular Receptor  Virus  Reference  Sialic acid  Reoviruses Influenza virus Polyomavirus  Paul etal., 1989; Choi etal., 1990 Weis etal, 1988 Fried etal., 1981  HIV Polioviruses Major serotype group of HRV FMDV Epstein-Barr viruses  Dalgleish etal., 1984 Mendelsohn et al., 1989 Greve etal., 1989; Staunton etal, 1989; Tomassini etal, 1989 Acharya etal, 1989; Mason etal., 1993 Moore etal., 1987; Tanner etal, 1987  Ecotropic murine leukemia virus Mouse hepatitis virus (a coronavirus) Human B19 Parvovirus Minor serotype group of HRV Transmissible gastroenteritis virus Human coronavirus, strain 229E  Kim  Immunoglobulin family: (i) CD4 (ii) Poliovirus receptor (iii) ICAM-1 Integrins Complement receptor type 2 (B lymphocyte surface glycoprotein) Amino acid permeases Carcinoembryonic antigen Erythrocyte P antigen LDL receptor Aminopeptidase N  etal, 1991; Wang etal, 1991  Williams etal, 1991 Browner al, 1993 Hoferefa/., 1994 Yeageref al, 1992 Vlasakef al, 1988  'Table is from Rossmann, 1994.  V i r u s r e c e p t o r s h a v e b e e n d e f i n e d as structures o n the c e l l s u r f a c e t o w h i c h v i r u s attachment is f o l l o w e d b y a b i o l o g i c a l l y r e l e v a n t r e s p o n s e ( T a r d i e u et al, 1982). T h i s r e s p o n s e i s u s u a l l y i n f e c t i o n o f the c e l l b y entry o f the v i r u s v i a e i t h e r m e m b r a n e f u s i o n o r r e c e p t o r - m e d i a t e d e n d o c y t o s i s ( P a u l s o n , 1985; M a r s h a n d H e l e n i u s , 1989). F r o m studies o f v i r u s - r e c e p t o r i n t e r a c t i o n s u s i n g t i s s u e c u l t u r e systems, s e v e r a l f a c t o r s , i n c l u d i n g temperature, i o n i c strength, p H , c o m p o s i t i o n o f the m e d i u m , a n d the p r e s e n c e o f s e r u m , h a v e m a j o r e f f e c t s o n v i r u s b i n d i n g ( L o n b e r g - H o l m , 1981).  45  Initial c h a r a c t e r i z a t i o n o f the i n t e r a c t i o n b e t w e e n a v i r u s a n d a p u t a t i v e r e c e p t o r i n v o l v e s the e s t a b l i s h m e n t o f s p e c i f i c i t y i n the i n t e r a c t i o n ( T a r d i e u et al., 1982). T h r e e c r i t e r i a w e r e d e s c r i b e d b y T a r d i e u et al. ( 1 9 8 2 ) as n e c e s s a r y f o r e s t a b l i s h i n g that a s p e c i f i c r e c e p t o r exists o n host cells. First, s p e c i f i c i t y o f b i n d i n g m u s t c o r r e l a t e w i t h the b i o l o g y o f the v i r u s - h o s t i n t e r a c t i o n . S e c o n d , b i n d i n g o f v i r u s to host c e l l m u s t be saturable, further d e f i n i n g the i n t e r a c t i o n as s p e c i f i c . T h i r d , u n l a b e l e d v i r u s m u s t be a b l e to c o m p e t e o f f l a b e l e d h o m o l o g o u s v i r u s , or h e t e r o l o g o u s v i r u s k n o w n to h a v e the s a m e b i o l o g i c a l i n t e r a c t i o n w i t h the host c e l l ( T a r d i e u et ah,  1982).  T w o further c r i t e r i a u s e d to c o n f i r m the i d e n t i t y o f a v i r a l r e c e p t o r are the d e m o n s t r a t i o n o f s p e c i f i c b i n d i n g o f a p u r i f i e d r e c e p t o r to the v i r u s a n d an a b i l i t y to confer, b y g e n e transfer, v i r u s b i n d i n g a c t i v i t y u p o n a c e l l that p r e v i o u s l y d i d not b i n d v i r u s ( W h i t e a n d L i t t m a n , 1989). S u c c e s s f u l a p p r o a c h e s f o r the i d e n t i f i c a t i o n o f receptors h a v e i n c l u d e d the use o f a n t i b o d i e s to c e l l - s u r f a c e antigens to b l o c k v i r a l a t t a c h m e n t or i n f e c t i v i t y a n d transfer o f D N A  f r o m p e r m i s s i v e to n o n - p e r m i s s i v e c e l l  l i n e s ( V i l e a n d W e i s s , 1991).  1.5 Summary and  Thesis Objectives  T h e o v e r a l l g o a l o f this thesis is to e x a m i n e , at the m o l e c u l a r l e v e l , the interactions between a plant virus, C N V ,  a n d its vector, O. bornovanus, to d e t e r m i n e i f  there are s p e c i f i c sites o n the v i r u s p a r t i c l e w h i c h interact w i t h s p e c i f i c m o l e c u l e s o n the f u n g a l z o o s p o r e a c c o r d i n g to the p r e v a i l i n g r e c e p t o r hypothesis. T h e thesis o b j e c t i v e s are d i v i d e d i n t o two parts, the first o b j e c t i v e p e r t a i n i n g to the v i r u s p a r t i c l e a n d p o t e n t i a l r e c e p t o r b i n d i n g sites o n its surface, a n d the s e c o n d o b j e c t i v e p e r t a i n i n g to the f u n g a l 46  z o o s p o r e s a n d the s e a r c h f o r p u t a t i v e r e c e p t o r m o l e c u l e s o n its surface. T h e s e o b j e c t i v e s are d e s c r i b e d i n m o r e d e t a i l b e l o w . 1. To determine if specific sequences or structures on the cucumber necrosis virus coat protein are involved in fungus transmission.  A . Analysis of naturally occurring CNV mutants deficient in transmission.  One  c o m m o n l y used approach for identifying regions o f proteins which have specific f u n c t i o n s i s to s e a r c h f o r n a t u r a l l y o c c u r r i n g m u t a n t s w h i c h h a v e l o s t that f u n c t i o n . T h e m u t a n t s c a n t h e n b e c l o n e d a n d s e q u e n c e d a n d the l o c a t i o n o f the m u t a t i o n i d e n t i f i e d . T h e s p e c i f i c a m i n o a c i d o r the r e g i o n i t i s l o c a t e d i n i s t h e n i d e n t i f i e d as b e i n g i m p o r t a n t i n that p a r t i c u l a r f u n c t i o n . T h i s a p p r o a c h was u s e d to l o c a t e a r e g i o n i n the C N V C P r e q u i r e d f o r f u n g u s t r a n s m i s s i o n . T h e r e s u l t s o f this s t u d y are d e s c r i b e d i n C h a p t e r 3. B. Primary amino acid sequence alignment. T h e a m i n o a c i d s e q u e n c e o f the C P o f s e v e r a l 0//?/ti/wra-transmissible v i r u s e s as w e l l as v i r u s e s w i t h n o k n o w n f u n g a l v e c t o r s w e r e a l i g n e d i n o r d e r to d e t e r m i n e i f s p e c i f i c r e g i o n s o f the C P are c o n s e r v e d ( a n d p o s s i b l y t h e r e f o r e r e q u i r e d ) i n 0//?/d/wm-transmitted v i r u s e s . R e s u l t s o f this a n a l y s i s are d e s c r i b e d i n C h a p t e r 4. C. Mutational analysis. S i t e - s p e c i f i c in vitro m u t a g e n e s i s c a n b e u s e d to i d e n t i f y r e g i o n s o f p r o t e i n s i m p o r t a n t to s p e c i f i c i n t e r a c t i o n s . H o w e v e r , I o p t e d to i n s t e a d a n a l y z e n a t u r a l l y o c c u r r i n g mutants. T h i s is b e c a u s e i t was a n t i c i p a t e d that m a n y artificial fungus transmission mutants may actually represent virus mutants d e f i c i e n t i n other aspects o f the v i r u s s u c h as p a r t i c l e f o r m a t i o n , e n c a p s i d a t i o n o f R N A , stability, r e p l i c a t i o n , etc.  47  To  assess the possible involvement of a receptor in the interaction between  cucumber necrosis virus and zoospores of its fungal vector, Olpidium  bornovanus.  A . In vitro zoospore/virus binding studies. I f a s p e c i f i c m o l e c u l e m e d i a t i n g v i r u s b i n d i n g o c c u r s o n the s u r f a c e o f Olpidium z o o s p o r e s , the b i n d i n g w i l l satisfy three criteria: (i) b i o l o g i c a l s p e c i f i c i t y o f t r a n s m i s s i o n w i l l c o r r e l a t e w i t h the b i n d i n g assay, (ii) v i r u s b i n d i n g to z o o s p o r e s w i l l b e saturable, a n d ( i i i ) l a b e l e d v i r u s w i l l be competitively inhibited by excess unlabeled h o m o l o g o u s virus. T h e e x p e r i m e n t s d e s c r i b e d i n C h a p t e r 5 address the p o s s i b l e i n v o l v e m e n t o f r e c e p t o r s i n C N V t r a n s m i s s i o n b y O. bornovanus. B. I d e n t i f i c a t i o n o f types o f sugars o n the s u r f a c e s o f Olpidium z o o s p o r e s . W o r k d o n e i n m y l a b o r a t o r y c o n c u r r e n t l y w i t h m y thesis r e s e a r c h has s u g g e s t e d the involvement o f a zoospore glycoprotein i n C N V / z o o s p o r e interactions. T h e sugar c o m p o s i t i o n o f the z o o s p o r e s u r f a c e o f Olpidium z o o s p o r e s was e x a m i n e d u s i n g f i v e F T T C - l a b e l e d lectins. A n i d e n t i f i c a t i o n o f p o t e n t i a l sugars f o r v i r u s b i n d i n g o n the s u r f a c e o f Olpidium w o u l d a i d i n f u r t h e r i n v e s t i g a t i o n s i n t o the nature o f putative receptors for fungus-transmitted viruses.  48  2 C H A P T E R T W O :  M A T E R I A L S A N D  M E T H O D S  2.1 Maintenance of virus cultures T h e c h e r r y strain o f Tomato bushy stunt tombusvirus ( T B S V - C h ) w a s a gift f r o m T . J . M o r r i s ( U n i v . o f N e b r a s k a , L i n c o l n ) ; Tobacco necrosis necrovirus ( T N V - N Z ) a n d Cucumber leaf spot aureusvirus ( C L S V ) w e r e gifts f r o m R.N. C a m p b e l l (U.C. D a v i s ,  C A ) . Melon necrotic spot carmovirus ( M N S V ) ( D u t c h isolate) was o r i g i n a l l y o b t a i n e d f r o m D.Z. M a a t . Cucumber necrosis virus ( C N V ) a n d C L S V i n f e c t i o n s w e r e i n i t i a t e d u s i n g transcripts o f f u l l - l e n g t h c D N A c l o n e s ( R o c h o n a n d J o h n s t o n , 1991; D. R o c h o n , u n p u b l i s h e d ) . C N V , C L S V , a n d M N S V w e r e c o n f i r m e d to b e t r a n s m i s s i b l e b y O. bornovanus a n d T N V - N Z b y O. brassicae. T B S V - C h was c o n f i r m e d to b e n o n -  t r a n s m i s s i b l e b y O. bornovanus a n d O. brassicae.  2.2 Purification of virus  2.2.1 C s C l purification  C N V and T B S V - C h were purified using isopycnic centrifugation through C s C l g r a d i e n t s ( R o c h o n et al., 1994). F r o z e n v i r u s - i n f e c t e d p l a n t m a t e r i a l w a s h o m o g e n i z e d i n a W a r i n g b l e n d e r u s i n g t w o v o l u m e s o f 100 m M N a O A c , p H 5.0/5 m M 2m e r c a p t o e t h a n o l , f i l t e r e d t h r o u g h M i r a c l o t h ( C a l b i o c h e m ) , a n d a l l o w e d t o s t a n d at 4 ° C f o r 1 hr. T h e s u s p e n s i o n was c e n t r i f u g e d at 9 0 0 0 x g f o r 15 m i n , after w h i c h the  49  supernatant w a s a d j u s t e d t o 8 % p o l y e t h y l e n e g l y c o l ( P E G ) ( M W = 8 0 0 0 ; S i g m a ) . T h e s u s p e n s i o n w a s stirred at 4 ° C f o r 1 h r a n d t h e n c e n t r i f u g e d f o r 2 0 m i n at 9 0 0 0 x g. T h e v i r u s p e l l e t w a s r e s u s p e n d e d i n 10 m M N a P 0 , p H 7.2, 1 5 0 m M N a C l a n d C s C l ( G i b c o ) 4  w a s a d d e d t o a f i n a l d e n s i t y o f 1.33 g / c m (0.45 g o f C s C l / m l o f f i n a l s o l u t i o n ) . C s C l 3  g r a d i e n t s w e r e c e n t r i f u g e d i n a B e c k m a n T i 8 0 r o t o r at 4 2 0 0 0 r p m f o r 16-24 h r at 2 0 ° C . T h e v i r u s b a n d w a s w i t h d r a w n u s i n g a 18 g a u g e n e e d l e a n d d i a l y z e d a g a i n s t three c h a n g e s o f 10 m M N a O A c , p H 5.0 at 4 ° C . T h e v i r u s s u s p e n s i o n w a s t h e n c e n t r i f u g e d at 1 4 0 0 0 r p m f o r 15 m i n i n a n E p p e n d o r f b e n c h t o p c e n t r i f u g e f o l l o w e d b y f i l t r a t i o n t h r o u g h a 0.2 p m M i l l i p o r e filter. V i r u s w a s q u a n t i f i e d s p e c t r o p h o t o m e t r i c a l l y a n d its i d e n t i t y a n d c o n c e n t r a t i o n c o n f i r m e d b y agarose g e l e l e c t r o p h o r e s i s u s i n g 1 % agarose a n d T B b u f f e r ( 8 8 m M T r i s , 8 8 m M b o r i c a c i d , p H 8.3) f o l l o w e d b y s t a i n i n g w i t h e t h i d i u m b r o m i d e (0.5 p g / m l ) i n T B b u f f e r c o n t a i n i n g 1 m M E D T A ( i n c l u s i o n o f E D T A i n t h e stain w a s f o u n d t o e n h a n c e s t a i n i n g o f R N A w i t h i n particles). A 1 m g / m l s o l u t i o n o f C N V or T B S V - C h has an A  2 6 0  o f 4.5.  2.2.2 Differential centrifugation  M N S V , T N V , a n d C L S V w e r e p u r i f i e d as d e s c r i b e d f o r C N V a n d T B S V a b o v e e x c e p t that t h e P E G p e l l e t w a s s u b j e c t e d t o a l t e r n a t i n g l o w s p e e d / h i g h  speed  centrifugation for virus purification. F o l l o w i n g P E G precipitation o f virus the pellet was r e s u s p e n d e d i n 10 m M N a O A c , p H 5.0. T h e v i r u s s u s p e n s i o n w a s t h e n c e n t r i f u g e d at 9 0 0 0 x g f o r 2 0 m i n f o l l o w e d b y c e n t r i f u g a t i o n o f the supernatant i n a B e c k m a n T i 50.2 r o t o r at 4 0 0 0 0 r p m f o r 2.5 hr. T h e v i r u s p e l l e t w a s r e s u s p e n d e d i n 10 m M N a O A c , p H 5.0 a n d t h e n c e n t r i f u g e d at 1 4 0 0 0 r p m f o r 15 m i n i n a n E p p e n d o r f b e n c h t o p c e n t r i f u g e  50  f o l l o w e d b y f i l t r a t i o n o f the s u p e r n a t a n t t h r o u g h a 0.2 u r n M i l l i p o r e filter. V i r u s w a s q u a n t i f i e d s p e c t r o p h o t o m e t r i c a l l y a n d its i d e n t i t y a n d p u r i t y c o n f i r m e d b y a g a r o s e g e l e l e c t r o p h o r e s i s as d e s c r i b e d f o r C s C l p u r i f i e d C N V a n d T B S V - C h . A 1 m g / m l s o l u t i o n o f M N S V has a n A o o f 5.0; T N V o f 5.2 a n d C L S V o f 5.63. 2 6  2.2.3 Miniprep purification  A m i n i p r e p p r o c e d u r e w a s e m p l o y e d to p a r t i a l l y p u r i f y v i r i o n s f r o m i n f e c t e d l e a v e s . O n e l e a f ( a p p r o x i m a t e l y 2 0 0 m g ) w a s g r o u n d i n l i q u i d n i t r o g e n a n d a d d e d to 1 m l o f 0.1 M N a O A c , p H 5.0/5 m M 2 - m e r c a p t o e t h a n o l i n a 1.5-ml m i c r o f u g e tube. T h e m i x t u r e w a s a l l o w e d to sit o n i c e f o r 10 m i n at 4 ° C . P E G ( M W = 8 0 0 0 ; S i g m a ) w a s t h e n a d d e d to the s u p e r n a t a n t to a f i n a l c o n c e n t r a t i o n o f 8 % a n d the s a m p l e s w e r e i n c u b a t e d w i t h g e n t l e s h a k i n g f o r 3 0 m i n at 4 ° C . V i r u s w a s p e l l e t e d b y c e n t r i f u g a t i o n i n a E p p e n d o r f b e n c h t o p c e n t r i f u g e at 1 4 0 0 0 r p m f o r 15 m i n at 4 ° C . P e l l e t s w e r e r e s u s p e n d e d i n 5 0 p i c o l d , sterile d e i o n i z e d w a t e r o r i n 10 m M N a O A c , p H 5.0. T h e concentration o f virus was estimated b y electrophoresis o f several dilutions through a 1 % a g a r o s e g e l b u f f e r e d i n T B ( H e a t o n , 1992) f o l l o w e d b y s t a i n i n g w i t h e t h i d i u m b r o m i d e (see s e c t i o n 2.2.1). C s C l - p u r i f i e d C N V o r T B S V w a s u s e d as a c o n c e n t r a t i o n s t a n d a r d .  2.3 Maintenance of fungal cultures O. bornovanus (isolate S S 1 9 6 ) w a s m a i n t a i n e d o n the r o o t s o f c u c u m b e r cv.  P o i n s e t t 7 6 a n d O. brassicae (lettuce isolate, S S 5 8 ) o n the r o o t s o f l e t t u c e c v . W h i t e B o s t o n as d e s c r i b e d b y C a m p b e l l et al. (1995). W h i t e s a n d (flint shot) w a s u s e d as the m e d i u m f o r a l l s t o c k c u l t u r e s a n d e x p e r i m e n t a l plants.  51  Seeds w h i c h d i d not have a fungicide coating were surface sterilized. C u c u m b e r seeds w e r e i m m e r s e d i n 7 0 % e t h a n o l f o r o n e m i n f o l l o w e d b y 1 5 % c o m m e r c i a l b l e a c h for 15 m i n . T h e seeds w e r e t h e n w a s h e d i n three c h a n g e s o f d i s t i l l e d w a t e r a n d t h e n p l a c e d o n sterile p a p e r t o w e l i n a u t o c l a v e d c l e a r p l a s t i c v e s s e l s to g e r m i n a t e . A f t e r 7-10 days, c u c u m b e r s e e d l i n g s w e r e t r a n s f e r r e d to o p a q u e p l a s t i c c u p s ( t w o s e e d l i n g s / c u p ) c o n t a i n i n g sterile sand. S e e d s w i t h a f u n g i c i d e c o a t i n g w e r e r i n s e d s e v e r a l t i m e s i n d i s t i l l e d w a t e r to r e m o v e the f u n g i c i d e f o l l o w e d b y g e r m i n a t i o n as d e s c r i b e d a b o v e . L e t t u c e seeds w e r e i m m e r s e d i n 15 % c o m m e r c i a l b l e a c h f o r f i v e m i n a n d t h e n p l a c e d just u n d e r the s u r f a c e o f wet, sterile s a n d to g e r m i n a t e . Olpidium s t o c k c u l t u r e s w e r e o b t a i n e d i n d r i e d r o o t s f r o m R. N . C a m p b e l l (U.C.,  Davis, C A ) . T o recover fungal infectivity, dried roots were cut into two m m pieces, i m m e r s e d i n sterile w a t e r a n d a l l o w e d to h y d r a t e f o r f i v e m i n . O. bornovanus s u s p e n s i o n s w e r e p o u r e d o n t o the r o o t s o f c u c u m b e r s e e d l i n g s o r o n t o u n g e r m i n a t e d c u c u m b e r seeds. O. bornovanus z o o s p o r e s c o u l d b e o b s e r v e d after a p p r o x i m a t e l y t h r e e w e e k s . T o m a i n t a i n Olpidium z o o s p o r e c u l t u r e s , z o o s p o r e s u s p e n s i o n s w e r e p o u r e d o n t o 6-10 d a y o l d c u c u m b e r s e e d l i n g s ( p a s s a g i n g o f the f u n g u s ) . A f t e r t h r e e to f i v e w e e k s sufficient zoospores were present i n cucumber root washes for use i n transmission/binding experiments. O. brassicae z o o s p o r e s u s p e n s i o n s w e r e p o u r e d o n t o l e t t u c e s e e d l i n g s o n the  s u r f a c e o f sterile sand. A p p r o x i m a t e l y s i x w e e k s o f g r o w t h w e r e n e c e s s a r y b e f o r e s u f f i c i e n t n u m b e r s o f O. brassicae z o o s p o r e s c o u l d b e o b s e r v e d i n r o o t w a s h e s f r o m lettuce. O. brassicae z o o s p o r e s w e r e n o r m a l l y p a s s e d o n t o 14 d a y o l d l e t t u c e s e e d l i n g s  52  a n d a l l o w e d a f u r t h e r 4-6 w e e k s f o r the a c c u m u l a t i o n o f s u f f i c i e n t n u m b e r s o f z o o s p o r e s for transmission and b i n d i n g experiments. Zoospore suspensions were prepared by immersing washed fungus-infected roots i n sterile d e i o n i z e d w a t e r f o r 15 m i n f o l l o w e d b y f i l t r a t i o n t h r o u g h M i r a c l o t h ( C a l b i o c h e m ) to r e m o v e r o o t debris. Z o o s p o r e s w e r e c o u n t e d u s i n g a B r i g h t l i n e haemocytometer and phase-contrast microscopy.  2.4 Production of antibodies  2.4.1 Polyclonal antibody production  T o p r o d u c e a n t i b o d i e s w h i c h w e r e r e a c t i v e i n b o t h W e s t e r n B l o t s a n d E L I S A , 100 |ig o f S D S - P A G E d e n a t u r e d a n d 100 \ig o f n a t i v e v i r i o n s i n P B S ( f o r 1 liter: 8 g N a C l , 1.44 g N a H P O - 2 H 0 , 0.2 g K H P 0 , 0.2 g K C 1 , p H 7.4) w e r e m i x e d i n a 1:1 ratio w i t h 2  4  2  2  4  Freund's incomplete adjuvant and injected subcutaneously into female N e w  Zealand  Giant White rabbits ( U B C A n i m a l Care Centre). F o l l o w i n g a standard boosting schedule, n o r m a l l y t w o b o o s t s at 21 d a y intervals, the r a b b i t was e x s a n g u i n a t e d a n d the b l o o d a l l o w e d t o c l o t at 3 7 ° C f o r 1 h r f o l l o w e d b y 4 ° C o v e r n i g h t . T h e s e r u m w a s t h e n s e p a r a t e d b y c e n t r i f u g a t i o n at 3 6 0 0 r p m i n a H e r m l e b e n c h t o p c e n t r i f u g e . S o d i u m a z i d e at a c o n c e n t r a t i o n o f 0 . 0 2 % w a s a d d e d b e f o r e s t o r a g e at 4 C .  2.4.2 Monoclonal antibody production  M o n o c l o n a l a n t i b o d i e s ( M A b s ) w e r e p r o d u c e d a c c o r d i n g to H a r l o w a n d L a n e (1989). C s C l - p u r i f i e d v i r i o n s ( 1 0 0 |0,g i n P B S ) w e r e m i x e d 1:1 w i t h F r e u n d ' s i n c o m p l e t e  53  a d j u v a n t a n d i n j e c t e d s u b c u t a n e o u s l y u s i n g a total v o l u m e o f 2 0 0 |il/mouse. A f t e r f o u r w e e k s , m i c e w e r e b o o s t e d i n t r a p e r i t o n e a l l y w i t h 1 0 0 u g o f v i r i o n s m i x e d 1:1 w i t h F r e u n d ' s i n c o m p l e t e a d j u v a n t u s i n g a total v o l u m e o f o n e m l p e r m o u s e . E L I S A o n a tail b l e e d three d a y s later i n d i c a t e d w h e t h e r a s u f f i c i e n t a n t i b o d y r e s p o n s e h a d b e e n m o u n t e d a n d w a s f o l l o w e d i m m e d i a t e l y b y h a r v e s t i n g o f the s p l e e n a n d f u s i o n w i t h m y e l o m a c e l l s ( H a r l o w a n d L a n e , 1989). C l o n e s p o s i t i v e f o r a n t i b o d y p r o d u c t i o n w e r e e x p a n d e d a n d purified using batch culture a n d a m m o n i u m sulfate precipitation f o l l o w e d b y dialysis a g a i n s t P B S . P u r i f i e d M A b s w e r e s t o r e d at 4 ° C i n the p r e s e n c e o f 0 . 0 2 % s o d i u m a z i d e .  2.4.3 Purification of IgG and production of alkaline phosphatase conjugate  I g G w a s p u r i f i e d u s i n g P r o t e i n G S e p h a r o s e 4 F F ( P h a r m a c i a ) a c c o r d i n g to the m a n u f a c t u r e r ' s i n s t r u c t i o n s . B r i e f l y , 2 0 m M s o d i u m phosphate, p H 7.0, a n d 0.1 M g l y c i n e , p H 2.7 w e r e u s e d f o r b i n d i n g a n d e l u t i o n o f I g G , r e s p e c t i v e l y . E a c h o n e m l f r a c t i o n w a s n e u t r a l i z e d w i t h the a d d i t i o n o f 5 0 p i o f 1 M T r i s - H C l , p H 9.0. F r a c t i o n s c o n t a i n i n g I g G (as d e t e r m i n e d b y a b s o r b a n c e at 2 8 0 n m ) w e r e c o m b i n e d a n d d i a l y z e d against three c h a n g e s o f P B S at 4 ° C f o r a total o f 6 h o u r s f o l l o w e d b y f i l t e r - s t e r i l i z a t i o n a n d storage at - 2 0 ° C . I g G c o n c e n t r a t i o n s w e r e q u a n t i f i e d s p e c t r o p h o t o m e t r i c a l l y (a 1 m g / m l s o l u t i o n o f I g G w i l l g i v e a n a b s o r b a n c e v a l u e o f 1.35 at 2 8 0 n m ) . C o n j u g a t i o n o f I g G to a l k a l i n e p h o s p h a t a s e ( A L P ) w a s p e r f o r m e d u s i n g B o e h r i n g e r M a n n h e i m A L P ( c a t a l o g # 5 6 7 7 4 4 ) . O n e m l o f 1 m g / m l I g G a n d 1 0 0 JLLI o f 10 m g / m l A L P w e r e p l a c e d t o g e t h e r i n d i a l y s i s t u b i n g a n d left to d i a l y z e f o r 5 h r at 4 ° C a g a i n s t P B S c o n t a i n i n g 0 . 0 6 % f r e s h l y d i l u t e d E M grade g l u t a r a l d e h y d e . F o l l o w i n g c o n j u g a t i o n , the I g G w a s d i a l y z e d a g a i n s t three c h a n g e s o f P B S ( 5 0 0 m l ) at 4 ° C o v e r a  54  p e r i o d o f 9 t o 18 h o u r s . S o d i u m a z i d e at 0 . 0 2 % w a s a d d e d t o the I g G - A L P c o n j u g a t e s w h i c h w e r e t h e n d i l u t e d 1:1 w i t h sterile 1 0 0 % g l y c e r o l a n d s t o r e d at 4 ° C a n d - 2 0 ° C . T h e p r e s e n c e o f g l y c e r o l w a s n e c e s s a r y t o k e e p the c o n j u g a t e s f r o m f r e e z i n g at - 2 0 ° C w h i c h w o u l d h a v e i n a c t i v a t e d the A L P .  2.5 Enzyme-linked Immunoassay (ELISA) D o u b l e antibody s a n d w i c h E L I S A ( D A S - E L I S A ) u s i n g polyclonal antisera raised to C N V , T B S V , C L S V , o r M N S V a n d the a p p r o p r i a t e A L P - I g G c o n j u g a t e s p r o d u c e d as d e s c r i b e d a b o v e w a s p e r f o r m e d as d e s c r i b e d b y C l a r k et al. ( 1 9 8 6 ) . B r i e f l y , I g G w a s diluted i n coating buffer (50 m M  s o d i u m c a r b o n a t e b u f f e r , p H 9.6) t o a f i n a l  c o n c e n t r a t i o n o f 1 p.g/ml a n d 100 jxl a l i q u o t s w e r e a d d e d t o t h e w e l l s o f 9 6 w e l l m i c r o t i t e r plates a n d i n c u b a t e d at 3 7 ° C f o r o n e h o u r . P l a t e s w e r e w a s h e d f o u r t i m e s u s i n g d e i o n i z e d water. W e l l s w e r e b l o c k e d u s i n g 2 0 0 \\\ o f 2 % s k i m m i l k i n P B S at 3 7 ° C f o r 3 0 m i n . A f t e r r e m o v a l o f the b l o c k i n g b u f f e r , 1 0 0 p J o f test s a m p l e s i n 5 0  mM  p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.0 w e r e a d d e d t o the w e l l s a n d the p l a t e s i n c u b a t e d at 3 7 ° C f o r o n e h o u r . A f t e r w a s h i n g t h e plates as a b o v e , 100 p i o f the a p p r o p r i a t e c o n j u g a t e d i l u t e d 1:250 t o 1:2000 i n 0 . 2 % s k i m m i l k i n P B S w a s a d d e d t o e a c h w e l l a n d i n c u b a t e d at 3 7 ° C f o r o n e h o u r f o l l o w e d b y w a s h i n g . T h e substrate, p - n i t r o p h e n y l p h o s p h a t e ( S i g m a ) , w a s a d d e d at a f i n a l c o n c e n t r a t i o n o f 1 m g / m l i n A P b u f f e r ( 1 0 0 m M N a C l , 5 0 m M M g C l , 100 m M T r i s - H C l , p H 9.5) at a v o l u m e o f 100 ixl/well a n d i n c u b a t e d at 3 7 ° C 2  f o r o n e h o u r . A b s o r b a n c e w a s m e a s u r e d at 4 0 5 n m u s i n g a D y n a t e c h M R X p l a t e reader.  55  2.6 Immunoblotting P r o t e i n s w e r e d e n a t u r e d i n 2 X L a e m m l i b u f f e r ( 2 0 % g l y c e r o l , 1 0 % 2m e r c a p t o e t h a n o l , 0 . 0 5 % b r o m o p h e n o l b l u e , 4 % s o d i u m d o d e c y l s u l p h a t e ( S D S ) , 125  mM  T r i s - H C l , p H 6.8) a n d e l e c t r o p h o r e s e d t h r o u g h 1 2 % p o l y a c r y l a m i d e g e l s c o n t a i n i n g S D S at 2 0 0 V f o r 4 5 m i n ( B i o - R a d M i n i P r o t e a n II) e s s e n t i a l l y a c c o r d i n g to L a e m m l i (1970). P r o t e i n standards, n o r m a l l y C s C l p u r i f i e d v i r i o n s i n c o n c e n t r a t i o n s f r o m 1 n g to 100 ng, w e r e i n c l u d e d o n b l o t s to e n a b l e q u a n t i t a t i o n o f v i r u s i n the s a m p l e s . P r o t e i n s w e r e t h e n e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to I m m o b i l o n P V D F m e m b r a n e s ( M i l l i p o r e ) at 100 V f o r 9 0 m i n u t e s at 4 ° C i n a s u b m a r i n e a p p a r a t u s ( B i o - R a d ) c o n t a i n i n g W e s t e r n t r a n s f e r b u f f e r (25 m M  T r i s , 192 m M  g l y c i n e , 0 . 0 5 % S D S a n d 2 0 % m e t h a n o l ) . B l o t s w e r e first b l o c k e d  f o r o n e h o u r i n a s o l u t i o n o f 5 % s k i m m i l k p o w d e r i n T B S (10 m M  T r i s - H C l , 150  N a C l , p H 7.4). B l o t s w e r e t h e n w a s h e d three t i m e s f o r 10 m i n e a c h i n T B S T ( 1 0 T r i s - H C l , 150 m M  mM mM  N a C l , 0 . 0 5 % T w e e n - 2 0 , p H 7.4) a n d i n c u b a t e d w i t h a 1:250 t o 1:2000  d i l u t i o n o f the a p p r o p r i a t e a n t i b o d y i n T B S T o v e r n i g h t at 4 ° C . A l l w a s h i n g a n d h y b r i d i z a t i o n steps, u n l e s s noted, w e r e c a r r i e d out at r o o m t e m p e r a t u r e w i t h c o n s t a n t agitation. T h e w e s t e r n b l o t was t h e n w a s h e d i n T B S T as d e s c r i b e d above.  The  s e c o n d a r y a n t i b o d y was h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d A f f i n i P u r e g o a t anti-rabbit o r a n t i - m o u s e I g G ( J a c k s o n I m m u n o R e s e a r c h ) , w h i c h was u s e d at 1:10 0 0 0 d i l u t i o n i n T B S T f o r o n e hour. T h e w e s t e r n w a s s u b s e q u e n t l y w a s h e d i n T B S T as a b o v e a n d b o u n d a n t i b o d y v i s u a l i z e d u s i n g the E C L W e s t e r n b l o t t i n g s y s t e m ( A m e r s h a m P h a r m a c i a B i o t e c h , Inc.) a n d H y p e r f i l m ™ - E C L ™  ( A m e r s h a m Life Sciences).  56  2.7 Isolation of transmission-deficient CNV  variants  C N V w a s m e c h a n i c a l l y p a s s a g e d 13 t i m e s t h r o u g h t h e s y s t e m i c host, Nicotiana clevelandii, b e g i n n i n g w i t h i n f e c t i o u s transcripts o f the w i l d - t y p e C N V c D N A c l o n e  p K 2 / M 5 (hereafter r e f e r r e d t o as W T C N V R N A ; R o c h o n a n d J o h n s t o n , 1991). I n addition, o u r laboratory culture o f C N V (Lc), w h i c h has been maintained f o r several years b y m e c h a n i c a l passage, w a s used. I n f e c t e d N. clevelandii l e a v e s f r o m t h e t h i r t e e n t h m e c h a n i c a l p a s s a g e w e r e t h e n u s e d t o i n o c u l a t e the l o c a l l e s i o n host, Chenopodium quinoa. I n d i v i d u a l l o c a l l e s i o n s f r o m this h o s t w e r e i s o l a t e d a n d t h e n f u r t h e r " p u r i f i e d "  b y a s e c o n d p a s s a g e o n C. quinoa. L o c a l l e s i o n s f r o m this p l a n t w e r e u s e d t o i n o c u l a t e N. clevelandii t o i n c r e a s e t h e a m o u n t o f v i r u s . C r u d e l e a f extracts o r p u r i f i e d v i r u s f r o m N. clevelandii w e r e u s e d i n f u n g u s t r a n s m i s s i o n assays.  2.8 Fungus transmission assay E i t h e r l e a f extracts o r p u r i f i e d v i r i o n s w e r e t e s t e d f o r in vitro a c q u i s i t i o n a n d t r a n s m i s s i o n t o c u c u m b e r s e e d l i n g s e s s e n t i a l l y as d e s c r i b e d p r e v i o u s l y ( C a m p b e l l et  al.,  1991, 1995; M c L e a n et al., 1994). S p e c i f i e d a m o u n t s o f p u r i f i e d v i r u s o r l e a f extracts (see v i r u s m i n i p r e p p u r i f i c a t i o n a b o v e ) w e r e a d d e d to z o o s p o r e s o f the S S 1 9 6 strain o f O. bornovanus ( 1 0 4 z o o s p o r e s / m l i n 5 0 m M  g l y c i n e - N a O H , p H 7.6) o r b u f f e r a l o n e t o a  f i n a l v o l u m e o f 10 m l . A f t e r a 15 m i n a c q u i s i t i o n p e r i o d , this s u s p e n s i o n w a s p o u r e d i n t o p o t s c o n t a i n i n g 13-16 d a y o l d c u c u m b e r s e e d l i n g s . S i x d a y s later, i n o c u l a t e d c u c u m b e r roots w e r e a s s a y e d f o r t h e p r e s e n c e o f v i r u s b y D A S - E L I S A u s i n g p o l y c l o n a l a n t i s e r a r a i s e d t o C N V , T B S V , C L S V , M N S V , o r T N V particles.  57  2.9 RT-PCR and cloning of the LL5 Virion RNA  coat protein  o f L L 5 was e x t r a c t e d f r o m p a r t i c l e s u s i n g p h e n o l / c h l o r o f o r m as  d e s c r i b e d ( R o c h o n a n d J o h n s t o n , 1991). D o u b l e - s t r a n d e d c D N A c o p i e s o f the c o a t p r o t e i n c o d i n g r e g i o n o f C N V v a r i a n t s w e r e o b t a i n e d b y u s i n g the r e v e r s e t r a n s c r i p t a s e / p o l y m e r a s e c h a i n r e a c t i o n ( R T - P C R ; S a m b r o o k et al., 1989). T h e p l u s sense p r i m e r ( C N V o l i g o n. 30; 5' A C G T G A A T T C G T G A C C C C T G A G G C A A3') c o r r e s p o n d s to C N V n u c l e o t i d e s 2 4 4 7 to 2 4 6 2 a n d lies 181 n u c l e o t i d e s u p s t r e a m o f the c o a t p r o t e i n O R F ( i t a l s o i n c l u d e s a n o n c o d e d £ c o R l site at its 5' t e r m i n u s ) . T h e m i n u s sense p r i m e r ( C N V o l i g o n. 24; 5 ' G G G A G T A A T G G T A C C T C C 3 ' ) c o r r e s p o n d s to the c o m p l e m e n t o f C N V n u c l e o t i d e s 3901 to 3 9 1 8 a n d lies 148 n u c l e o t i d e s d o w n s t r e a m o f the C P O R F .  T h e L L 5 C P R T - P C R p r o d u c t was l i g a t e d i n t o p T 7 B l u e ( N o v a g e n ) .  DNA  f r o m t h i s c l o n e was d i g e s t e d w i t h Bsu36l a n d Ncol, w h i c h c u t at u n i q u e sites f l a n k i n g the C P O R F .  T h e 1.4-kb f r a g m e n t was l i g a t e d i n t o s i m i l a r l y d i g e s t e d p K 2 / M 5 (a f u l l -  l e n g t h C N V c D N A c l o n e ; see R o c h o n a n d J o h n s t o n , 1991) to p r o d u c e p M 5 / L L 5 . T h e s e q u e n c e o f the e n t i r e t r a n s f e r r e d r e g i o n o f the L L 5 C P g e n e was d e t e r m i n e d b y the d i d e o x y c h a i n t e r m i n a t i o n p r o c e d u r e ( S a n g e r et al.,  1977).  2.10 In vitro transcription and inoculation of plants T 7 R N A p o l y m e r a s e r u n - o f f transcripts a n d i n o c u l a t i o n o f p l a n t s w e r e as d e s c r i b e d p r e v i o u s l y ( R o c h o n a n d J o h n s t o n , 1991).  58  2.11 In vitro mutagenesis In v i t r o m u t a g e n e s i s w a s u s e d to p r o d u c e the C N V C P m u t a n t s L L 5 s a n d L L 5 , a  w h i c h , r e s p e c t i v e l y , c o n t a i n the s i n g l e n u c l e o t i d e s u b s t i t u t i o n s f o u n d i n the s h e l l a n d a r m d o m a i n s o f p M 5 / L L 5 . A 1.55-kb s u b c l o n e o f p K 2 / M 5 , w h i c h e n c o m p a s s e s t h e C N V  CP  a n d f l a n k i n g r e g i o n s , w a s u s e d as a t e m p l a t e f o r o l i g o n u c l e o t i d e - d i r e c t e d in vitro m u t a g e n e s i s as d e s c r i b e d b y K u n k e l et al. ( 1 9 8 7 ) . T h e m u t a g e n i c o l i g o n u c l e o t i d e ( m u t a t i o n s h o w n u n d e r l i n e d ) u s e d to p r o d u c e L L 5 was: S  5'GATGATAAGGTCTGG3'  ( C N V n u c l e o t i d e s 3 1 4 4 to 3 1 6 6 ) a n d the o n e to p r o d u c e L L 5  a  was:  5 ' C A C C C A C A G G C T T G T C C T G G G G C T A T C 3 ' ( C N V n u c l e o t i d e s 2 8 1 1 to 2 8 3 7 ) . M u t a n t s w e r e s c r e e n e d b y s e q u e n c i n g a n d t h e p l a s m i d D N A w a s d i g e s t e d w i t h BgWBglQ to o b t a i n a 6 9 8 - b p f r a g m e n t c o n t a i n i n g the m u t a t i o n . T h i s g e l - p u r i f i e d 6 9 8 - b p f r a g m e n t w a s l i g a t e d i n t o the s i m i l a r l y d i g e s t e d i n t e r m e d i a t e v e c t o r p C N V C P [a C N V  EcoRUKpnl  s u b c l o n e i n the p h a g e m i d B l u e s c r i p t ( S t r a t a g e n e ) e n c o m p a s s i n g the C P O R F ; M c L e a n  et  al., 1994] to f o r m L L 5 / p C N V C P a n d L L 5 / p C N V C P . B o t h L L 5 / p C N V C P a n d s  a  s  L L 5 / p C N V C P w e r e d i g e s t e d w i t h Bsu36VNcoI as d e s c r i b e d a b o v e a n d l i g a t e d i n t o a  s i m i l a r l y d i g e s t e d p K 2 / M 5 to f o r m L L 5 s / M 5 a n d L L 5 / M 5 . T h e s e q u e n c e b e t w e e n the a  BglVBgUl sites w a s c o n f i r m e d to e n s u r e that n o o t h e r m u t a t i o n s o c c u r r e d as a r e s u l t o f  the o l i g o n u c l e o t i d e - d i r e c t e d m u t a g e n e s i s .  2.12 Binding Assays A s t a n d a r d b i n d i n g a s s a y w a s as f o l l o w s . O. bornovanus o r O. brassicae z o o s p o r e s (4 X 1 0 to 1 X 10 ) w e r e i n c u b a t e d w i t h C N V , T B S V - C h , M N S V , T N V , o r 5  6  C L S V (see f i g u r e l e g e n d s f o r a m o u n t s o f v i r u s u s e d ) i n o n e m l 5 0 m M 59  NaPC>4 b u f f e r , p H  7.6 f o r o n e hour. T h e s u s p e n s i o n was c e n t r i f u g e d i n an E p p e n d o r f b e n c h t o p c e n t r i f u g e at 5 0 0 0 r p m f o r s e v e n m i n to p e l l e t z o o s p o r e s a n d a n y b o u n d v i r u s . I m m e d i a t e l y f o l l o w i n g c e n t r i f u g a t i o n , the supernatant was r e m o v e d b y g e n t l e a s p i r a t i o n , the p e l l e t w a s h e d i n 1.5 m l o f b u f f e r a n d z o o s p o r e s p e l l e t e d as b e f o r e . T h e z o o s p o r e p e l l e t was r e s u s p e n d e d i n 10 (J.1 o f sterile w a t e r a n d 2.5 p i was a s s a y e d f o r the p r e s e n c e o f v i r u s u s i n g W e s t e r n b l o t a n a l y s i s a n d v i r u s - s p e c i f i c antisera. T h e E C L W e s t e r n b l o t t i n g s y s t e m ( A m e r s h a m P h a r m a c i a B i o t e c h , Inc.) was u s e d f o r d e t e c t i o n as d e s c r i b e d a b o v e . T h e q u a n t i t y o f v i r u s r e p r e s e n t e d b y a b a n d was d e t e r m i n e d b y d e n s i t o m e t r y u s i n g the I m a g e Q u a n t p r o g r a m ( M o l e c u l a r D y n a m i c s ) . F o r C N V d e t e c t i o n a m o n o c l o n a l a n t i b o d y (57-2) was p r e p a r e d (see a b o v e ) w h i l e r a b b i t p o l y c l o n a l a n t i b o d i e s w e r e u s e d f o r C L S V a n d T N V d e t e c t i o n (see above). T h e p r e p a r a t i o n o f M N S V p o l y c l o n a l a n t i b o d i e s was p r e v i o u s l y d e s c r i b e d ( R i v i e r e , 1989) a n d T B S V - C h a n t i b o d i e s w e r e o b t a i n e d f r o m the A g r i c u l t u r e a n d A g r i F o o d C a n a d a v i r u s a n t i s e r a c o l l e c t i o n . D A S - E L I S A was u s e d to d e t e r m i n e the a m o u n t o f T B S V - C h binding.  2.13 Introduction of a frameshift mutation into the CLSV CP  ORF  J R 3 , a f u l l - l e n g t h i n f e c t i o u s C L S V c D N A c l o n e (D. R o c h o n a n d J. M i l l e r , u n p u b l i s h e d ) was d i g e s t e d w i t h Xhol, w h i c h c l e a v e s at C L S V nt p o s i t i o n 2 5 0 8 , 132 nt 3' to the C L S V C P start c o d o n . F o l l o w i n g d i g e s t i o n , the f o u r base 5' o v e r h a n g was f i l l e d i n w i t h K l e n o w ( B R L ) a n d p l a s m i d r e c i r c u l a r i z e d u s i n g ligase. T h e r e s u l t i n g C L S V m u t a n t , J R 3 A X h o , was c o n f i r m e d b y r e s t r i c t i o n e n z y m e a n a l y s i s a n d s e q u e n c i n g to c o n t a i n a f r a m e s h i f t at the N - t e r m i n u s o f the C P c o d i n g r e g i o n .  60  2.14 Analysis of JR3AXho B o t h the w i l d - t y p e C L S V i n f e c t i o u s c l o n e J R 3 a n d the C P m u t a n t J R 3 A X h o w e r e d i g e s t e d w i t h Smal to l i n e a r i z e the D N A a n d t h e n T 7 R N A p o l y m e r a s e r u n - o f f transcripts w e r e p r o d u c e d as d e s c r i b e d p r e v i o u s l y b y R o c h o n a n d J o h n s t o n ( 1 9 9 1 ) . T r a n s c r i p t s (ca. 1-2 p g p e r leaf) w e r e r u b - i n o c u l a t e d o n t o the l e a v e s o f y o u n g N. benthamiana (three l e a f stage). R N A extracts w e r e o b t a i n e d u s i n g p h e n o l / c h l o r o f o r m as d e s c r i b e d ( R o c h o n a n d J o h n s t o n , 1991).  2.15 Lectin labelling of fungal zoospores F I T C - l a b e l l e d l e c t i n s w e r e p u r c h a s e d i n l y o p h i l i z e d f o r m a n d r e s u s p e n d e d at a f i n a l c o n c e n t r a t i o n o f 1 m g / m l i n P B S a c c o r d i n g to the m a n u f a c t u r e r ' s i n s t r u c t i o n s a n d s t o r e d at - 2 0 ° C ( S i g m a ) . Z o o s p o r e s w e r e w a s h e d o n c e ( i n 10 m M  sodium phosphate  b u f f e r , p H 7.3) b y c e n t r i f u g a t i o n f o r 5 m i n at 5 0 0 0 r p m i n a n E p p e n d o r f b e n c h t o p c e n t r i f u g e a n d r e s u s p e n d e d i n 1 m l w a s h b u f f e r to g i v e a f i n a l z o o s p o r e c o n c e n t r a t i o n o f 2 x 1 0 to 2 x 1 0 z o o s p o r e s / m l . F I T C - l e c t i n s w e r e a d d e d to z o o s p o r e s at the 5  6  m a n u f a c t u r e r s r e c o m m e n d e d c o n c e n t r a t i o n f o r the a g g l u t i n a t i o n o f f r e s h h u m a n e r y t h r o c y t e s . T h e z o o s p o r e - l e c t i n s u s p e n s i o n w a s a l l o w e d to i n c u b a t e i n the d a r k f o r 10 m i n f o l l o w e d b y c e n t r i f u g a t i o n a n d r e s u s p e n s i o n o f the z o o s p o r e p e l l e t i n 5 0 p i o f 10 mM  s o d i u m p h o s p h a t e b u f f e r , p H 7.3. In s u g a r c o m p e t i t i o n e x p e r i m e n t s , a p p r o p r i a t e  s u g a r s at 100 p g / m l w e r e a d d e d to F I T C - l e c t i n s f o r 3 0 m i n i n the d a r k b e f o r e i n c u b a t i o n with zoospores. Images were observed using a Zeiss A x i o P h o t with D I C and e p i f l u o r e s c e n c e o p t i c s . Z e i s s filter set 4 8 7 9 1 0 ( e x c i t a t i o n 4 5 0 to 4 9 0 n m ) w a s used.  61  3 C H A P T E R  T H R E E :  I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N  M O L E C U L A R  O FN A T U R A L C N V  W I T H R E D U C E D  V A R I A N T S  T R A N S M I S S I B I L I T Y  3.1 Introduction E f f e c t i v e d i s s e m i n a t i o n o f p l a n t v i r u s e s i n nature o f t e n i n v o l v e s s p e c i f i c i n v e r t e b r a t e o r f u n g a l vectors. A l t h o u g h a c o n s i d e r a b l e a m o u n t o f i n f o r m a t i o n i s a v a i l a b l e r e g a r d i n g b i o l o g i c a l aspects o f v e c t o r m e d i a t e d t r a n s m i s s i o n , r e l a t i v e l y l i t t l e i n f o r m a t i o n i s a v a i l a b l e about v i r u s - v e c t o r i n t e r a c t i o n s at the m o l e c u l a r l e v e l . T h e i n v o l v e m e n t o f the c o a t p r o t e i n ( C P ) i n t r a n s m i s s i o n i s w e l l e s t a b l i s h e d ( f o r r e v i e w s , see C a m p b e l l , 1996; G r a y , 1996 a n d P i r o n e a n d B l a n c , 1996). In p o t y v i r u s e s , a p h i d t r a n s m i s s i b i l i t y r e q u i r e s a s p e c i f i c s e q u e n c e ( D A G ) i n the e x p o s e d N - t e r m i n a l r e g i o n o f the c a p s i d ( A t r e y a et al, 1990; A t r e y a et al, 1991; G a l - o n et al, 1992; A t r e y a et al, 1995). A s i m i l a r s e q u e n c e is a l s o r e q u i r e d f o r a p h i d t r a n s m i s s i o n o f mosaic virus ( B a u l c o m b e et al,  Potato aucuba  1993). T h e r e a d t h r o u g h p o r t i o n o f the C P s o f  bymoviruses, s o m e furoviruses and several luteoviruses have been implicated i n vector t r a n s m i s s i o n ( T a m a d a a n d K u s u m e , 1991; S h i r a k o a n d B r a k k e , 1984; S h i r a k o a n d W i l s o n , 1993; J o l l y a n d M a y o , 1994; B r a u l t et al, 1995; D e s s e n s a n d M e y e r , 1995; Jacobi  et al,  1995; C h a y  et al,  1996). In  Beet necrotic yellow vein virus ( B N Y V V ) ,  a  K T E R m o t i f i n the C P r e a d t h r o u g h d o m a i n has b e e n s h o w n to b e i m p o r t a n t f o r e f f i c i e n t transmission by  Polymyxae betae  (Tamada  et al,  62  1996). C e r t a i n v i r u s e s , s u c h as  p o t y v i r u s e s a n d Cauliflower mosaic virus ( C a M V ) e n c o d e a d d i t i o n a l n o n - v i r i o n associated "helper factors" w h i c h aid i n aphid transmission probably by bridging an i n t e r a c t i o n b e t w e e n the v i r i o n a n d s p e c i f i c sites i n the f o o d c a n a l (see P i r o n e a n d B l a n c , 1996; G r a y , 1996). R e c e n t l y , it has b e e n s u g g e s t e d that t o b r a v i r u s e s m a y a l s o e n c o d e h e l p e r f a c t o r s w h i c h i n f l u e n c e the e f f i c i e n c y o f n e m a t o d e t r a n s m i s s i o n ( M a c F a r l a n e  and  B r o w n , 1995). Cucumber necrosis virus ( C N V )  i s o n e o f s e v e r a l m e m b e r s o f the f a m i l y  Tombusviridae k n o w n to b e f u n g a l l y t r a n s m i t t e d ( C a m p b e l l , 1996). C N V  is transmitted  to c u c u m b e r t h r o u g h the s o i l v i a z o o s p o r e s o f the c h y t r i d f u n g u s Olpidium bornovanus ( D i a s , 1970a; C a m p b e l l a n d S i m , 1994; C a m p b e l l etal, 1995). Z o o s p o r e s a n d v i r u s are r e l e a s e d i n d e p e n d e n t l y i n t o the s o i l f r o m i n f e c t e d roots. C N V p a r t i c l e s are t h e n t h o u g h t to a d h e r e to the z o o s p o r e p l a s m a l e m m a ( T e m m i n k et al., 1970) a n d enter p l a n t r o o t s f o l l o w i n g z o o s p o r e e n c y s t m e n t i n r o o t c e l l s (see s e c t i o n s 1.2.3.1 a n d 1.2.3.2 f o r m o r e detail). R e c i p r o c a l e x c h a n g e o f the C P g e n e o f C N V  a n d that o f a n o n - t r a n s m i s s i b l e  t o m b u s v i r u s , the c h e r r y strain o f Tomato bushy stunt virus ( T B S V - C h ) , h a v e d e m o n s t r a t e d that the C N V C P c o n t a i n s the d e t e r m i n a n t s f o r the s p e c i f i c i t y o f f u n g u s t r a n s m i s s i o n ( M c L e a n et al.,  1994).  L o n g t e r m m e c h a n i c a l p a s s a g e o f p l a n t v i r u s e s f r e q u e n t l y results i n the l o s s o f v e c t o r t r a n s m i s s i b i l i t y (see C a m p b e l l , 1996; G r a y , 1996). W e are i n t e r e s t e d i n d e t e r m i n i n g the s p e c i f i c a m i n o a c i d s e q u e n c e s o r s t r u c t u r a l features o f the C N V  CP  i n v o l v e d i n f u n g u s t r a n s m i s s i o n . T o w a r d this end, t r a n s m i s s i o n d e f e c t i v e m u t a n t s present i n m e c h a n i c a l l y p a s s a g e d C N V h a v e b e e n c h a r a c t e r i z e d . S e v e r a l C N V  variants  d e f i c i e n t i n t r a n s m i s s i o n w e r e i d e n t i f i e d . O n e variant, L L 5 , was a n a l y z e d i n d e t a i l a n d it  63  was f o u n d that a s i n g l e G l u to L y s c h a n g e i n the c o a t p r o t e i n s h e l l d o m a i n i s r e s p o n s i b l e f o r the r e d u c e d t r a n s m i s s i b i l i t y . F u r t h e r m o r e , in vitro b i n d i n g assays w e r e d e v e l o p e d  and  i t was f o u n d that the l o s s o f t r a n s m i s s i b i l i t y is at least p a r t i a l l y d u e to i n e f f i c i e n t z o o s p o r e binding.  3.2 Results  3.2.1 Mechanically passaged CNV contains variants deficient in fungus transmission.  Individual local lesion isolates o f C N V  (see s e c t i o n 2.7) w e r e a m p l i f i e d i n N.  clevelandii a n d i n f e c t e d l e a v e s w e r e u s e d i n a f u n g u s t r a n s m i s s i o n assay. L e a v e s w e r e  m a c e r a t e d i n b u f f e r , m i x e d w i t h 10 m l o f O. bornovanus z o o s p o r e s a n d t h e n p o u r e d i n t o s o i l c o n t a i n i n g c u c u m b e r s e e d l i n g s . S i x d a y s later, i n o c u l a t e d c u c u m b e r r o o t s w e r e a s s a y e d f o r the p r e s e n c e o f v i r u s b y b i o a s s a y u s i n g C. quinoa. U s i n g this assay, l e a f extracts o f W T C N V i n v a r i a b l y p r o d u c e d o v e r 100 l o c a l l e s i o n s / l e a f o n C. quinoa f o l l o w i n g t r a n s m i s s i o n . H o w e v e r , s e v e r a l C N V  local lesion  isolates r e p r o d u c i b l y g a v e f e w e r l o c a l l e s i o n s (less than 10 p e r l e a f ) a n d t h e r e f o r e t r a n s m i s s i o n o f these i s o l a t e s a p p e a r e d to b e less e f f i c i e n t . F o u r o f these v a r i a n t s w e r e f u r t h e r c h a r a c t e r i z e d b y s y m p t o m f o r m a t i o n , b y e x a m i n i n g l e a f extracts f o r the p r e s e n c e o f v i r u s p a r t i c l e s a n d f o r the a b u n d a n c e a n d s i z e o f v i r a l R N A  species. C h a r a c t e r i s t i c s o f  these m u t a n t s are s u m m a r i z e d i n T a b l e 3.1. O n e m u t a n t , L L 2 , e x h i b i t e d d e l a y e d s y s t e m i c s y m p t o m s a n d d i d n o t p r o d u c e v i r u s p a r t i c l e s o r c o a t p r o t e i n as d e t e r m i n e d  by  e l e c t r o n m i c r o s c o p y a n d w e s t e r n b l o t a n a l y s i s (not s h o w n ) . A g a r o s e g e l e l e c t r o p h o r e s i s o f total l e a f R N A  extracts (not s h o w n ) r e v e a l e d an apparent f u l l - l e n g t h g e n o m i c 64  RNA  s p e c i e s s u g g e s t i n g that the l a c k o f c o a t p r o t e i n w a s d u e t o a m i n o r c h a n g e i n t h e c o a t p r o t e i n gene. A n o t h e r mutant, L L A 1 6 , a l s o s h o w e d d e l a y e d s y s t e m i c s y m p t o m s a n d c o n t a i n e d a g e n o m i c R N A d e l e t i o n o f a p p r o x i m a t e l y 1.1 kb. A l t h o u g h n o t d i r e c t l y d e t e r m i n e d , t h i s d e l e t i o n is m o s t l i k e l y i n the C P g e n e s i n c e d e l e t i o n s o f this s i z e i n o t h e r parts o f the C N V g e n o m e w o u l d p r e c l u d e v i r a l R N A r e p l i c a t i o n o r m o v e m e n t ( R o c h o n a n d J o h n s t o n , 1991; R u s s o et al., 1994). T h e i n a b i l i t y o f these t w o m u t a n t s t o b e t r a n s m i t t e d is c o n s i s t e n t w i t h the r o l e o f the C N V C P i n t r a n s m i s s i o n ( M c L e a n et  al.,  1994). O n e o t h e r mutant, L L A 1 , s h o w e d attenuated s y m p t o m s a n d w a s f o u n d a s s o c i a t e d with defective interfering (DI) R N A s and a corresponding l o w level o f viral genomic R N A ( n o t s h o w n ) . T h e i n e f f i c i e n t t r a n s m i s s i o n o f this m u t a n t w a s t h e r e f o r e l i k e l y d u e t o the l o w l e v e l o f v i r u s i n i n f e c t e d l e a v e s . P r o p e r t i e s o f L L 5 w e r e s i m i l a r t o W T e x c e p t f o r the r e d u c t i o n i n f u n g u s t r a n s m i s s i o n . A d e t a i l e d d e s c r i p t i o n o f L L 5 transmissibility and other properties is provided below.  65  CNV  Table 3.1  Phenotypes o f C N V transmission mutants. a  Virus  b  d  Symptoms  >100  w i l d type  +  W i l d type  LL2  0  Delayed  -  Mutated C P gene  LL5  9  Systemic  +  Mutated C P gene  LLA1  0  Attenuated  +  LLA16  0  Delayed  WT CNV  Particles  c  + Fungus  ND  Genotype  DI  RNA  Deleted C P gene  Average number of local lesions per leaf in bioassay on Ch. quinoa following fungus transmission.  b  Symptoms were monitored 4 to 21 dpi. In LL2 and LLA16 symptoms were delayed 7 days beyond the normal 4 days that symptoms appear in WT CNV infection.  c  Presence of particles were determined by examination of leaf extracts by electron microscopy (see McLean et ai, 1994) and/or by agarose gel electrophoresis following "mini-prep" purification from a single infected leaf (see Materials and Methods).  d  Western blots of total protein and agarose gel electrophoresis of total RNA were performed using leaf extracts of inoculated plants. For LL2, the absence of coat protein in Western blots indicated no CP was being produced. A defective interfering RNA was found in LLA1 total RNA. LLA16 virion RNA from total RNA contained a deletion which is presumed to have occurred in the CP gene. 3.2.2 A Glu to Lys mutation in the L L 5 coat protein S domain is responsible for reduced fungal transmission.  T h e C P g e n e o f L L 5 was a m p l i f i e d u s i n g R T - P C R (see F i g u r e 3.1) a n d t h e n c l o n e d i n t o o u r W T C N V c D N A c l o n e i n o r d e r to assess its r o l e i n the r e d u c e d t r a n s m i s s i b i l i t y o f L L 5 . T 7 R N A p o l y m e r a s e r u n - o f f transcripts o f o n e s u c h c l o n e ( d e s i g n a t e d p M 5 / L L 5 ) w e r e u s e d to i n o c u l a t e N. clevelandii a n d p u r i f i e d v i r u s o b t a i n e d f r o m i n f e c t e d l e a v e s was u s e d i n a f u n g u s t r a n s m i s s i o n assay. C u c u m b e r r o o t s f r o m the t r a n s m i s s i o n a s s a y w e r e e x a m i n e d f o r the p r e s e n c e o f M 5 / L L 5 v i r u s u s i n g D A S - E L I S A . F i g u r e 3.2 s h o w s that l e v e l s o f M 5 / L L 5 v i r u s w e r e s i g n i f i c a n t l y l o w e r t h a n W T v i r u s i n i n o c u l a t e d c u c u m b e r roots. T h i s suggests that the L L 5 c o a t p r o t e i n c o n t a i n s the alterations w h i c h r e d u c e f u n g u s t r a n s m i s s i b i l i t y .  66  A amber cod on  p20  iriiTTiSfaitan l T T - rr p33  p92  I  p41  m  CNV  genomic RNA  2P21  B R |a|  t nt 2824 Phe to Cys (LL5.)  S  P  i nt 3156 Glu to Lys (LL5 )  |  I  nt 3674 (silent)  S  F i g u r e 3.1 L o c a t i o n o f m u t a t i o n s i n the L L 5 C P gene. A ) . S t r u c t u r e o f the C N V g e n o m e s h o w i n g the l o c a t i o n s o f the t w o p r i m e r s (1 a n d 2) u s e d to a m p l i f y the L L 5 C P gene. B ) S t r u c t u r e o f the C N V C P g e n e s h o w i n g the R, S a n d P d o m a i n s a n d the c o n n e c t i n g a r m (a) a n d h i n g e (h). T h e g e n o m i c l o c a t i o n s o f the 3 n u c l e o t i d e s u b s t i t u t i o n s i n L L 5 are g i v e n a l o n g w i t h the a m i n o a c i d c h a n g e s they cause.  T h e M 5 / L L 5 C P g e n e was s e q u e n c e d a n d f o u n d to c o n t a i n three n u c l e o t i d e s u b s t i t u t i o n s (see F i g u r e 3.1): a T to G s u b s t i t u t i o n at C N V n u c l e o t i d e 2 8 2 4 c h a n g e d a P h e to C y s i n the a r m (a) r e g i o n , a G to A s u b s t i t u t i o n at n u c l e o t i d e 3 1 5 6 c h a n g e d a G l u to L y s ( a m i n o a c i d 177) i n the s h e l l (S) d o m a i n a n d a G to T s u b s t i t u t i o n at 3 6 7 4 i n the P d o m a i n c o d i n g r e g i o n was silent. T h e r e w e r e n o o t h e r c h a n g e s o u t s i d e o f the C P  67  ORF.  F i g u r e 3.2 S u m m a r y o f f u n g u s t r a n s m i s s i o n assays. T r a n s m i s s i o n a s s a y s w e r e c o n d u c t e d u s i n g 1 o r 5 u g o f v i r u s p l u s ( + F ) o r m i n u s f u n g u s (-F) o r f u n g u s a l o n e . S i x d a y s after t r a n s m i s s i o n , r o o t s w e r e g r o u n d a n d a s s a y e d f o r v i r u s u s i n g D A S - E L I S A a n d C N V p o l y c l o n a l a n t i s e r u m . OD405 v a l u e s f o r f u n g u s o n l y c o n t r o l s w e r e s u b t r a c t e d f r o m a l l v a l u e s . A l l v a l u e s w e r e n o r m a l i z e d a g a i n s t W T C N V (arbitrarily a s s i g n e d OD405 o f 1.0) w i t h i n e a c h i n d i v i d u a l e x p e r i m e n t ( w h i c h c o n t a i n e d 1 o r 2 r e p l i c a t e s ) a n d t h e n o r m a l i z e d v a l u e s f r o m 5 separate e x p e r i m e n t s w e r e a v e r a g e d . B a r s r e p r e s e n t s t a n d a r d d e v i a t i o n s f o r e a c h treatment. T h e n u m b e r o f i n f e c t e d pots/total n u m b e r o f p o t s were: W T C N V (10/10), M 5 / L L 5 (2/10), M 5 / L L 5 s (5/10), a n d M 5 / L L 5 a (10/10).  Site-directed mutagenesis was used to introduce the L L 5 a r m a n d S d o m a i n m u t a t i o n s i n d i v i d u a l l y i n t o W T C N V c D N A t o assess t h e p o s s i b l e i n d e p e n d e n t r o l e s o f the t w o m u t a t i o n s i n t h e L L 5 l o w t r a n s m i s s i b i l i t y p h e n o t y p e . W h e n v i r u s f r o m p l a n t s i n f e c t e d w i t h T 7 R N A p o l y m e r a s e r u n - o f f transcripts o f t h e s e t w o c l o n e s w e r e t e s t e d f o r t r a n s m i s s i b i l i t y ( F i g u r e 3.2), i t w a s f o u n d that t h e a r m d o m a i n m u t a n t ( d e s i g n a t e d 68  M 5 / L L 5 ) was h i g h l y t r a n s m i s s i b l e b u t that the s h e l l d o m a i n m u t a n t ( M 5 / L L 5 s ) was a  t r a n s m i s s i b l e at o n l y a v e r y l o w l e v e l ( 0 - 2 0 % o f W T ) . that the G l u to L y s s u b s t i t u t i o n i n the C N V  W e c o n c l u d e f r o m these s t u d i e s  S d o m a i n is l a r g e l y r e s p o n s i b l e f o r the l o w  transmission phenotype. 3.2.3 The Glu to Lys substitution in the LL5 the loss of transmissibility.  shell domain is directly responsible for  O u r data h a v e s u g g e s t e d that a s p e c i f i c a m i n o a c i d c h a n g e i n the L L 5 C P i s r e s p o n s i b l e f o r the r e d u c t i o n i n t r a n s m i s s i b i l i t y o f this C N V  variant. T h i s m u t a t i o n  may  reduce transmission directly by interfering with zoospore recognition or binding. A l t e r n a t i v e l y , the m u t a t i o n m a y a f f e c t t r a n s m i s s i o n i n d i r e c t l y b y p r o d u c i n g a v i r u s w h i c h is e i t h e r less stable, less c a p a b l e o f i n i t i a t i n g an i n f e c t i o n o r less c a p a b l e o f a c c u m u l a t i n g i n i n f e c t e d tissue. T h e f o l l o w i n g d e s c r i b e s the results o f e x p e r i m e n t s d e s i g n e d to d i s t i n g u i s h b e t w e e n these v a r i o u s p o s s i b i l i t i e s .  69  4?  ^  r  <? #  Jf  F i g u r e 3.3 A g a r o s e g e l e l e c t r o p h o r e s i s o f m u t a n t a n d W T v i r u s . T w o | i g o f C s C l p u r i f i e d v i r u s w a s e l e c t r o p h o r e s e d t h r o u g h a 1 % a g a r o s e g e l b u f f e r e d i n 10 m M T r i s / 7 5 m M g l y c i n e , p H 8.0 at 9 V / c m f o r 1.2 hr. G e l s w e r e s t a i n e d w i t h e t h i d i u m b r o m i d e .  M 5 a n d M 5 / L L 5 S p a r t i c l e s c o n t a i n intact R N A b u t h a v e a l t e r e d e l e c t r o p h o r e t i c m o b i l i t i e s o n a g a r o s e gels. 3.2.3.1  E l e c t r o n m i c r o s c o p y w a s u s e d to v i s u a l i z e a n d c o m p a r e p a r t i c l e s p r o d u c e d i n W T C N V and mutant virus infections. Differences i n morphology or staining characteristics w e r e n o t a p p a r e n t (not s h o w n ) . P a r t i c l e s w e r e a l s o e x a m i n e d b y a g a r o s e g e l e l e c t r o p h o r e s i s to assess p a r t i c l e i n t e g r i t y a n d r e l a t i v e m o b i l i t y . It c a n b e s e e n i n F i g u r e 3.3 that p a r t i c l e p r e p a r a t i o n s o f W T C N V , M 5 / L L 5 , M 5 / L L 5 a n d M 5 / L L 5 are intact b u t S  a  that M 5 / L L 5 a n d M 5 / L L 5 m i g r a t e s l i g h t l y s l o w e r t h a n e i t h e r W T o r M 5 / L L 5 . T h e G l u S  a  to L y s s u b s t i t u t i o n i n the s h e l l d o m a i n s o f M 5 / L L 5 a n d M 5 / L L 5 s l i k e l y c o n t r i b u t e s to the altered m o b i l i t y b y i n c r e a s i n g the net p o s i t i v e c h a r g e o f the particle. In a d d i t i o n , s i n c e this m u t a t i o n i s i m m e d i a t e l y a d j a c e n t to an A s p r e s i d u e i n v o l v e d i n C a 70  + 2  ion mediated  s u b u n i t c o n t a c t s ( R i v i e r e et al., 1989), t h e m u t a t i o n m a y a l s o l o w e r e l e c t r o p h o r e t i c m o b i l i t y b y p r o d u c i n g a p a r t i c l e w i t h a s w o l l e n c o n f o r m a t i o n (see s e c t i o n 3.3).  F i g u r e 3.4 A g a r o s e g e l e l e c t r o p h o r e s i s o f R N A e x t r a c t e d f r o m m u t a n t a n d W T v i r i o n s . V i r i o n s were purified f r o m leaves using a "mini-prep" procedure, a n d R N A extracted using phenol/chloroform. E q u a l concentrations (200 ng) o f R N A were electrophoresed t h r o u g h a n o n - d e n a t u r i n g agarose g e l a n d s t a i n e d w i t h e t h i d i u m b r o m i d e . T h e results o f two independent experiments are s h o w n for M 5 / L L 5 s and M 5 / L L 5 . T h e position o f g e n o m i c R N A is i n d i c a t e d . T h e a r r o w p o i n t s t o the f l u o r e s c e n t m a t e r i a l present i n the wells loaded with M 5 / L L 5 and M 5 / L L 5 R N A . a  a  V i r u s p a r t i c l e s w i t h a s w o l l e n c o n f o r m a t i o n are k n o w n to b e m o r e s u s c e p t i b l e to R N A a s e d e g r a d a t i o n ( R o e n h o r s t et al, 1989; H e a t o n , 1992). W e w i s h e d t o d e t e r m i n e i f p a r t i c l e s o f M 5 / L L 5 o r M 5 / L L 5 S c o n t a i n d e g r a d e d R N A s i n c e this w o u l d e x p l a i n the l o s s of fungus transmissibility. T h e integrity o f viral R N A extracted f r o m W T and mutant p a r t i c l e s w a s e x a m i n e d b y agarose g e l e l e c t r o p h o r e s i s . F i g u r e 3.4 s h o w s that approximately equivalent amounts o f largely intact R N A were extracted from W T a n d M 5 / L L 5 particles. H o w e v e r , less R N A w a s e x t r a c t e d f r o m M 5 / L L 5 a n d M 5 / L L 5 S  71  a  particles. A s w i l l b e d e s c r i b e d b e l o w , M 5 / L L 5 a n d M 5 / L L 5 are as i n f e c t i o u s as W T a n d a  M 5 / L L 5 s . Therefore, the actual amount o f viral R N A i n particles should b e equivalent. It i s p o s s i b l e that t h e f l u o r e s c i n g m a t e r i a l p r e s e n t i n t h e w e l l s o f l a n e s c o n t a i n i n g M 5 / L L 5 a n d M 5 / L L 5 R N A (but absent i n W T a n d M 5 / L L 5 w e l l s ; F i g u r e 3.4) c o r r e s p o n d s t o a  S  virion R N A w h i c h was not readily released f r o m viral C P during phenol/chloroform e x t r a c t i o n (see s e c t i o n 3.3). 3.2.3.2 M 5 / L L 5 a n d M 5 / L L 5 s p a r t i c l e s are s t a b l e a n d as i n f e c t i o u s as W T . M u t a n t a n d W T p a r t i c l e s w e r e a l s o e x a m i n e d f o r t h e i r r e l a t i v e stabilities a n d i n f e c t i v i t i e s u s i n g b i o a s s a y s a n d s e v e r a l l o c a l l e s i o n hosts (Cucumis sativis, Phaseolus vulgaris, Vigna unguiculata, C. amaranticolor a n d C. quinoa). E q u a l p a r t i c l e  concentrations o f each virus produced approximately equal numbers o f local lesions over a r a n g e o f d i l u t i o n s (data n o t s h o w n ) i n d i c a t i n g that a l l v i r u s e s w e r e e q u a l l y i n f e c t i o u s . F o r the stability assay, e q u i v a l e n t a m o u n t s o f W T C N V , M 5 / L L 5 , M 5 / L L 5 a n d M 5 / L L 5 S  p a r t i c l e s w e r e i n c u b a t e d f o r 0, 1, 3 o r 18 hrs i n t r a n s m i s s i o n b u f f e r ( 5 0 m M g l y c i n e , p H 7.6) a n d i n f e c t i v i t y a n a l y z e d u s i n g C. quinoa as a l o c a l l e s i o n host. T h e r e w a s n o d e c r e a s e i n t h e n u m b e r o f l o c a l l e s i o n s f o r a n y o f the v i r u s p a r t i c l e p r e p a r a t i o n s o v e r t h i s t i m e p e r i o d (data n o t s h o w n ) , i n d i c a t i n g that the r e d u c e d t r a n s m i s s i b i l i t y o f M 5 / L L 5 a n d M 5 / L L 5 s i s n o t d u e t o p a r t i c l e instability.  72  a  /////  V  ^-Genomic  RNA  F i g u r e 3.5 A c c u m u l a t i o n o f v i r a l R N A o r v i r u s p a r t i c l e s i n i n f e c t e d l e a v e s . ( A ) R N A a c c u m u l a t i o n . T o t a l R N A w a s e x t r a c t e d f r o m i n o c u l a t e d N. clevelandii l e a v e s i n f e c t e d with WT, M 5 / L L 5 , M 5 / L L 5 s or M 5 / L L 5 and equal volumes o f R N A were e l e c t r o p h o r e s e d t h r o u g h a n o n - d e n a t u r i n g 1% agarose gel. T h e p o s i t i o n o f g e n o m i c R N A is i n d i c a t e d . ( B ) V i r u s w a s p u r i f i e d f r o m l e a v e s i n f e c t e d as a b o v e u s i n g a " m i n i - p r e p " p r o c e d u r e . A l i q u o t s o f e a c h v i r u s p r e p a r a t i o n (0.2 a n d 0.5 p i ) w e r e e l e c t r o p h o r e s e d t h r o u g h a 1% agarose g e l a n d s t a i n e d w i t h e t h i d i u m b r o m i d e . a  3.2.3.3 M 5 / L L 5 a n d M 5 / L L 5 a c c u m u l a t e t o W T l e v e l s i n i n f e c t e d plants. S  T h e l e v e l o f a c c u m u l a t i o n o f g e n o m i c R N A i n i n f e c t e d N. clevelandii l e a v e s as c o m p a r e d to W T R N A w a s d e t e r m i n e d f o r M 5 / L L 5 , M 5 / L L 5 a n d M 5 / L L 5 . T o t a l R N A a  S  was extracted f r o m single inoculated leaves a n d the levels o f g e n o m i c R N A assessed b y e t h i d i u m b r o m i d e s t a i n i n g o f e q u i v a l e n t v o l u m e s o f total R N A ( F i g u r e 3.5 A ) . T h e r e w e r e n o m a j o r d i f f e r e n c e s i n t h e l e v e l o f g e n o m i c R N A as c o m p a r e d t o W T i n f e c t i o n s 73  s u g g e s t i n g that e a c h v i r u s a c c u m u l a t e s t o a p p r o x i m a t e l y e q u i v a l e n t l e v e l s d u r i n g infection. Similar experiments were conducted c o m p a r i n g the level o f virions w h i c h c o u l d be extracted f r o m single leaves a n d no differences i n the amounts o f virus were f o u n d ( F i g u r e 3.5 B ) . T h u s a r e d u c e d a b i l i t y t o a c c u m u l a t e i n i n f e c t e d p l a n t s d o e s n o t account for the l o w E L I S A values obtained for L L 5 / M 5 and L L 5 / M 5  S  following  transmission. 1.2  E a  IT)  ©  0.8  CU U  e at  u ©  0.6 G virus - F  c«  < >  0 virus + F 0.4  "a* OH  0.2  WT CNV -0.2  TBSV  M5/LL5  M5/LL5  C  M5/LL5.,  Virus  F i g u r e 3.6 S u m m a r y o f in vitro b i n d i n g assays. B i n d i n g a s s a y s w e r e c o n d u c t e d u s i n g v i r u s p l u s ( + F ) o r m i n u s f u n g u s (-F). T h e z o o s p o r e p e l l e t w a s r e s u s p e n d e d i n b u f f e r a n d the a m o u n t o f v i r u s p r e s e n t d e t e r m i n e d b y D A S - E L I S A u s i n g e i t h e r a C N V o r T B S V - C h p o l y c l o n a l antisera. A l l v a l u e s w e r e n o r m a l i z e d a g a i n s t W T C N V (OD405 o f 1.0) w i t h i n each individual experiment a n d the n o r m a l i z e d values were averaged across experiments. Buffer controls were subtracted f r o m virus only samples while fungus only controls were subtracted f r o m the virus plus fungus samples. W T C N V corresponds to seven independent experiments, T B S V to five experiments and M 5 / L L 5 , M 5 / L L 5 s and M 5 / L L 5 to t w o e x p e r i m e n t s . A l l e x p e r i m e n t s c o n t a i n e d at least t w o replicates. a  74  3.2.4 M5/LL5 and M5/LL5s particles bind zoospores less efficiently than WT  virus.  W e d e v e l o p e d a p r e l i m i n a r y b i n d i n g a s s a y t o assess i f the r e d u c e d a b i l i t y o f M 5 / L L 5 a n d M 5 / L L 5 s p a r t i c l e s t o b e t r a n s m i t t e d b y O. bornovanus z o o s p o r e s i s d u e t o the i n a b i l i t y o f v i r u s t o b i n d z o o s p o r e s o r i n s t e a d to a d e f e c t i n a n o t h e r stage o f the t r a n s m i s s i o n p r o c e s s . T o i n i t i a l l y v a l i d a t e t h e s e studies, w e d e t e r m i n e d i f p a r t i c l e s o f C N V a n d t h e n o n - t r a n s m i s s i b l e T B S V - C h b i n d O. bornovanus z o o s p o r e s d i f f e r e n t i a l l y . O n e h u n d r e d p g o f W T C N V o r T B S V - C h p a r t i c l e s w e r e i n c u b a t e d f o r 15 m i n (the a c q u i s i t i o n p e r i o d u s e d i n o u r t r a n s m i s s i o n a s s a y s ) w i t h o r w i t h o u t z o o s p o r e s (1 X 1 0  6  p e r m l ) i n 10 m l o f a c q u i s i t i o n b u f f e r . F o l l o w i n g i n c u b a t i o n , z o o s p o r e s w e r e c e n t r i f u g e d and w a s h e d two times a n d any pelleted material was then e x a m i n e d f o r the presence a n d l e v e l o f v i r u s u s i n g D A S - E L I S A a n d either a C N V o r T B S V p o l y c l o n a l a n t i b o d y . U s i n g this assay, i t w a s d e t e r m i n e d that a p p r o x i m a t e l y 15 n g o f C N V p a r t i c l e s i s a s s o c i a t e d w i t h t h e z o o s p o r e p e l l e t ( T a b l e 3.2) a n d that l i t t l e o r n o C N V c o u l d b e d e t e c t e d w h e n z o o s p o r e s w e r e o m i t t e d f r o m t h e assay. F u r t h e r m o r e , T B S V - C h p a r t i c l e s w e r e o n l y d e t e c t e d at o r n e a r b a c k g r o u n d l e v e l s . T h e s e e x p e r i m e n t s s u g g e s t that C N V s p e c i f i c a l l y a s s o c i a t e s w i t h z o o s p o r e s a n d that t h e l a c k o f t r a n s m i s s i b i l i t y o f T B S V - C h m a y b e d u e t o the i n a b i l i t y o f T B S V - C h p a r t i c l e s t o r e c o g n i z e o r b i n d z o o s p o r e s . S i m i l a r b i n d i n g experiments were then conducted using purified preparations o f M 5 / L L 5 , M 5 / L L 5 s or M 5 / L L 5 particles. T h e results o f s e v e r a l r e p e t i t i o n s o f t h e s e b i n d i n g a s s a y s a r e a  s u m m a r i z e d i n F i g u r e 3.6. It c a n b e s e e n that M 5 / L L 5 a n d M 5 / L L 5 s p a r t i c l e s a r e p r e s e n t i n the z o o s p o r e p e l l e t at o n l y a p p r o x i m a t e l y 2 0 - 6 0 % t h e l e v e l o f W T v i r u s , w h e r e a s M 5 / L L 5 b o u n d at 7 5 - 1 1 0 % t h e e f f i c i e n c y o f C N V . T h e s e results s u g g e s t that t h e a  75  i n e f f i c i e n t t r a n s m i s s i o n s o f M 5 / L L 5 a n d M 5 / L L 5 s are at least p a r t l y d u e to t h e i r i n a b i l i t y to r e c o g n i z e o r s t a b l y b i n d z o o s p o r e s . Table 3.2 B i n d i n g o f C N V t o O. bornovanus z o o s p o r e s in vitro. +Fungus  -Fungus  CNV  a) 0.978 b) 0.451  b ng v i r u s 15 15  TBSV-Ch  a) 0.118 b) 0.025  3 2  a) 0.084 b) 0.000  2 <1  no virus  a) 0.050 b) 0.012  <1 <1  a) 0.055 b) 0.019  <1 <1  Virus  DAS-ELISA  3  DAS-ELISA  ng virus  a) 0.055 b) 0.011  <1 <1  Values correspond to absorbance at 405 nm. The values in (a) and (b) correspond to the average values obtained from two separate experiments. Experiment (a) contained two replicates and experiment (b) three replicates.  b Determined by comparing the D A S - E L I S A value of the virus in the zoospore pellet to that of a dilution standard of C N V or T B S V - C h .  3.3 Discussion  R e p e a t e d m e c h a n i c a l p a s s a g e o f p l a n t v i r u s e s f r e q u e n t l y r e s u l t s i n the l o s s o f v e c t o r t r a n s m i s s i b i l i t y . I n s o m e cases, the l o s s o f t r a n s m i s s i b i l i t y i s a s s o c i a t e d w i t h d e l e t i o n s i n g e n o m i c R N A , p r e s u m a b l y i n the g e n e s s p e c i f y i n g t r a n s m i s s i o n (see C a m p b e l l , 1 9 9 6 ; G r a y , 1996). T h i s s t u d y d e m o n s t r a t e s that m e c h a n i c a l l y p a s s a g e d  CNV  c o n t a i n s v a r i a n t s w h i c h h a v e l o s t the a b i l i t y t o b e t r a n s m i t t e d b y O. bornovanus. A c o m p r e h e n s i v e a n a l y s i s o f the l e v e l o f s u c h m u t a n t s w a s not u n d e r t a k e n , b u t f o u r o f 4 0 l o c a l l e s i o n s w e r e a n a l y z e d . T w o o f the f o u r v a r i a n t s ( L L A 1 6 a n d L L 2 ; T a b l e 3.1) d i d not p r o d u c e v i r a l C P e i t h e r b e c a u s e the C P g e n e w a s d e l e t e d o r b e c a u s e the C P w a s n o t expressed. Another variant ( L L A 1 ) contained D I R N A s with a corresponding decreased  76  level of genomic R N A  a n d thus the l e a f e x t r a c t u s e d as i n o c u l u m was l i k e l y d e f i c i e n t i n  v i r u s particles. A l t e r n a t i v e l y , i t i s p o s s i b l e that L L A 1 t r a n s m i t s w e l l b u t that D I  RNA  i n t e r f e r e d w i t h v i r u s a c c u m u l a t i o n i n roots. H o w e v e r , p r e v i o u s s t u d i e s h a v e s h o w n that D I R N A s are p a c k a g e d i n e f f i c i e n t l y , i f at a l l , i n C N V c a p s i d s ( R o c h o n et ah,  1994).  Isolates f r o m s e v e r a l o t h e r l o c a l l e s i o n s w e r e a l s o p o o r l y transmitted. M o s t o f these m u t a n t s a p p e a r e d to b e e i t h e r C P d e l e t i o n m u t a n t s o r c o n t a i n e d D I R N A s . I n d i v i d u a l C N V d e l e t i o n m u t a n t s l a c k i n g the C P g e n e d o not b e c o m e d o m i n a n t i n m e c h a n i c a l l y p a s s a g e d i n o c u l u m (data n o t s h o w n ) , d e s p i t e the f a c t that the C N V C P i s n o t r e q u i r e d f o r v i r u s a c c u m u l a t i o n o r s p r e a d ( M c L e a n et al., 1993, S i t et al., 1995). T h e p r e s e n c e o f the C P g e n e m u s t t h e r e f o r e p r o v i d e s o m e s e l e c t i v e a d v a n t a g e o v e r its a b s e n c e . T h e c u r r e n t s t u d y e m p h a s i z e s that t r a n s m i s s i b i l i t y c a n b e a f f e c t e d b y s e v e r a l d i f f e r e n t g e n e t i c traits, i n c l u d i n g l o s s o f the c o a t p r o t e i n g e n e ( L L A 1 6 ) o r its p r o d u c t ( L L 2 ) , an a l t e r a t i o n i n c a p s i d s e q u e n c e o r structure ( L L 5 ) o r r e d u c e d a b i l i t y to r e p l i c a t e o r a c c u m u l a t e ( L L A 1 ) . In this study, w e w i s h e d to f o c u s o n m u t a t i o n s w h i c h d i r e c t l y a f f e c t the a b i l i t y o f C N V to b e transmitted, s u c h as those that a f f e c t b i n d i n g to the vector. W e h a v e d e m o n s t r a t e d that the G l u to L y s s u b s t i t u t i o n i n the L L 5 C P g e n e i s l a r g e l y r e s p o n s i b l e f o r the r e d u c t i o n i n f u n g u s t r a n s m i s s i b i l i t y o f this C N V  variant.  M 5 / L L 5 s p a r t i c l e s are stable, h i g h l y i n f e c t i o u s a n d a c c u m u l a t e to W T l e v e l s i n m e c h a n i c a l l y i n o c u l a t e d plants. In a d d i t i o n , M 5 / L L 5 s v i r i o n R N A RNA  is intact and g e n o m i c  a c c u m u l a t e s to W T l e v e l s ( F i g u r e s 3.3-3.5). T h u s the G l u to L y s m u t a t i o n i n the  C N V C P s h e l l d o m a i n appears to d i r e c t l y a f f e c t t r a n s m i s s i b i l i t y rather than m e r e l y i n d i r e c t l y a f f e c t i n g t r a n s m i s s i o n t h r o u g h an e f f e c t o n s o m e o t h e r aspect o f v i r u s i n f e c t i o n . In vitro " b i n d i n g " assays f u r t h e r s u p p o r t the n o t i o n that the G l u to L y s m u t a t i o n d i r e c t l y  77  affects t r a n s m i s s i o n . V i r a l p a r t i c l e s c o n t a i n i n g t h i s m u t a t i o n b i n d O. bornovanus z o o s p o r e s l e s s e f f i c i e n t l y ( F i g u r e 3.6) s u g g e s t i n g that the d e c r e a s e d t r a n s m i s s i b i l i t y i s d u e to a d e c r e a s e i n the a b i l i t y o f v i r a l c a p s i d s to r e c o g n i z e o r s t a b l y b i n d z o o s p o r e s (see b e l o w ) . T h e b i n d i n g assays ( F i g u r e 3.6) i n d i c a t e that M 5 / L L 5 a n d M 5 / L L 5 s b i n d at a p p r o x i m a t e l y 2 0 - 6 0 % the e f f i c i e n c y o f W T particles. T h e s e v a l u e s are s l i g h t l y h i g h e r than t h o s e o b t a i n e d u s i n g the f u n g u s t r a n s m i s s i o n a s s a y ( 0 - 2 0 % ; see F i g u r e 3.2). T h i s result m a y suggest that f a c t o r s o t h e r t h a n b i n d i n g c o n t r i b u t e to the l o w t r a n s m i s s i o n phenotype. A l t h o u g h a s i n g l e a m i n o a c i d c h a n g e i n the C N V C P has b e e n i d e n t i f i e d w h i c h affects t r a n s m i s s i o n , other c h a n g e s e l s e w h e r e i n the C P w o u l d a l s o b e e x p e c t e d to h a v e an affect. T h e G l u to L y s c h a n g e i n L L 5 o c c u r s i n the " E F " l o o p o f the v i r a l c a p s i d s h e l l d o m a i n . T h i s l o o p i s i n v o l v e d i n s u b u n i t i n t e r a c t i o n s a n d i s a l s o p a r t i a l l y e x p o s e d o n the s u r f a c e o f the s h e l l ( H a r r i s o n , 1983). E x p o s e d r e g i o n s o f the p o t y v i r u s c a p s i d a n d l u t e o v i r u s c a p s i d h a v e p r e v i o u s l y b e e n s h o w n to b e i n v o l v e d i n t r a n s m i s s i o n (see P i r o n e a n d B l a n c , 1996; G r a y , 1996). L L 5 and M 5 / L L 5 particles migrate slightly slower than W T particles i n agarose gels ( F i g u r e 3.3). T h e G l u to L y s s u b s t i t u t i o n w o u l d i n c r e a s e the net p o s i t i v e c h a r g e o f the p a r t i c l e a n d t h e r e f o r e s l o w its m i g r a t i o n . I n t e r s u b u n i t c o n t a c t s i n m a n y s m a l l s p h e r i c a l p l a n t v i r u s e s are s t a b i l i z e d b y C a  + 2  i o n s ( H a r r i s o n , 1983; H e a t o n a n d M o r r i s ,  1992). W h e n these i o n s are s e q u e s t e r e d w i t h E D T A the p a r t i c l e takes o n a s w o l l e n c o n f o r m a t i o n a n d this r e s u l t s i n l o w e r e d e l e c t r o p h o r e t i c m o b i l i t y i n a g a r o s e g e l s ( H e a t o n , 1992). T h e G l u to L y s s u b s t i t u t i o n i n L L 5 o c c u r s i m m e d i a t e l y a d j a c e n t to a n A s p r e s i d u e w h i c h i s i n v o l v e d i n C a + 2 i o n m e d i a t e d s u b u n i t contacts. T h u s , i t i s p o s s i b l e that the  78  lowered mobilities o f L L 5 a n d M 5 / L L 5 s might be partially attributed to their m o r e s w o l l e n states. W e s h o w that M 5 / L L 5 s v i r i o n R N A is l a r g e l y i n t a c t a n d p r e s e n t at n o r m a l W T l e v e l s a n d t h e r e f o r e t h e r e d u c e d t r a n s m i s s i b i l i t y o f these p a r t i c l e s i s n o t d u e t o d e g r a d a t i o n o r l o s s o f the i n f e c t i o u s c o m p o n e n t . Interestingly, t h e y i e l d o f M 5 / L L 5 a n d M 5 / L L 5 v i r i o n R N A i s s i g n i f i c a n t l y l o w e r t h a n that o f W T o r M 5 / L L 5 ( F i g u r e 3.4). a  S  F i g u r e 3.4 s h o w s that a l a r g e a m o u n t o f f l u o r e s c i n g m a t e r i a l is p r e s e n t i n t h e w e l l s o f lanes c o n t a i n i n g M 5 / L L 5 R N A ( a n d t o a l e s s e r extent M 5 / L L 5 ) . It is p o s s i b l e that t h e a  m a t e r i a l i n t h e w e l l s c o r r e s p o n d s t o v i r a l C P / R N A aggregates. I f so, t h e L L 5 a r m mutation (Phe to C y s ) is likely responsible for this unusual p h e n o m e n o n . W e h a v e d e v e l o p e d a p r e l i m i n a r y in vitro b i n d i n g a s s a y t o assess t h e r o l e o f b i n d i n g i n t h e s p e c i f i c i t y o f C N V t r a n s m i s s i o n a n d t o f u r t h e r assess t h e d i r e c t r o l e o f v i r a l C P i n f u n g u s t r a n s m i s s i o n . O u r assays ( T a b l e 3.2 a n d F i g u r e 3.6) s h o w that b i n d i n g i s s p e c i f i c (i.e., C N V b i n d s b u t t h e n o n - t r a n s m i s s i b l e T B S V - C h b i n d s at o n l y a v e r y l o w l e v e l ) a n d that M 5 / L L 5 a n d M 5 / L L 5 p a r t i c l e s b i n d l e s s e f f i c i e n t l y t h a n W T C N V . T h e s e S  d a t a s u g g e s t that t h e s p e c i f i c i t y o f t r a n s m i s s i o n lies at t h e l e v e l o f O. bornovanus zoospore recognition or binding. Similar observations and conclusions were previously m a d e b y T e m m i n k et al. ( 1 9 7 0 ) f o r t o b a c c o n e c r o s i s v i r u s a n d its v e c t o r O. brassicae a n d a l s o f o r C N V a n d O. bornovanus u s i n g e l e c t r o n m i c r o s c o p y o f v i r u s / z o o s p o r e interactions. T h e s e b i n d i n g d a t a r a i s e t h e i n t r i g u i n g p o s s i b i l i t y that t h e i n t e r a c t i o n b e t w e e n particles a n d zoospores is mediated b y a specific receptor o n the zoospore p l a s m a l e m m a , p e r h a p s s i m i l a r i n s o m e r e s p e c t s to v i r u s / r e c e p t o r i n t e r a c t i o n s i n a n i m a l s y s t e m s  79  ( H a y w o o d , 1994). T h e p o s s i b l e i n v o l v e m e n t o f s p e c i f i c receptors i n p l a n t v i r u s t r a n s m i s s i o n has b e e n p r e v i o u s l y s u g g e s t e d f o r Barley yellow dwarf luteovirus ( G i l d o w , 1993) a n d Tomato spotted wilt tosposvirus ( B a n d l a et al., 1998). In a d d i t i o n , it has b e e n s u g g e s t e d that d i f f e r e n c e s i n s u r f a c e c h a r g e o f v e c t o r a n d n o n - v e c t o r i s o l a t e s o f O. brassicae m a y be r e s p o n s i b l e f o r the d i f f e r e n t i a l t r a n s m i s s i o n o f T N V  perhaps by  i n f l u e n c i n g b i n d i n g b e t w e e n z o o s p o r e s a n d v i r u s ( M o w a t , 1968). It is p o s s i b l e that the G l u to L y s a l t e r a t i o n i n the L L 5 C P s h e l l d o m a i n affects the a b i l i t y o f v i r u s to interact stably with a putative receptor perhaps by altering specific ionic interactions with a c o m p o n e n t o f the z o o s p o r e m e m b r a n e . A l t e r n a t i v e l y , o r a d d i t i o n a l l y , a  conformational  c h a n g e i n the v i r u s p a r t i c l e , w h i c h is a result o f a l t e r e d s u b u n i t i n t e r a c t i o n s , m a y efficient virus binding.  80  preclude  4 C H A P T E R  F O U R :  C O M P A R A T I V E S E Q U E N C E A N A L Y S I S O F T H E C O A T  P R O T E I N S O F F O U R OLPIDIUM T R A N S M I T T E D  BORNOVANUS  V I R U S E S  4.1 Introduction O n e o b j e c t i v e o f m y thesis i s to d e t e r m i n e r e g i o n s o f the Cucumber necrosis virus ( C N V ) c a p s i d w h i c h are i n v o l v e d i n a t t a c h m e n t to O. bornovanus z o o s p o r e s . In C h a p t e r 3, n a t u r a l l y o c c u r r i n g C N V  m u t a n t s d e f i c i e n t i n t r a n s m i s s i o n w e r e e x a m i n e d w i t h the  a i m o f i d e n t i f y i n g s p e c i f i c c o a t p r o t e i n ( C P ) a m i n o a c i d s r e q u i r e d f o r t r a n s m i s s i o n . In this chapter, an a d d i t i o n a l a p p r o a c h has b e e n taken, n a m e l y c o m p a r a t i v e s e q u e n c e a n a l y s i s o f the C P s o f t r a n s m i s s i b l e a n d n o n - t r a n s m i s s i b l e m e m b e r s o f the Tombusviridae.  A t the t i m e this part o f m y thesis w o r k was i n i t i a t e d the C P s e q u e n c e s o f t w o other O. bornovanus t r a n s m i t t e d v i r u s e s w e r e k n o w n , i.e. those o f Melon necrotic spot carmovirus ( M N S V ) ( f a m i l y Tombusviridae; R i v i e r e et al., 1989) a n d Red clover necrotic mosaic dianthovirus ( R C N M V ) ( f a m i l y Tombusviridae; X i o n g a n d L o m m e l ,  1989). In a d d i t i o n , the C P s e q u e n c e s o f Tobacco necrosis necrovirus ( T N V ) ("strains" A a n d D; f a m i l y Tombusviridae) w e r e a l s o k n o w n ( M e u l e w a e t e r et al., 1989). T h e  CP  s e q u e n c e s o f s e v e r a l other a p p a r e n t l y n o n - t r a n s m i s s i b l e m e m b e r s o f the Tombusviridae w e r e a l s o k n o w n (see T a b l e 4.1). Interestingly, c o m p a r i s o n s o f the C N V  and M N S V  CPs  s h o w e d that t h e i r a m i n o a c i d s e q u e n c e s are m o r e s i m i l a r to e a c h other t h a n they are to the  81  r e s p e c t i v e m e m b e r s o f t h e i r o w n g e n u s ( R i v i e r e et al, 1989). M o r e o v e r , C N V a n d M N S V w e r e s h o w n to share a r e g i o n o f a m i n o a c i d s e q u e n c e s i m i l a r i t y i n a short r e g i o n o f the o t h e r w i s e n o n - c o n s e r v e d p r o t r u d i n g (P)-domains.  T h i s l e d to the s u g g e s t i o n that  the s e q u e n c e s i m i l a r i t i e s o b s e r v e d b e t w e e n these v i r u s e s m a y b e d u e to the c o n s e r v a t i o n o f s e q u e n c e s r e q u i r e d f o r t r a n s m i s s i o n b y O. bornovanus ( R i v i e r e a n d R o c h o n , 1989). T o f u r t h e r assess this h y p o t h e s i s , the C P s e q u e n c e o f another O. bornovanus t r a n s m i t t e d v i r u s , Cucumber leaf spot virus ( C L S V ) ( C a m p b e l l et ah, 1991) w a s d e t e r m i n e d ( M i l l e r et al, 1997). T h i s c h a p t e r d e s c r i b e s the results o f c o m p a r a t i v e s e q u e n c e a n a l y s i s o f C P s o f C N V , M N S V , C L S V , R C N M V and several other related but non-transmissible v i r u s e s . T h e data s h o w that the C P s o f these f o u r v i r u s e s are m o r e e v o l u t i o n a r i l y c l o s e l y r e l a t e d to e a c h other than t h e y are to a n y o f the o t h e r v i r u s C P s c o m p a r e d . M o s t plant viruses require a C P for efficient long distance m o v e m e n t (Carrington et al, 1996). H o w e v e r , c e r t a i n v i r u s e s , s u c h as the t o m b u s v i r u s e s ( M c L e a n et al, 1 9 9 3 ; S c h o l t o f et al, 1993; D a l m a y et al, 1992), c a n m o v e ( a l t h o u g h l e s s e f f i c i e n t l y ) w i t h o u t a C P . A n i n v e s t i g a t i o n i n t o the r o l e o f the C L S V C P g e n e i n v i r u s m o v e m e n t w a s a l s o c o n d u c t e d as part o f a l a r g e r g o a l o f a s s e s s i n g the r o l e o f the C P i n aspects o f the C L S V m u l t i p l i c a t i o n c y c l e . T h e s e results are d e s c r i b e d i n the l a s t part o f this chapter.  4.1.1 Properties of O. bornovanus transmitted viruses used in this study  4.1.1.1 Cucumber leaf spot virus  C L S V , o r i g i n a l l y c l a s s i f i e d as a c a r m o v i r u s ( R u s s o et al, 1994) has r e c e n t l y b e e n r e - c l a s s i f i e d as a m e m b e r o f a n e w g e n u s (Aureusvirus) o f the f a m i l y Tombusviridae ( M i l l e r et al, 1997; M a r t e l l i et al, 1998). C L S V i s k n o w n to i n f e c t s e v e r a l c u l t i v a r s o f 82  Cucumis sativis ( c u c u m b e r s ) i n a d d i t i o n t o a w i d e r a n g e o f h e r b a c e o u s h o s t s b u t is f o u n d  n a t u r a l l y o n l y i n c u c u m b e r s ( W e b e r , 1986). Y o u n g l e a v e s o f C L S V - i n f e c t e d c u c u m b e r s are l i g h t g r e e n t o y e l l o w , c o n t a i n i n g i r r e g u l a r l y s h a p e d c l e a r i n g s w i t h b r o w n n e c r o t i c centres. P l a n t s i n f e c t e d w i t h C L S V b e c o m e s e v e r e l y s t u n t e d a n d e v e n t u a l l y d i e ( W e b e r , 1986). C L S V v i r i o n s are i c o s a h e d r a l i n s h a p e a n d m e a s u r e 2 8 n m i n d i a m e t e r ( W e b e r , 1986). T h e y are c o m p r i s e d o f a s i n g l e C P o f 4 1 k D a a n d c o n t a i n p o s i t i v e - s e n s e , s i n g l e s t r a n d e d R N A o f 4.4 k b ( M i l l e r et al., 1997) a n d are t r a n s m i t t e d i n t h e in vitro m a n n e r b y z o o s p o r e s o f O. bornovanus ( C a m p b e l l et ah, 1991). 4.1.1.2 Melon necrotic spot virus  M N S V is a c a r m o v i r u s w i t h a p p r o x i m a t e l y 3 0 n m s p h e r i c a l p a r t i c l e s w h i c h is t r a n s m i t t e d i n t h e in vitro m a n n e r b y z o o s p o r e s o f O. bornovanus ( F u r u k i , 1981; R i v i e r e et al., 1989; C a m p b e l l et al., 1991). M N S V c a u s e s s i g n i f i c a n t l o s s o f y i e l d o f g r e e n h o u s e g r o w n m e l o n s a n d c u c u m b e r s ( R i v i e r e a n d R o c h o n , 1990). A s m e n t i o n e d a b o v e , the C P s e q u e n c e o f M N S V is k n o w n ( R i v i e r e et al., 1989) a n d s h o w s s i g n i f i c a n t s e q u e n c e s i m i l a r i t y w i t h that o f C N V . 4.1.1.3 Red clover necrotic mosaic virus  R C N M V is a d i a n t h o v i r u s w h i c h i n f e c t s c a r n a t i o n . D i a n t h o v i r u s p a r t i c l e s are isometric, 33 n m i n diameter and consist o f a single capsid protein o f approximately 37 k D a . V i r i o n s e n c a p s i d a t e t w o m a j o r g e n o m i c ( g ) R N A s p e c i e s ; R N A 1 c o n t a i n s the o p e n r e a d i n g f r a m e ( O R F ) f o r t h e C P ( X i o n g a n d L o m m e l , 1989). R C N M V is t r a n s m i t t e d i n the in vitro m a n n e r b y z o o s p o r e s o f O. bornovanus ( C a m p b e l l , 1996).  83  4.1.1.4 Cucumber necrosis virus  P r o p e r t i e s o f C N V w e r e p r e v i o u s l y d e s c r i b e d (see s e c t i o n 1.3).  4.2 Results  4.2.1 Comparative sequence analysis of the CNV, MNSV, and RCNMV coat proteins  The C P a m i n o acid sequences of several small spherical plant viruses were c o m p a r e d to d e t e r m i n e i f those v i r u s e s k n o w n to b e t r a n s m i t t e d b y O. bornovanus (i.e. C N V , C L S V , M N S V , a n d R C N M V ) share features i n c o m m o n . A d e n d o g r a m ( F i g u r e 4.1) b a s e d o n these a l i g n m e n t s suggests that the C P s o f three o f the f o u r v i r u s e s ( C N V , C L S V , a n d M N S V ) t r a n s m i t t e d b y O. bornovanus are e v o l u t i o n a r y m o r e c l o s e l y r e l a t e d to o n e another than to a n y o f the o t h e r ten v i r u s C P s c o m p a r e d . It i s s u r p r i s i n g that R C N M V d o e s n o t c l u s t e r w i t h the o t h e r O. bornovanus t r a n s m i t t e d v i r u s e s i n the d e n d o g r a m ( F i g u r e 4.1). In a seperate d e n d o g r a m u t i l i z i n g a m i n o a c i d s e q u e n c e s f r o m the C P P - d o m a i n s o n l y , i t w a s f o u n d that C N V , C L S V , M N S V , a n d R C N M V w e r e present i n the s a m e c l u s t e r b u t seperate f r o m the o t h e r v i r u s e s u s e d i n the a n a l y s i s ( D . Rochon, personal communication).  T h e C P s o f T N V - A , w h i c h i s t r a n s m i t t e d b y O.  brassicae, a n d Oat chlorotic stunt virus ( O C S V ) , w h i c h has b e e n s u g g e s t e d to b e  t r a n s m i t t e d b y Olpidium ( B o o n h a m et al., 1995; C a m p b e l l , 1996), s h o w o n l y a d i s t a n t e v o l u t i o n a r y r e l a t i o n s h i p to C L S V , M N S V , a n d C N V . A s w i l l b e d i s c u s s e d , the o v e r a l l sequence similarity b e t w e e n C N V , C L S V , and M N S V reflects their similar transmissions.  84  T a b l e 4.1 V i r u s n a m e , g e n u s , a c c e s s i o n n u m b e r a n d r e f e r e n c e f o r v i r u s e s u s e d i n t h e c o a t p r o t e i n s e q u e n c e a l i g n m e n t s a n d d e n d o g r a m f o r F i g u r e s 4.1, 4.2, a n d 4.3. Virus  Genus Tombusvirus  Accession N u m b e r S05457  Reference G r i e c o etal., 1 9 8 9  Aureusvirus  CAB59794  R u b i n o etal., 1 9 9 5  PI 1689  Hillmanera/., 1989  Tombusvirus  P14836  Grieco and Gallitelli, 1990  Cucumber necrosis Tombusvirus virus ( C N V ) Cucumber leaf spot Aureusvirus virus ( C L S V ) Melon necrotic spot Carmovirus virus ( M N S V ) Red clover necrotic Dianthovirus mosaic virus  P15183  Rochon and Tremaine, 1989 M i l l e r etal, 1 9 9 7  P19899  R i v i e r e s ai, 1 9 8 9  P22955  Xiong and Lommel, 1989  Carmovirus  AAA96971  Carmovirus  P04383  C a r r i n g t o n et al., 1989a G u i l l e y e r a / . , 1985  Unclassified  CAA58798  Necrovirus  P22959  Machlomovirus  PI 1642  3  Cymbidium ringspot  virus ( C y R S V ) Pothos latent virus  (PoLV)  Tomato bushy stunt Tombusvirus virus, cherry strain  (TBSV-Ch) Artichoke mottle crinkle virus  (AMCV)  (RCNMV) Turnip crinkle virus  (TCV) Carnation mottle  virus ( C a r M V ) Oat chlorotic stunt virus ( O C S V ) Tobacco necrosis virus strain A  (TNV-A) Maize chlorotic mottle virus  (MCMV) 'The  virus name is followed by its acronym in parenthesis.  85  B o o n h a m et al., 1995 M e u l e w a e t e r et al., 1990 N u t t e r etal, 1 9 8 9  CyRSV CP (Tombus)  PoLV CP (Aureus)  ~"  I  .  I  I  .  AMCV CP (Tombus)  I  TBSV-Ch CP (Tombus)  *CNV CP (Tombus)  .  *MNSV CP (Carmo)  I  *CLSV CP (Aureus)  *RCNMV CP (Diantho)  .  CarMV CP (Carmo)  I  TCV CP (Carmo)  OCSV CP (Unclassified)  •  MCMV CP (Machlomo)  TNV-A CP (Necro)  F i g u r e 4.1 D e n d o g r a m d e p i c t i n g r e l a t i o n s h i p s a m o n g the C P s o f s e v e r a l i c o s a h e d r a l v i r u s e s . B r a n c h l e n g t h s are p r o p o r t i o n a l to the d i s t a n c e s b e t w e e n the r e s p e c t i v e sequences. See T a b l e 1 for full virus names, accession numbers and references. T h e asterisks i n d i c a t e the f o u r v i r u s e s k n o w n to b e t r a n s m i t t e d b y O. bornovanus. T h e d e n d o g r a m was o b t a i n e d u s i n g " G r o w t r e e " a v a i l a b l e t h r o u g h " G e n e w o r k s " .  T h e a l i g n m e n t u s e d to p r o d u c e the d e n d o g r a m s h o w n i n F i g u r e 4.1 ( F i g u r e 4.3) was e x a m i n e d i n o r d e r to f u r t h e r assess s p e c i f i c s e q u e n c e r e l a t i o n s h i p s a m o n g the f o u r O. bornovanus t r a n s m i t t e d v i r u s e s . It c a n b e s e e n that there are s e v e r a l r e g i o n s i n the  86  a l i g n m e n t w h e r e s e q u e n c e s i m i l a r i t y (at least three o u t o f f o u r r e s i d u e s , see a s t e r i s k s ) o c c u r s a m o n g C N V , C L S V , M N S V a n d R C N M V b u t not g e n e r a l l y a m o n g the n i n e o t h e r v i r u s e s e x a m i n e d . O n e s o m e w h a t s t r i k i n g r e g i o n o c c u r s i n the P - d o m a i n , the d o m a i n o f the t o m b u s v i r u s c o a t p r o t e i n w h i c h is least c o n s e r v e d ( R i v i e r e et al., 1989). I n t e r e s t i n g l y , this r e g i o n c o r r e s p o n d s to a l o o p a n d its f l a n k i n g r e g i o n s at a distal p o r t i o n o f the Pd o m a i n . M o r e o v e r , a C N V t r a n s m i s s i o n m u t a n t r e c e n t l y i d e n t i f i e d i n t h i s l a b o r a t o r y (K. K a k a n i a n d D . R o c h o n , p e r s o n a l c o m m u n i c a t i o n ) m a p s to a s e r i n e r e s i d u e i n a n a d j a c e n t l o o p i n the C N V particle. T h i s t r a n s m i s s i o n m u t a n t a l s o fails to b i n d to O. bornovanus z o o s p o r e s i n in vitro b i n d i n g assays. T h e s e o b s e r v a t i o n s s u g g e s t that t h i s r e g i o n o f the C N V p a r t i c l e ( a n d p e r h a p s C L S V , M N S V , a n d R C N M V p a r t i c l e s ) m a y c o r r e s p o n d to a v i r u s attachment site.  87  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  ME NKGEKIAMNPTVQTLAQKGDKLAVKL VTR--GWASLSTNQKR ME NDPRVRKFASDGAQWAIKWQKKGWST LT SRQKQTARAAM MAMVKRN1SDSITGLIPVSTKQLMALGAAAGASALQGFVRNNGAAIVGKWDV MAMVKRNNNTGMIPVSTKQLLALGAAAGATALQGFVKNNGMAIVEGAVDL MAMTTTNNNRAMTRAAKQALPALGALATSGMGQQLF MSGVNYAIEK MALVSRNNNMRTLAKLAAPLATAGTRTIVDNKEAIW--NGVKWIWGKLPK MAMVKRINNLPTVKLAKQALPLLANPKLVNKAIDW- - PLWQGGRKLSK ME - IARTNKNS WKYVPAAVG-AAYQMGKSIVPYAP - - TIVDALGNWSR MS - - SKAPKKSKQRSQPRN RTPNT MALVKRNNNMALIASEAGAAIKAGQVMLSPAGRELIW-NGVNWVRRKLGR MTLNLRKVPAYLPGKVDGALTNLVHAAVDHWPGLGKAEKAAAVYNIKQV MAASSRSTRGR KQRGRSVEAKSRAIR ANPPVPR P MAGKKNNNNGQYIILRTPEQQVEIDQRNARRAQMGRMKKARQPVQRYLQQ MA...R.NN L -V  42 41 50 50 46 48 48 46 22 49 50 34 50 50  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  RAE ML. -AGYTPA ILAFTPRRPRMTNP GIK QKVTRLSAPVALAY LS- -PVAQPV GQK GGTGGAIMA-VAVTR - VYKAVKKRGGKKQ- -QQIKHV TKR GGTGGAIMAPVAVTR -AYKAVRRRGGKKQ- -QMINHV GKQ - -F-VPNRRGGKKN- -RNTDMVAHPGALSGSMAAPVAISR GKK GKNG--NG- -ALIAHP QAFPGAIAAPISYAY AAK RLLGAYGG- -NISYTE GAKPGAISAPVAISR ATG RKKKSKGK- -EVQNQI VGGIGAIAAPVSITK SVK TVAIPFAKTQ SKK S--DVILHV LPGAIAAPVANTR VKKLGTYTEQGVKKIAKKTLGELGYLNYTPSSHLGMAITGRGTKQINMSR NPQRN R PPPAGTTCSMSEI--LLAVSATT ADQILEIPV HGLRNGLSGRGGYIVAPTSGGWTRPIVPKFSNRGDSTIVRNTEILNNQI .K...G..... G. . .APVA. .  67 66 86 87 84 78 80 78 35 72 100 70 100 100  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  PPRTSRNS PG-QAGKSMTMSKTREVSTQPRVS-TARDGITRSGSQLTGSKPKFTGKTSGSVTVTHRQLVGSKPKFTGRTSGSVTVTHRIVRGSKPRFI-RSKGSVTITHRAVKGRKPRFQ-TAKGSVRITHR-  ELLST- -VKGTTG --VI ELITT- -LKKNTD --TE EYLSQ- -VNMSTG-F--QVN EYLSQ- -VNNSTG-F--QVN ELVGQ- -FNSSSA-L--WN EYVSV- -LSGTNGEF--LRN EFIAS- -VLP-SSDL--TVN RVRGMRPSFR-QTKGKVRIVHR ELVTS- -VINLVGNF--RVN IIKTVNP PPKPARGILHT QLVMS- -WGSVQ-M--RTN IIRASKPKFT-RSKGSVTIAHR ELLGQ- -FS--SG-L--WN STNAGGFALGGTTAAPVSISRNINRRSKPSIKMMGDAWISHSEMLGAIN CAGID-FPAGTSPRYIGAAKWLAAQSQMWNTIVFNSVRITWETFTADTTS LAALGAFNTTNSALIAAAPSWLASIADLYSKYRWLSCEIIYIPKCPTTTS P.F.-.. .GSV.I.HR E --V -.-.VN  101 100 123 124 120 115 116 115 68 106 150 119 150 150  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  PSF - EDWWS PRNVAVFPQLSLLATNFNKYRITALTVKYS P PKY - TTAVLNP SEPGTFNQLIKEAAQYEKYRFTSLRFRYSP GGI-VG-NLLQLNPLNGTLFSWLPAIASNFDQYSFNSVLLHYVP GGI-VG-NLLQLNPLNGTLFSWLPAIASNFDQYTFNSWLHYVP GGI-TG-NLYKINPANAVLFPWLQTLASNFDQYMFNTLRLQYVP NGTGPN NDFSINPLNPFLFPWLVNIAANFDQYKFNSLRFEYVP NG-DVN IGKYRVNPSNNALFTWLQGQAQLYDMYRFTRLRITYIP NNVSAQ IGQFRINPSNSSLFTWLPTIASNFDSYRFTSIRFVYVP NGK-SN QRFRLNPSNPALFPTLAYEAANYDMYRLKKLTLRYVP GGV-SG NVYRINPSNP WFPWLQGIAASFDQYKFDQ - - LQYVP SGTPSSNVTAFRCTGYRANPGMSTIFPWLSATAVNYEKYKFRRLSFTLVP GYISMAFLSDYMLSIPTGVEDVARIVPSATI-ALKNRGPSIVMPQNRTVF GSIAMAFTYDRNDAAPTARAQLSQSYKAINFPPYAGYDGAAYLNSNQGAG . G. - . . NP.N.. LFPWL ...A.NFD.Y.F..L... YVP  141 140 165 166 162 158 159 159 110 146 200 168' 200 200  S->*  88  ** *  **  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  ACSFETNGRVALGFNDDASDTPPTTKVGFYDLGKHVETAAQTAKDLVIPV MSPSTTGGKVALAFDRDAAKPPPNDLASLYNIEGCVSSVPWTGFILTVPT LCATTEVGRVAMYFDKDSEDPEPADRVELANYSVLAETAPWAERALWVPT LCSTTEVGRVAIYFDKDSEDPEPADRVELANYSVLKETAPWAEAMLRVPT MCATTETGRVAIYFDKDSQDLEPVDRIELANMRHLTETAPWCEGSLRVPV LVNTTTNGRVALYFDKDSEDPGPDDRAALANYAHLSEISPWAITKLTVPT TTGSTSTGRVSLLWDRDSQDPLPIDRAAISSYAHSADSAPWAENVLWPC LCATTETGRVSLFWDKDSQDPLPVDRAALSSYGHSNEGPPWAETTLNVPT LVTVQNSGRVAMIWDPDSQDSAPQSRQEISAYSRSVSTAVYEKCSLTIPA MCATTETGRVAIYFDKDSQDVEPADRDELAIMAH--ESAPWCESTLSIPV LVSTNYSGRIGVGFDYDSSDLVPGNRQEFYALSNHCENMPWQESTVEIKC RCIQAG-QFAALG SAADKQMYSPGRFIVAIPKASATQAVGQIKISY SAIAVQLDVTKLDKPWYPTISSAGFGALSVLDQNQFCPASLWASDGGPA .C.TT..GRVAL.FD.DS.D..P.DR..L E.APW.E..L.VP.  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP MCMV CP TNV-A CP Consensus  DGKTRFIRDSASDDAKLVDFGRIVL- STYGFDKADTV--VG--ELFIQYT DSTDRFVADGIS-DPKLVDFGKLIM- ATYGQGANDAAQLG- -EVRVEYT DRIKRFCDDSSTLDHKLIDLGQLGV- ATYG--GAGTNA--VG--DIFISYS DKIKRFCDDSSTSDHKLIDLGQLGI- ATYG--GAGTNA--VG--DIFISYS DSVKRFMNDNSTVDPKLIDLGQVGL- ATYG--GPGTNA--VG--DLFIHYT DNVKRFISDTSSGDPKLINLGQFGW- VAYS--GP-TAE--LG- -DIFVEYT DNTWRYMNDTNAVDRKLVDFGQFLF- ATYS--GAGSTA--HG- -DLYVEYA DGKQRFVTDSNTTDRKLVDLGQFAF- ATYA--GGSNNQ--IG- -DIYVEYG DNQWRFVADNTTVDRKLVDFGQLLF- VTHS--GSDGIE--TG- -DIFLDCE DNIKRFMNDNTTTDPKLVDLGQIGL- ATGG--S--TNP--VG--DLFIHYT DNAYRFTGTHVAADNKLIDLGQVWMSDSVSNGGTISAALPLFDLIVNYT SVSYRGAAILQ--PALV-PGPGLANH 236 TATPAGDLFIKYVIEFIEPINPTMNV 27 6 D...RF..D....D.KL.DLGQ. -ATY.-G -.G--D.F. .Y.  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP Consensus  IVLSDPTKT -AKIS QASN-DKVSDG-PTYW-PSVNG-NELVQLKNRTGS -TSDA QIGQFAGVKDG-PRLVSWSKTKGTAGWVTLYFPQPT -NTLLS--TRRLDLAGTPVTASG-PGYI--LLTRTPTVLVTLYFPQPT -NTLLS--TRRLDLAGALVTASG-PGYL--LVSRTATVLVTFYEPQPS SGLTS--T--LQ-TGTGSANAG-PTLV--AVATTATTTVDLFEAQPT SPLLE--SLFRESASSVQTRMGLPYFS--LEVASATDLVEFKDPQPI AGMVCMFDRLVSLSEVGSTIKGVNYIADRDVITTGGNIVEFSEAQPA GGLTQYITK-- SVGATAST-TGPSYWDANINVNATTAVEFKGPQPT ASIVQ--KTVIDLGGTLTSFEGPSYLMPPDAFITSSSFITLFEPQPL ASLVE - - T EQTGTGAAPFG LV- - TVS SNAT IT VELIEPQQALFSSQLYSGSTTFTSGIPLGTGADTTTWGPTWNSTTVTN V.L. .PQP.--G....V T..-  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP Consensus  -QLRWAA-GKWCII-VRGTVE--GGFTKPTLIG—PGISGDVDYESARP -EHDCHFL-GTGNFS-LTLFYEK-APVSGLENAD--ASDFSVLGEAAAGS -TMTFRAT-GTFVIS-GAYRCLTSTVLGLTGGVN—VNSITWDNVGTSS -TMTFRAT-GTFVIS-GTYRCLTATTLGLAGGVN--VNSITWDNIGTDS -TVTFRSP-GTYLVS-MVQRATTFTGVT-PIALT--FNSNTNTTAAGTNY -VWQARVP-GTYWT-IIFNSTVGGLTPSISGGG—TINSSFSVSTAGSS -GVNINIP-GTYLVT-IVLNATSIGPLTFTGNSKLVGNSLNLTSSGASAL -NVEFFSP-GTFLIT-AWYGSTIASPSMAGGNGTLIGDLPWGGSNASI -GLFVDVA-GTYLLT-LWTCSTTGSVTVGGNSTLVGDGRAAYGSSNYIA -IYTFEGP-GVYLLA-LSQRAASFTTFVTAGGVN--SHTTTITSGPAYQS CWTFKLPAGVFEVSYFIAWSTGTAAWPTVPTTGAGSKLSNTSTGSNSY -GT. . . . —  315 318 346 347 339 339 347 344 296 318 400 400  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP Consensus  IAVCELV-TQ MEGQILKIT--KTSAEQPL QWWYRM VQWAGVK-VA ERGQGVKMV--TTEEQPKG KLQALRI SFFINCT-VS NLPSVITFT-TTGITSATI QCNRRTRQNDVS AFFINCT-VS NLPSWTFT-STGITSATV HCVRATRQNDVS SANYNVT-VP VPGAQMRFV-GTGFGNYTL QVTRAKITNAAT AYVANIT-IR VNANLSLSG-LTGATNAQL FAVRAITENAVQ TFTLNST-GV PNSSDSSFSVGTWALTRV RMTITRCSPETA WTCVFST-TG VSTSVPTFT-QAGTGLTRV QYTITRVNSQTA SIVFTSSGVL STTPSVQFSGSSGVSRVQM NICRCKQGNTFI ICEYYCC-YT WGS—ITYN-GTLFGNYTL TRAKISNNAT GVCFINSPVECDLLLTATVLLIIPTLPSSTCVFHAPARRCTTPMCHRLLT -V. -.T  348 351 385 386 378 378 387 383 337 353 450 450  *  *  *  191 190 215 216 212 208 209 209 160 194 250 213 250 250  236 235 259 260 256 251 253 253 204 236 300  300  P->  89  272 274 301 302 295 294 300 297 249 273 350 350  CarMV CP TCV CP AMCV CP TBSV-Ch CP CyRSV CP CNV CP MNSV CP CLSV CP RCNMV CP PoLV CP OCSV CP Consensus  -LI-LI-LL-WYLAYQV-LG-LISLAG -L. -  348 351 387 388 380 380 390 386 339 355 454 454  Figure 4.2 A l i g n m e n t o f the C P s o f s e v e r a l s m a l l s p h e r i c a l v i r u s e s u s i n g p r o g r a m s  a v a i l a b l e t h r o u g h G C G ( D e v e r e u x et al., 1984). A s t e r i s k s (*) i n d i c a t e aa c o n s e r v e d (at least three o f f o u r ) a m o n g the O. ^ornova/zMS-transmitted v i r u s e s ( C N V , C L S V , M N S V , a n d R C N M V ) b u t not g e n e r a l l y s h a r e d a m o n g the o t h e r n o n - t r a n s m i t t e d v i r u s e s . S e e T a b l e 4.1 f o r f u l l v i r u s n a m e s , a c c e s s i o n n u m b e r s a n d r e f e r e n c e s . T h e b o l d letters f o l l o w e d b y a r r o w s i n d i c a t e the b e g i n n i n g s o f the R N A b i n d i n g d o m a i n ( R ) , the a r m (a), the s h e l l d o m a i n (S) a n d the p r o t r u d i n g d o m a i n (P).  CNV CP MNSV CP CLSV CP RCNMV CP Consensus  --AYVANITI FTLNSTGVPN TCVFSTTGVS SIVFTSSGVL  RVNANLSLSG SSDSSFSVGT TSVPTFT-QA STTPSVQFSG  LTGATNAQLF WALTRVRMT GTGLTRVQYT SSGVSRVQMN G.TRVQ. .  AVRAITENAV ITRCSPETAY ITRVNSQTAY ICRCKQGNTF I.R A.  QW -LA -QV ILG ...  380 3 90 3 86 339 393  Figure 4.3 A l i g n m e n t o f the C P a m i n o a c i d s e q u e n c e s o f f o u r O. bornovanus-  transmitted viruses ( C N V , M N S V , C L S V , and R C N M V ) using programs available t h r o u g h G C G ( D e v e r e u x et al., 1984) s h o w i n g the t e r m i n a l r e g i o n o f the C P ( P - d o m a i n ) . S i m i l a r a m i n o a c i d s (at least three o f f o u r ) w h i c h d o not o c c u r i n the a l i g n m e n t i n F i g u r e 4.2 are h i g h l i g h t e d i n b o l d type. S e e T a b l e 4.1 f o r f u l l v i r u s n a m e s , a c c e s s i o n n u m b e r s a n d references.  4.2.2 The CLSV coat protein is not required for cell-to-cell movement but is required for systemic movement in A , benthamiana. 7  A f r a m e s h i f t m u t a t i o n w a s i n t r o d u c e d i n the 5' t e r m i n a l r e g i o n o f the C L S V C P O R F ( C L S V nt 2 5 9 1 ) i n a f u l l - l e n g t h i n f e c t i o u s C L S V c D N A c l o n e , J R 3 , to p r o d u c e m u t a n t J R 3 A X h o . T h e m u t a t i o n r e s u l t s i n a t r u n c a t e d (81 aa) C P . F o l l o w i n g i n o c u l a t i o n o f J R 3 a n d J R 3 A X h o T 7 R N A p o l y m e r a s e r u n - o f f transcripts o n t o N. benthamiana, p r o g r e s s i o n o f s y m p t o m s w a s o b s e r v e d o v e r the c o u r s e o f 2 4 days. W i l d - t y p e C L S V 90  ( J R 3 ) p r o d u c e d faint c h l o r o s i s o n i n o c u l a t e d l e a v e s t w o d a y s p o s t - i n o c u l a t i o n ( d p i ) w i t h c h l o r o t i c l e s i o n s a p p e a r i n g three d p i . C h l o r o t i c l e s i o n s w e r e v i s i b l e o n J R 3 s y s t e m i c a l l y infected leaves (not inoculated) six d p i a c c o m p a n i e d b y severe stunting and necrosis o f the p l a n t at 12 d p i a n d d e a t h b y 2 4 d p i ( F i g u r e 4.4 A , B, a n d C ) . In three s e p a r a t e e x p e r i m e n t s i t w a s f o u n d that the C P f r a m e s h i f t m u t a n t J R 3 A X h o p r o d u c e d c h l o r o s i s o n the i n o c u l a t e d l e a v e s w i t h i n t w o to three d p i s i m i l a r to J R 3 b u t n o s y s t e m i c s y m p t o m s o c c u r r e d as late as 2 4 d p i ( F i g u r e 4.4 A , B, a n d C ) . Interestingly, b l a c k - r i m m e d n e c r o t i c l e s i o n s w e r e o b s e r v e d o n the i n o c u l a t e d l e a v e s o f J R 3 A X h o at s i x d p i b u t n o t o n J R 3 i n o c u l a t e d leaves, w h i c h s t i l l d i s p l a y e d c h l o r o t i c l e s i o n s ( F i g u r e 4.5 A a n d B ) . J R 3 A X h o - i n f e c t e d p l a n t s d i s p l a y e d n o n e o f the s t u n t i n g o r d e a t h s e e n i n J R 3 i n f e c t i o n s ( F i g u r e 4.4 C ) , a s i d e f r o m d e a t h o f the o r i g i n a l t r a n s c r i p t - i n o c u l a t e d l e a v e s ( F i g u r e 4.4 B a n d C ) . S a p f r o m J R 3 A X h o - i n o c u l a t e d l e a v e s w a s i n f e c t i o u s w h e n p a s s e d to n e w  N.  benthamiana plants, a l t h o u g h f e w e r l e s i o n s w e r e o b s e r v e d f r o m J R 3 A X h o as c o m p a r e d  to J R 3 a n d n o s y s t e m i c i n f e c t i o n o c c u r r e d ( n o t s h o w n ) .  91  F i g u r e 4.4 S y m p t o m s o n N. benthamiana i n o c u l a t e d w i t h transcripts o f J R 3 (left pot) J R 3 A X h ( r i g h t pot) at ( A ) s i x d p i , ( B ) 12 d p i , a n d ( C ) 24 dpi. 0  92  Figure 4.5 S y m p t o m s o n K benthamiana o f l e a v e s i n o c u l a t e d w i t h transcripts o f ( A ) J R 3 A X h o o r ( B ) J R 3 ( w i l d - t y p e C L S V ) . P h o t o g r a p h s w e r e t a k e n s i x dpi.  93  Total R N A was extracted from inoculated and systemically infected leaves of JR3 and JR3AXho-inoculated N. benthamiana plants and elecrophoresed through agarose gels (Figure 4.6). Genomic length (g)RNA was readily observed in both inoculated plants (Figure 4.6) demonstrating that JR3AXho infects and moves cell-to-cell in transcriptinoculated leaves. JR3 gRNA was observed in total R N A extracts of systemically infected leaves of JR3 transcript-inoculated plants but JR3AXho gRNA was not detected in total R N A extracts of similar leaves of plants inoculated with JR3AXho transcripts (Figure 4.6). These results demonstrate that the C L S V CP is not required for cell-to-cell movement but is required for systemic movement. Similar observations were made following sap transmission of JR3AXho-transcript-inoculated leaves (not shown).  JR3  mock  tx  JR3AXho  syst  tx  syst  Figure 4.6 Agarose gel of total R N A extracted from single leaves of N. benthamiana. Equal volumes of total leaf R N A extracted from a single leaf were loaded in wells. Lane 1 contains total R N A from a mock-inoculated leaf. Lanes 2 and 3 contain total R N A from JR3 transcript-inoculated and systemically infected leaves, respectively. Lanes 4 and 5 contain total R N A from JR3AXho transcript-inoculated and systemically infected leaves, respectively. The arrow indicates the location of the viral genomic (g)RNA band.  94  4.3 Discussion  4.3.1 Comparative sequence analysis  In t h i s c h a p t e r c o m p a r a t i v e a m i n o a c i d s e q u e n c e a n a l y s i s h a s s h o w n that t h e C P s o f three O. bornovanus-transmitted v i r u s e s ( C N V , C L S V , a n d M N S V ) a r e m o r e s i m i l a r to e a c h o t h e r t h a n t o t h e t e n o t h e r s m a l l s p h e r i c a l v i r u s e s u s e d i n t h e p h y l o g e n e t i c a n a l y s i s ( M i l l e r et al, 1997; F i g u r e 4.1). T h e c o n s e r v a t i o n o f s e q u e n c e a m o n g t h e C P s o f these three v i r u s e s i s p a r t i c u l a r l y s t r i k i n g i n v i e w o f the a f f i n i t i e s o f these v i r u s e s w i t h d i s t i n c t g e n e r a i n t h e f a m i l y Tombusviridae ( T a b l e 4.1). T o e x a m i n e f u r t h e r t h e s e q u e n c e s i m i l a r i t y that o c c u r s b e t w e e n C N V , M N S V , C L S V , and R C N M V , a n a m i n o acid sequence alignment w a s performed using the C P a m i n o a c i d s e q u e n c e s o f t h i r t e e n s m a l l s p h e r i c a l p l a n t v i r u s e s f r o m t h e Tombusviridae ( F i g u r e 4.2). S e v e r a l r e g i o n s o f a m i n o a c i d s e q u e n c e s i m i l a r i t y w e r e i d e n t i f i e d , i n c l u d i n g a r e g i o n i n t h e o t h e r w i s e v a r i a b l e p r o t r u d i n g d o m a i n . T h e c o n s e r v e d r e g i o n i n t h e Pd o m a i n was previously identified f r o m a m i n o acid sequence alignments between the C P s o f C N V a n d M N S V ( R i v i e r e et al, 1989). A n e x a m i n a t i o n o f t w o - d i m e n s i o n a l s t r u c t u r a l representations o f C N V a n d M N S V , based o n the k n o w n T B S V P-domain structure (see s e c t i o n 1.3.5), i n d i c a t e d that t h e a m i n o a c i d s c o r r e s p o n d i n g t o t h e c o n s e r v e d r e g i o n o c c u r i n a l o o p a n d f l a n k i n g r e g i o n s i n t h e P - d o m a i n s o f these v i r u s e s . M u t a t i o n s w e r e m a d e i n this r e g i o n i n C N V c h a n g i n g a S e r t o A l a ( C N V C P a a 3 1 2 ) a n d a T h r t o A l a ( C N V C P a a 3 1 3 ) i n t w o separate C N V c D N A c l o n e s ( M . R o b b i n s , u n p u b l i s h e d results). H o w e v e r , C N V p a r t i c l e s c o n t a i n i n g these t w o m u t a t i o n s a p p e a r e d t o b e t r a n s m i t t e d as e f f i c i e n t l y as w i l d - t y p e C N V . It w o u l d b e i n t e r e s t i n g t o p e r f o r m f u r t h e r m u t a g e n e s i s o f these a m i n o  95  a c i d s a n d others i n this r e g i o n i n o r d e r to f u r t h e r assess t h e i r r o l e i n f u n g u s t r a n s m i s s i o n . F o r e x a m p l e , the S e r a n d T h r c o u l d b e m u t a t e d e i t h e r to G l y ( r e m o v i n g the s i d e - c h a i n s ) o r to c h a r g e d o r a r o m a t i c a m i n o a c i d s t o alter the p u t a t i v e i n t e r a c t i o n s w i t h a z o o s p o r e receptor. A n o t h e r r e g i o n o f a m i n o a c i d s e q u e n c e s i m i l a r i t y b e t w e e n the P - d o m a i n s o f the O. £>0ra0vanws-transmitted v i r u s e s w a s i d e n t i f i e d f r o m a m i n o a c i d s e q u e n c e a l i g n m e n t s o f these f o u r v i r u s e s a l o n e ( G . T R V Q ) ( F i g u r e 4.3). B a s e d o n a m i n o a c i d s e q u e n c e c o m p a r i s o n s b e t w e e n C N V a n d T B S V , this s e q u e n c e is l o c a t e d i n a l o o p o n the Pd o m a i n , a g a i n adjacent to the t w o other i d e n t i f i e d l o o p s . T h e f a c t that t h i s r e g i o n o f s i m i l a r i t y is n o t o b s e r v e d i n the a l i g n m e n t c o n t a i n i n g the 13 m e m b e r s o f the Tombusviridae i n d i c a t e s that c a u t i o n s h o u l d b e t a k e n i n i n t e r p r e t a t i o n o f the results.  M u t a g e n e s i s o f this r e g i o n w o u l d h e l p e s t a b l i s h its r o l e i n f u n g u s t r a n s m i s s i o n .  4.3.2 Role of the CLSV CP in virus movement  T o f u r t h e r e x a m i n e the r o l e o f the C L S V C P i n the m u l t i p l i c a t i o n - c y c l e o f this v i r u s , a f r a m e s h i f t m u t a t i o n w a s i n t r o d u c e d i n t o the 5' t e r m i n a l r e g i o n o f the C P O R F . T r a n s c r i p t s o f this m u t a n t w e r e i n o c u l a t e d o n t o N. benthamiana a n d e x a m i n e d f o r t h e i r a b i l i t y to c a u s e a n i n f e c t i o n , t o m o v e c e l l - t o - c e l l , a n d to m o v e s y s t e m i c a l l y . It w a s f o u n d that l o s s o f the C P d i d n o t a f f e c t c e l l - t o - c e l l m o v e m e n t b u t d i d r e s u l t i n the i n a b i l i t y o f the v i r u s t o m o v e s y s t e m i c a l l y i n these p l a n t s ( F i g u r e 4.4 a n d 4.5). A n o t h e r Aureusvirus, Pothos latent virus ( P o L V ) , h a s b e e n r e p o r t e d to b e a b l e t o m o v e s y s t e m i c a l l y i n p l a n t s i n  the a b s e n c e o f a f u n c t i o n a l C P ( R u b i n o a n d R u s s o , 1997). I n these studies, s e v e r a l d i f f e r e n t C P - m u t a n t s o f P o L V w e r e p r o d u c e d ( R u b i n o a n d R u s s o , 1997). T h e s e m u t a n t s  96  a l l h a d the a b i l i t y t o m o v e c e l l - t o - c e l l b u t s h o w e d v a r i a b i l i t y i n t h e i r a b i l i t y to m o v e s y s t e m i c a l l y . O n e d e l e t i o n mutant, D B g / B s , c o n t a i n e d a l a r g e d e l e t i o n i n the P o L V C P O R F ( i t r e t a i n e d the C P R - d o m a i n b u t n o t the S- o r P - d o m a i n s ) . D B g / B s c o u l d i n f e c t t r a n s c r i p t - i n o c u l a t e d l e a v e s o f N. benthamiana b u t w a s u n a b l e t o m o v e s y s t e m i c a l l y ( R u b i n o a n d R u s s o , 1997). S e v e r a l o t h e r m u t a n t s w e r e p r o d u c e d w h i c h l a c k e d a Pd o m a i n o r w h i c h c o n t a i n e d m u t a t i o n s i n the S - d o m a i n . T h e s e m u t a n t s w e r e i n f e c t i o u s a n d s p r e a d s y s t e m i c a l l y i n N. benthamiana w i t h the a p p e a r a n c e o f s y s t e m i c s y m p t o m s v a r y i n g f r o m 4 t o 21 d p i d e p e n d i n g o n the m u t a n t ( R u b i n o a n d R u s s o , 1997). Interestingly, s y m p t o m s e l i c i t e d b y t h e s e P o L V C P m u t a n t s w e r e m o r e s e v e r e t h a n w i l d type, w i t h n e c r o s i s o f v a s c u l a r tissues i n d u c i n g r a p i d d e a t h o f the w h o l e plant. It w a s n o t e d that c o n c e n t r a t i o n s o f the 0.8 k b s g R N A ( w h i c h e n c o d e s t h e s y m p t o m - i n d u c i n g p r o t e i n ) w e r e h i g h e r t h a n that o f w i l d - t y p e v i r u s ( R u b i n o a n d R u s s o , 1997). It is p o s s i b l e that e x c e s s p r o d u c t i o n o f the n e c r o s i s - i n d u c i n g 14 k D a p r o t e i n w a s r e s p o n s i b l e f o r the l e t h a l p h e n o t y p e s o f t h e s e m u t a n t s ( R u b i n o a n d R u s s o , 1997). T h e J R 3 A X h o m u t a n t w a s f o u n d to p r o d u c e d a r k - r i m m e d n e c r o t i c l e s i o n s 6 d p i w h i c h w e r e s t r i k i n g l y d i f f e r e n t f r o m the u s u a l c h l o r o t i c l e s i o n s o b s e r v e d s i x d p i w i t h w i l d - t y p e i n f e c t i o n s . It m a y b e that J R 3 A X h o i n f e c t i o n s p r o d u c e m o r e o f the n e c r o s i s i n d u c i n g 14 k D a p r o t e i n , as w a s h y p o t h e s i z e d f o r C P m u t a n t s o f P o L V ( R u b i n o a n d R u s s o , 1997). T h e s e m u t a n t s w e r e u s u a l l y d e l a y e d i n their p r o d u c t i o n o f s y s t e m i c s y m p t o m s ( R u b i n o a n d R u s s o , 1997). T h e n e c r o s i s c a u s e d b y J R 3 A X h o m a y h a v e b e e n s o s e v e r e that this m u t a n t c o u l d n o t e s c a p e the n e c r o t i c a r e a t o e n t e r the p h l o e m f o r l o n g d i s t a n c e transport.  97  5 C H A P T E R  F I V E :  E V I D E N C E T H A T F U N G A L Z O O S P O R E S S P E C I F I C R E C E P T O R S  C O N T A I N  F O RT R A N S M I S S I O N  O F  C N V  5.1 Introduction Infection of cells by animal, insect and bacterial viruses requires specific cellular r e c e p t o r s that, i n part, d e t e r m i n e host r a n g e a n d t i s s u e t r o p i s m . ( M a r s h a n d H e l e n i u s , 1989; R o s s m a n n , 1994). P l a n t v i r u s e s , h o w e v e r , g e n e r a l l y g a i n e n t r y to p l a n t c e l l s t h r o u g h s p e c i f i c i n v e r t e b r a t e o r f u n g a l v e c t o r s w h i c h b r e a c h the c e l l w a l l a n d e n a b l e access o f the v i r u s to the p l a n t c y t o s o l w h e r e u n c o a t i n g a n d r e p l i c a t i o n o c c u r s ( M a t t h e w s , 1991). C e l l - t o - c e l l s p r e a d o f the v i r u s b e y o n d the i n i t i a l site o f i n f e c t i o n t h e n o c c u r s v i a p r e - e x i s t i n g i n t e r c e l l u l a r j u n c t i o n s ( f o r r e v i e w see C a r r i n g t o n et al., 1996). T h e r e are s e v e r a l i n d i c a t i o n s i n the literature that h i g h l y s p e c i a l i z e d i n t e r a c t i o n s e x i s t b e t w e e n p l a n t v i r u s e s a n d t h e i r v e c t o r s ( C a m p b e l l et al., 1 9 9 5 ; ) . In m a n y cases the s p e c i f i c i t y o f i n t e r a c t i o n has b e e n f o u n d to reside, at least i n part, i n p r o t e i n s e n c o d e d b y the v i r a l g e n o m e . S u c h p r o t e i n s i n c l u d e the v i r a l c o a t p r o t e i n , h e l p e r c o m p o n e n t , a n d c e r t a i n o t h e r n o n - s t r u c t u r a l p r o t e i n s ( f o r r e v i e w s see G r a y , 1996; P i r o n e a n d B l a n c , 1996). H o w e v e r , there h a v e o n l y b e e n a f e w reports o f the p o s s i b l e i n v o l v e m e n t o f r e c e p t o r s i n o r o n v e c t o r s w h i c h m e d i a t e t r a n s m i s s i o n . G i l d o w (1993), u s i n g t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y , s h o w e d that r e c e p t o r m e d i a t e d e n d o c y t o s i s m a y b e i n v o l v e d i n b a r l e y y e l l o w d w a r f l u t e o v i r u s m o v e m e n t i n t o a p h i d gut cells. In a d d i t i o n , B a n d l a et al. ( 1 9 9 8 ) h a v e r e c e n t l y i d e n t i f i e d a p r o t e i n f r o m the t h r i p s m i d g u t w h i c h is a p o t e n t i a l r e c e p t o r f o r  98  t o m a t o s p o t t e d w i l t t o s p o v i r u s . S y m b i o n i n , a h o m o l o g u e o f the Escherichia coli G r o E L c h a p e r o n i n p r o t e i n w h i c h is h i g h l y a b u n d a n t i n a p h i d v e c t o r h e m o l y m p h , has b e e n s h o w n to b i n d l u t e o v i r u s e s a n d p l a y a c r u c i a l r o l e i n t r a n s m i s s i o n . H o w e v e r , s y m b i o n i n d o e s n o t a p p e a r to p l a y a r e c e p t o r r o l e i n t r a n s m i s s i o n ( v a n d e n H e u v e l et al.,  1997).  Cucumber necrosis tombusvirus ( C N V ) is o n e o f s e v e r a l s m a l l i c o s a h e d r a l v i r u s e s  i n the f a m i l y Tombusviridae k n o w n to b e t r a n s m i t t e d b y Olpidium spp. ( C a m p b e l l ,  1996).  T r a n s m i s s i o n o c c u r s f o l l o w i n g i n d e p e n d e n t r e l e a s e o f z o o s p o r e s a n d v i r u s i n t o the s o i l a n d s u b s e q u e n t a d s o r p t i o n o f v i r u s p a r t i c l e s o n t o the s u r f a c e m e m b r a n e o f m o t i l e zoospores. V i r u s particles gain entry into root cells f o l l o w i n g encystment o f fungal z o o s p o r e s ( C a m p b e l l , 1996; A d a m s 1991). E l e c t r o n m i c r o s c o p y s t u d i e s h a v e s u g g e s t e d that a d s o r p t i o n o f v i r u s to the z o o s p o r e p l a s m a l e m m a i s v e r y s p e c i f i c ; Tobacco necrosis virus ( T N V ) b i n d s O. brassicae z o o s p o r e s b u t not O. bornovanus z o o s p o r e s a n d  CNV  b i n d s O. bornovanus z o o s p o r e s b u t not O. brassicae z o o s p o r e s ( T e m m i n k et al, 1970). In a d d i t i o n , b i o l o g i c a l s t u d i e s h a v e s h o w n that there are strains o f Olpidium spp. w h i c h v a r y i n t h e i r a b i l i t y to t r a n s m i t a g i v e n v i r u s ( T e m m i n k et al., 1970; C a m p b e l l et  al.,  1994; C a m p b e l l et al., 1991). T h i s s p e c i f i c i t y o f i n t e r a c t i o n has s u g g e s t e d that b o t h v i r u s a n d z o o s p o r e s p o s s e s s s p e c i f i c c o m p o n e n t s w h i c h d e t e r m i n e t r a n s m i s s i b i l i t y (see s e c t i o n 1.2.3.3 f o r m o r e details). In p r e v i o u s w o r k , w e h a v e s h o w n that the C N V c o a t p r o t e i n g e n e c o n t a i n s d e t e r m i n a n t s f o r the s p e c i f i c i t y o f t r a n s m i s s i o n b y O. bornovanus ( M c L e a n et al., 1994). In a d d i t i o n , s t u d i e s o f a n a t u r a l l y o c c u r r i n g C N V m u t a n t d e f i c i e n t i n t r a n s m i s s i o n ( L L 5 ) h a v e l e d to the i d e n t i f i c a t i o n o f a s p e c i f i c a m i n o a c i d i n the C N V c o a t p r o t e i n s h e l l d o m a i n w h i c h i s c r i t i c a l f o r e f f i c i e n t f u n g u s t r a n s m i s s i o n ( C h a p t e r 3; R o b b i n s et  99  al.,  1997). Interestingly, in vitro b i n d i n g assays s h o w e d that L L 5 b o u n d z o o s p o r e s to o n l y a p p r o x i m a t e l y 5 0 % o f the l e v e l o f C N V .  T h i s s u g g e s t e d that at least part o f the r e d u c e d  t r a n s m i s s i o n o f L L 5 c a n b e a t t r i b u t e d to i n e f f i c i e n t z o o s p o r e b i n d i n g . W e w i s h e d to d e t e r m i n e w h e t h e r a c q u i s i t i o n a n d s u b s e q u e n t t r a n s m i s s i o n o f  CNV  b y O. bornovanus i n v o l v e s s p e c i f i c z o o s p o r e receptors. T o d o this, w e h a v e d e v e l o p e d a v i r u s / z o o s p o r e in vitro b i n d i n g assay. W e s h o w that b i n d i n g o f Olpidium t r a n s m i t t e d v i r u s to z o o s p o r e s is s p e c i f i c a n d saturable a n d that C N V c a n c o m p e t i t i v e l y i n h i b i t b i n d i n g o f t w o o t h e r v i r u s e s . T h e s e f i n d i n g s suggest that C N V  attachment to O.  bornovanus z o o s p o r e s i n v o l v e s s p e c i f i c z o o s p o r e receptors.  5.2 Results  5.2.1 Development of an in vitro assay for binding of CNV zoospores.  to O. bornovanus  T o o p t i m i z e the b i n d i n g assay, w e first e x a m i n e d the e f f e c t o f t i m e a n d p H o n the a b i l i t y o f C N V to b i n d z o o s p o r e s . F o r the t i m e c o u r s e e x p e r i m e n t , C N V  (6.77 X 1 0  p a r t i c l e s ) was i n c u b a t e d w i t h 4 X 1 0 O. bornovanus z o o s p o r e s i n 1 m l 5 0 m M 5  p H 7.6 f o r 0 to 120 m i n . F o l l o w i n g l o w s p e e d c e n t r i f u g a t i o n to p e l l e t z o o s p o r e s  12  NaPC>4,  and  w a s h i n g to r e m o v e u n b o u n d v i r u s , the a m o u n t o f b o u n d v i r u s was d e t e r m i n e d u s i n g W e s t e r n b l o t a n a l y s i s f o l l o w e d b y d e n s i t o m e t r y . T h e t i m e c o u r s e a n a l y s i s i n F i g u r e 5.1 s h o w s that a p p r o x i m a t e l y 5 0 % o f total b i n d i n g o c c u r s w i t h i n 10 m i n a n d that b i n d i n g i n c r e a s e s u n t i l 4 0 m i n after w h i c h the rate o f b i n d i n g a p p r o a c h e s e q u i l i b r i u m .  100  5.5  5  4.5  0.5  0*  0  , 20  , 40  , 60  , 80  , 100  , 120  , 140  Time in minutes  Figure 5.1 E f f e c t o f t i m e o n b i n d i n g o f C N V to O. bornovanus z o o s p o r e s . O. bornovanus z o o s p o r e s ( 4 X 1 0 ) w e r e i n c u b a t e d w i t h 6.77 X 10 p a r t i c l e s o f C N V f o r 0 to 120 m i n i n 50 m M NaPC>4 b u f f e r , p H 7.6. T h e l e v e l o f b i n d i n g was d e t e r m i n e d u s i n g  W e s t e r n b l o t a n a l y s i s f o l l o w e d b y d e n s i t o m e t r y . E a c h d a t a p o i n t represents the a v e r a g e o f three e x p e r i m e n t s u s i n g triplicate s a m p l e s f o r e a c h t i m e p o i n t . E r r o r bars r e p r e s e n t s t a n d a r d error. T h e a m o u n t o f v i r u s b o u n d to w a s h e s o f u n i n f e c t e d r o o t s was a l s o d e t e r m i n e d a n d t h i s v a l u e was s u b t r a c t e d f r o m the a m o u n t o f v i r u s b o u n d to z o o s p o r e s to g i v e the v a l u e s s h o w n .  W e a l s o w i s h e d to e x a m i n e the e f f e c t o f b u f f e r a n d p H o n b i n d i n g o f C N V to O. bornovanus. F u n g u s t r a n s m i s s i o n assays u s i n g C N V  c u s t o m a r i l y u s e d 50 m M  a n d O. bornovanus h a v e  g l y c i n e b u f f e r , p H 7.6 ( R o b b i n s et al, 1997; C a m p b e l l et al,  1991). W e w i s h e d to e x a m i n e t h i s b u f f e r as w e l l as o t h e r b u f f e r s at p H 7.6 f o r t h e i r  101  e f f e c t o n C N V b i n d i n g . It was d e t e r m i n e d that 50 m M  N a P C v b u f f e r ( p H 7.6) g a v e  c o n s i s t e n t l y h i g h b i n d i n g v a l u e s (data not s h o w n ) . T h e e f f e c t o f v a r y i n g the p H b e t w e e n 5.6 a n d 8.2 u s i n g 5 0 m M  NaPC>4 b u f f e r was t h e n e x a m i n e d . F i g u r e 5.2 s h o w s that  b i n d i n g o f C N V to O. bornovanus z o o s p o r e s i s p H d e p e n d e n t w i t h the h i g h e s t l e v e l o f b i n d i n g o c c u r r i n g a r o u n d p H 7.2. S i g n i f i c a n t l y l o w e r l e v e l s o f b i n d i n g o c c u r r e d at p H v a l u e s l e s s t h a n 7.0 a n d greater t h a n p H 8.0. 25  i  0 J  5  , 5.5  , 6  , 6.5  7  , 7.5  , 8  , 8.5  pH  F i g u r e 5.2 E f f e c t o f p H o n b i n d i n g o f C N V to O. bornovanus z o o s p o r e s . O. bornovanus z o o s p o r e s (1 X 10 ) w e r e i n c u b a t e d w i t h 6.77 X 1 0 p a r t i c l e s o f C N V f o r o n e h o u r i n 50 m M N a P C v b u f f e r at p H 5.6, 6.2, 6.8, 7.2, 7.6, o r 8.2 a n d the l e v e l o f b i n d i n g d e t e r m i n e d u s i n g W e s t e r n b l o t a n a l y s i s as i n F i g . 5.1. E a c h d a t a p o i n t represents the a v e r a g e o f three r e p l i c a t e s a n d the e r r o r bars the s t a n d a r d error. T h e a m o u n t o f v i r u s b o u n d to w a s h e s o f u n i n f e c t e d r o o t s was d e t e r m i n e d i n triplicate at e a c h p H a n d t h i s v a l u e was s u b t r a c t e d f r o m the a m o u n t o f v i r u s b o u n d to z o o s p o r e s to g i v e the v a l u e s s h o w n . 6  12  102  D u r i n g the d e v e l o p m e n t o f the b i n d i n g assay i t w a s n o t e d that a l o w l e v e l o f C N V (ca. 5 % o f the total b o u n d to z o o s p o r e s ) c o u l d b e p e l l e t e d i n N a P C U b u f f e r i n the a b s e n c e o f z o o s p o r e s . In a d d i t i o n , a l o w l e v e l o f b i n d i n g (ca. 2 0 % o f total b i n d i n g to z o o s p o r e s ) a l s o o c c u r r e d to c u c u m b e r r o o t w a s h e s f r o m p l a n t s n o t c o n t a i n i n g z o o s p o r e s .  Therefore  e a c h b i n d i n g e x p e r i m e n t i n c l u d e d a r o o t w a s h c o n t r o l to a c c o u n t f o r b a c k g r o u n d l e v e l s o f v i r u s p r e c i p i t a t i o n a n d n o n - s p e c i f i c b i n d i n g to r o o t w a s h m a t e r i a l . T h e f i n a l b i n d i n g a s s a y a d o p t e d u s e d 4 X 1 0 to 1 X 1 0 z o o s p o r e s , 5 0 m M 5  6  s o d i u m p h o s p h a t e , p H 7.6 i n a o n e m l f i n a l v o l u m e a n d a o n e h o u r a c q u i s i t i o n time. U s i n g these c o n d i t i o n s a n d 6.77 X 1 0 p a r t i c l e s o f C N V , i t w a s f o u n d that a p p r o x i m a t e l y 1 2  3 X 1 0 to 1 X 1 0 9  10  p a r t i c l e s o f C N V a s s o c i a t e d w i t h the z o o s p o r e p e l l e t (see F i g u r e s 5.1  a n d 5.2). T h i s c o r r e s p o n d s to 4 0 to 1 3 3 t i m e s the a m o u n t o f b i n d i n g o b s e r v e d i n o u r p r e v i o u s a s s a y ( R o b b i n s et al, 1997). 5.2.2 B i n d i n g o f v i r u s t o z o o s p o r e s i s s p e c i f i c . T o assess the p o s s i b i l i t y that a s p e c i f i c r e c e p t o r i s i n v o l v e d i n t r a n s m i s s i o n b y Olpidium spp., three v i r u s e s k n o w n to b e t r a n s m i t t e d b y O. bornovanus, [ C N V , Melon necrotic spot carmovirus ( M N S V ) a n d Cucumber leaf spot aureusvirus ( C L S V ) ;  C a m p b e l l , 1996], a n d o n e k n o w n to b e t r a n s m i t t e d b y O. brassicae ( T N V ;  Campbell,  1996) w e r e e x a m i n e d f o r their a b i l i t y to b i n d to z o o s p o r e s o f O. bornovanus a n d O. brassicae u s i n g the a b o v e d e s c r i b e d b i n d i n g assay. F i g u r e 5.3 A s h o w s that C N V ,  M N S V a n d C L S V e a c h b i n d O. bornovanus z o o s p o r e s a n d that b i n d i n g o f C N V a n d M N S V o c c u r s to a p p r o x i m a t e l y t w o t i m e s the l e v e l that o c c u r s w i t h T N V . T h e l e v e l o f b i n d i n g o f C L S V w a s the greatest b e i n g a p p r o x i m a t e l y f i v e - f o l d h i g h e r t h a n that o f e i t h e r  103  C N V o r M N S V . F i g u r e 3 B s h o w s that the l e v e l o f b i n d i n g o f T N V to O. brassicae w a s a l w a y s greater t h a n that o f C N V , C L S V , o r M N S V . 5.2.3 B i n d i n g o f v i r u s t o z o o s p o r e s i s s a t u r a b l e . If a p u t a t i v e r e c e p t o r m o l e c u l e i s l o c a t e d o n the s u r f a c e o f Olpidium z o o s p o r e s , the b i n d i n g o f v i r u s e s to the z o o s p o r e s s h o u l d b e s a t u r a b l e ( T a r d i e u et ah, 1982). T o d e t e r m i n e w h e t h e r C N V b i n d i n g to O. bornovanus z o o s p o r e s ' i s saturable, i n c r e a s i n g a m o u n t s o f C N V p a r t i c l e s w e r e i n c u b a t e d w i t h 4 X 1 0 O. bornovanus z o o s p o r e s a n d the 5  amount o f b o u n d virus determined using W e s t e r n blot analysis f o l l o w e d b y densitometry. F i g u r e 5.4 A s h o w s that O. bornovanus z o o s p o r e s e x h i b i t s a t u r a b l e b i n d i n g at h i g h c o n c e n t r a t i o n s o f C N V . A t saturation, a p p r o x i m a t e l y 7 x 1 0 C N V p a r t i c l e s w e r e b o u n d 9  to 4 x 1 0 z o o s p o r e s . T h i s c o r r e s p o n d s to 1.75 x 1 0 p a r t i c l e s o f C N V b o u n d p e r 5  4  z o o s p o r e at s a t u r a t i o n . B i n d i n g o f T N V o f to O. bornovanus d i d n o t a p p e a r s a t u r a b l e ( F i g u r e 5.4 A ) .  104  14  12  cn  10  u « a.  u  X-  B. CNV  MNSV  *CLSV  TNV  CNV  MNSV  CLSV  TNV  O X  •c  4  a cn  fl u  3  F i g u r e 5.3 S p e c i f i c i t y o f v i r u s b i n d i n g t o Olpidium spp. O n e h u n d r e d m i c r o g r a m s o f either C N V , M N S V , C L S V , T N V o r T B S V - C h w a s i n c u b a t e d w i t h 1 X 1 0 O. bornovanus ( A ) o r O. brassicae z o o s p o r e s ( B ) i n 5 0 m M NaP04, p H 7.6 f o r 1 h r a n d t h e amount o f b o u n d virus determined.by W e s t e r n blotting for C N V , M N S V , C L S V a n d T N V or b y E L I S A for T B S V - C h . T h e amount o f virus b o u n d to washes o f uninfected roots o f c u c u m b e r o r lettuce w a s also determined a n d this value w a s subtracted f r o m the a m o u n t o f v i r u s b o u n d t o z o o s p o r e s to g i v e t h e v a l u e s s h o w n . A l l v a l u e s r e p r e s e n t t h e a v e r a g e o f triplicate treatments f r o m t w o separate e x p e r i m e n t s . * T h e a c t u a l l e v e l o f C L S V b i n d i n g i s f i v e f o l d greater t h a n that i n d i c a t e d i n t h e g r a p h (i.e., 5.5 x 1 0 p a r t i c l e s / 10 zoospores). 6  10  6  105  F i g u r e 5.4 S a t u r a t i o n b i n d i n g o f v i r u s t o Olpidium spp. I n c r e a s i n g c o n c e n t r a t i o n s o f either C N V o r T N V w e r e a d d e d to 4 X 1 0 (A)<9. bornovanus o r ( B j O. brassicae z o o s p o r e s a n d t h e a m o u n t o f b o u n d v i r u s d e t e r m i n e d u s i n g W e s t e r n b l o t a n a l y s i s as d e s c r i b e d i n F i g u r e 1. T h e a m o u n t o f v i r u s b o u n d t o w a s h e s o f u n i n f e c t e d r o o t s w a s a l s o d e t e r m i n e d a n d this v a l u e w a s s u b t r a c t e d f r o m t h e a m o u n t o f v i r u s b o u n d t o z o o s p o r e s t o g i v e t h e v a l u e s h o w n . A l l v a l u e s r e p r e s e n t t h e a v e r a g e o f triplicate treatments f r o m o n e representative experiment. 5  106  Similar saturation binding experiments were conducted using  O.  brassicae  z o o s p o r e s ( F i g u r e 5.4 B ) . It c a n b e seen that b i n d i n g o f T N V i s saturable. A t s a t u r a t i o n a p p r o x i m a t e l y 5 x 1 0 p a r t i c l e s b o u n d t o 4 x 1 0 z o o s p o r e s . T h i s c o r r e s p o n d s t o 1.25 x 9  5  1 0 T N V p a r t i c l e s b o u n d p e r z o o s p o r e at saturation. C N V b i n d i n g t o O. brassicae 4  z o o s p o r e s w a s s i g n i f i c a n t l y less than that a c h i e v e d b y T N V a n d d i d n o t a p p e a r t o b e saturable. 5.2.4 C N V c a n c o m p e t e w i t h M N S V a n d T N V b u t n o t w i t h C L S V f o r b i n d i n g s i t e s . A n o t h e r c r i t e r i o n f o r r e c e p t o r s p e c i f i c i t y is b a s e d o n c o m p e t i t i o n f o r r e c e p t o r s b y r e l a t e d o r u n r e l a t e d v i r u s e s ( T a r d i e u et al, 1982). M N S V (3.39 x 1 0 p a r t i c l e s ) w a s 1 2  incubated with increasing amounts o f C N V and the a m o u n t o f M N S V b o u n d w a s d e t e r m i n e d . F i g u r e 5.5 A s h o w s that a t e n - f o l d e x c e s s o f c o m p e t i t o r C N V results i n a n approximate 6 2 % reduction i n the level o f b o u n d M N S V . C N V w a s also capable o f c o m p e t i t i v e l y i n h i b i t i n g T N V . I n this c a s e a t e n f o l d e x c e s s o f c o m p e t i t o r r e s u l t e d i n about 9 0 % r e d u c t i o n o f T N V b i n d i n g ( F i g u r e 5.5 B ) . I n b o t h cases c o m p e t i t i o n w a s e v i d e n t u s i n g as l i t t l e as a 2 - f o l d e x c e s s o f C N V . N o c o m p e t i t i o n w a s o b s e r v e d b e t w e e n C N V a n d C L S V e v e n w h e n a 2 0 f o l d e x c e s s o f C N V w a s u s e d as c o m p e t i t o r ( F i g u r e 5.5 C).  107  0  33.9  6.8  1.4  Number of CNV particles x 10' added 2  Number of CNV particles x 10 added 12  F i g u r e 5.5 C o m p e t i t i o n b i n d i n g e x p e r i m e n t s u s i n g C N V a g a i n s t M N S V , T N V o r C L S V . I n c r e a s i n g a m o u n t s (0-3.4 x 1 0 p a r t i c l e s ) o f C N V w e r e a d d e d c o n c u r r e n t l y t o e i t h e r ( A ) M N S V (3.4 x 1 0 particles), ( B ) T N V (4.5 x 1 0 p a r t i c l e s ) o r ( C ) C L S V (1.4 x 1 0 particles), a n d 4 X 1 0 O. bornovanus z o o s p o r e s . T h e a m o u n t o f v i r u s b o u n d w a s determined u s i n g W e s t e r n blot analysis. T h e amount o f virus b o u n d to washes o f uninfected roots was determined and subtracted f r o m virus b o u n d to zoospores to give the v a l u e s h o w n . V a l u e s s h o w n f o r M N S V , a n d T N V , a r e f r o m triplicate t r e a t m e n t s o f a r e p r e s e n t a t i v e e x p e r i m e n t w h i l e v a l u e s f o r C L S V are t h e a v e r a g e o f f o u r t r e a t m e n t s f r o m a s i n g l e e x p e r i m e n t . E r r o r b a r s r e p r e s e n t s t a n d a r d error. 1 3  1 2  1 2  5  108  1 2  5.3 Discussion W e h a v e p r e v i o u s l y s h o w n that C N V v i r i o n s b i n d O. bornovanus z o o s p o r e s in vitro ( C h a p t e r 3; R o b b i n s et al, 1997). W e w i s h e d t o f u r t h e r d e v e l o p the in vitro b i n d i n g  assay i n o r d e r to assess the p o s s i b l e i n v o l v e m e n t o f s p e c i f i c z o o s p o r e r e c e p t o r s i n C N V attachment to z o o s p o r e s . It w a s f o u n d that b i n d i n g o f C N V to z o o s p o r e s o f O. bornovanus i n c r e a s e s w i t h time, r e a c h i n g 5 0 % o f the f i n a l l e v e l o f b i n d i n g w i t h i n 10 m i n  a n d e q u i l i b r i u m at a p p r o x i m a t e l y 6 0 min. T h e t i m e d e p e n d e n c e o f b i n d i n g as w e l l as t h e a b i l i t y to r e a c h e q u i l i b r i u m i s s u g g e s t i v e o f a s p e c i f i c i n t e r a c t i o n b e t w e e n v i r u s a n d z o o s p o r e s . I n a d d i t i o n , the r a p i d a d s o r p t i o n o f C N V to z o o s p o r e s is c o n s i s t e n t w i t h e a r l i e r f i n d i n g s b y D i a s ( 1 9 7 0 ) w h o s u g g e s t e d that a n a c q u i s t i o n t i m e o f b e t w e e n 5 a n d 10 m i n w a s s u f f i c i e n t f o r C N V t r a n s m i s s i o n . It i s n o t e d that i n s o m e e x p e r i m e n t s b i n d i n g l e v e l s d e c r e a s e d u p t o 5 0 % after 8 0 m i n (not s h o w n ) . T h e b a s i s f o r the d e c r e a s e i n b i n d i n g i s n o t k n o w n b u t it m a y b e r e l a t e d to c e l l m e m b r a n e c h a n g e s that take p l a c e p r i o r to z o o s p o r e e n c y s t m e n t w h i c h g e n e r a l l y o c c u r s 1-2 hrs after z o o s p o r e r e l e a s e ( S t o b b s et al, 1982). A t i m e o f 6 0 m i n . w a s c h o s e n f o r the s t a n d a r d b i n d i n g assay s i n c e e q u i l i b r i u m w a s a c h i e v e d b y this t i m e a n d s i n c e it p r e c e d e d a d e c r e a s e i n b i n d i n g observed i n s o m e experiments. T h e p H d e p e n d e n c e o f b i n d i n g suggests that s p e c i f i c i n t e r a c t i o n s are r e q u i r e d f o r v i r u s / z o o s p o r e b i n d i n g . I n a d d i t i o n , the p H d e p e n d e n c e o f b i n d i n g r e f l e c t s e a r l i e r t r a n s m i s s i o n studies. D i a s ( 1 9 7 0 b ) s h o w e d that O. bornovanus t r a n s m i t s C N V at p H 6, 7, a n d 8 b u t fails t o t r a n s m i t at p H 4, 9, a n d 10. A l t h o u g h it w a s s u g g e s t e d that the l a c k o f t r a n s m i s s i o n w a s d u e t o p o o r z o o s p o r e m o t i l i t y ( D i a s , 1 9 7 0 b ) , o u r in vitro b i n d i n g d a t a  109  suggests that t h e f a i l u r e t o t r a n s m i t at t h e latter p H v a l u e s c a n a l s o b e a t t r i b u t e d to inefficient binding o f C N V to zoospores. T h r e e m a j o r c r i t e r i a f o r v i r a l r e c o g n i t i o n sites as r e c e p t o r s a r e saturability, s p e c i f i c i t y , a n d c o m p e t i t i o n ( T a r d i e u et al, 1982). P r e v i o u s w o r k h a s s h o w n that s p e c i f i c i n t e r a c t i o n s o c c u r b e t w e e n Olpidium spp. a n d t h e v i r u s e s it transmits. O. brassicae a n d O. bornovanus e a c h t r a n s m i t v i r u s e s that a r e n o t t r a n s m i t t e d b y t h e o t h e r s p e c i e s  ( T e m m i n k , 1970; C a m p b e l l etal., 1995). I n t h e c a s e o f T N V that i s t r a n s m i t t e d b y O. brassicae a n d C N V t r a n s m i t t e d b y O. bornovanus, e l e c t r o n m i c r o s c o p y h a s s h o w n that  the r e s p e c t i v e v i r u s a d s o r b s o n l y t o t h e z o o s p o r e o f the v e c t o r s p e c i e s ( T e m m i n k et al., 1970). F u r t h e r m o r e , there are strains o f e a c h s p e c i e s that v a r y i n a b i l i t y t o t r a n s m i t a g i v e n v i r u s ( T e m m i n k et al., 1970; C a m p b e l l et al, 1995, C a m p b e l l et al, 1991). T h i s b i o l o g i c a l s p e c i f i c i t y suggests that a s p e c i f i c r e c e p t o r m a y b e i n v o l v e d i n t r a n s m i s s i o n b y Olpidium spp. U s i n g t h e b i n d i n g a s s a y d e v e l o p e d i n C h a p t e r 3 w e h a v e d e t e r m i n e d that  C N V , M N S V a n d C L S V , w h i c h a r e t r a n s m i t t e d b y O. bornovanus, e a c h b i n d O. bornovanus z o o s p o r e s a n d that b i n d i n g o f C N V a n d M N S V o c c u r s t o a p p r o x i m a t e l y t w o  t i m e s t h e l e v e l that o c c u r s w i t h T N V . B i n d i n g o f C L S V o c c u r s at a p p r o x i m a t e l y 10 t i m e s t h e l e v e l as that f o r T N V . A d d i t i o n a l l y , b i n d i n g t o O. brassicae z o o s p o r e s i s greatest u s i n g T N V , w h i c h i s t r a n s m i t t e d b y this f u n g u s . T h e s e b i n d i n g e x p e r i m e n t s s h o w that s p e c i f i c i t y o c c u r s i n b i n d i n g o f these v i r u s e s t o Olpidium z o o s p o r e s . M o r e o v e r , t h e o b s e r v e d s p e c i f i c i t y reflects t h e p r e v i o u s l y d e s c r i b e d b i o l o g i c a l s p e c i f i c i t y ( C a m p b e l l , 1996). T h i s o b s e r v a t i o n s t r o n g l y suggests that s p e c i f i c z o o s p o r e r e c e p t o r s a r e i n v o l v e d i n a c q u i s i t i o n a n d t r a n s m i s s i o n o f these v i r u s e s i n nature.  110  A n o t h e r c r i t e r i o n f o r the p r e s e n c e o f s p e c i f i c r e c e p t o r s o n c e l l s i s that b i n d i n g s h o u l d b e s a t u r a b l e ( T a r d i e u et al., 1982). S e v e r a l r e p e t i t i o n s o f the s a t u r a t i o n b i n d i n g e x p e r i m e n t w i t h C N V p a r t i c l e s a n d O. bornovanus z o o s p o r e s i n d i c a t e that b i n d i n g o f C N V to O. bornovanus i s saturable, i n d i c a t i n g that b i n d i n g to O. bornovanus o c c u r s v i a a s p e c i f i c , l i m i t e d n u m b e r o f receptors. T h e l i n e a r i n c r e a s e o b s e r v e d w i t h T N V i n d i c a t e s that b i n d i n g o f T N V to O. bornovanus z o o s p o r e s m a y b e l a r g e l y n o n - s p e c i f i c . S a t u r a b l e b i n d i n g o f T N V to O. brassicae z o o s p o r e s i s s u g g e s t i v e o f a s p e c i f i c r e c e p t o r i n t e r a c t i o n w h e r e a s the l i n e a r c u r v e o b t a i n e d w i t h C N V i s i n d i c a t i v e o f a n o n - s p e c i f i c a s s o c i a t i o n . T h e s a t u r a t i o n b i n d i n g assays i n d i c a t e that s a t u r a t i o n o f C N V b i n d i n g sites o n O. bornovanus o c c u r s w h e n a p p r o x i m a t e l y  1.75 X 1 0 C N V p a r t i c l e s are b o u n d p e r 4  z o o s p o r e . T h i s suggests that there are a p p r o x i m a t e l y 1.75 X 1 0 p u t a t i v e C N V r e c e p t o r 4  m o l e c u l e s p e r z o o s p o r e . T h e n u m b e r o f T N V p a r t i c l e s b o u n d p e r O. brassicae z o o s p o r e is c a l c u l a t e d to b e 1.25 X 10 . It i s n o t e d that w e h a v e not yet d e t e r m i n e d the l e v e l o f 4  n o n - s p e c i f i c b i n d i n g o f C N V o r T N V to z o o s p o r e s s o these v a l u e s s h o u l d b e c o n s i d e r e d to b e the m a x i m u m n u m b e r o f p a r t i c l e s b o u n d . T h e a b i l i t y o f C N V to c o m p e t e w i t h M N S V a n d T N V f o r z o o s p o r e b i n d i n g p r o v i d e s f u r t h e r e v i d e n c e f o r the s p e c i f i c i t y o f i n t e r a c t i o n b e t w e e n these v i r u s e s a n d O. bornovanus z o o s p o r e s .  In a d d i t i o n , these data suggest that C N V shares r e c e p t o r ( s ) i n  c o m m o n w i t h M N S V a n d p e r h a p s T N V o n O. bornovanus z o o s p o r e s . E x p e r i m e n t s  have  not b e e n c o n d u c t e d to d e t e r m i n e i f C L S V , M N S V , o r T N V c a n c o m p e t i t i v e l y i n h i b i t b i n d i n g o f C N V , p a r t l y d u e to the v e r y l a r g e a m o u n t s o f p u r i f i e d v i r u s r e q u i r e d to d o these e x p e r i m e n t s . A t t e m p t s to l a b e l C N V p a r t i c l e s h a v e b e e n u n s u c c e s s f u l . T h e r e f o r e , c o m p e t i t i o n e x p e r i m e n t s u s i n g l a b e l e d C N V w e r e n o t c o n d u c t e d . S u c h e x p e r i m e n t s are  111  r e q u i r e d to m o r e f u l l y assess the s p e c i f i c i t y o f C N V b i n d i n g to z o o s p o r e s as w e l l as the level of background binding. W e h a v e r e c e n t l y i d e n t i f i e d a d e t e r m i n a n t i n the s h e l l d o m a i n o f the C N V p r o t e i n w h i c h i s r e q u i r e d f o r e f f i c i e n t t r a n s m i s s i o n a n d b i n d i n g ( R o b b i n s et al, A d d i t i o n a l studies h a v e s h o w n that t w o p a r t i c u l a r r e g i o n s w i t h i n the C N V  coat 1997).  protruding  d o m a i n a l s o c o n t a i n t r a n s m i s s i o n d e t e r m i n a n t s (D. R o c h o n a n d K. K a k a n i , u n p u b l i s h e d o b s e r v a t i o n s ) . T N V l a c k s a p r o t r u d i n g d o m a i n so the T N V attachment site is m o s t l i k e l y i n e x p o s e d r e g i o n s o f the s h e l l d o m a i n . It i s p o s s i b l e that the m o r e e f f i c i e n t b i n d i n g o f C N V , M N S V a n d C L S V to O. bornovanus i s at least i n part d u e to their p o s s e s s i o n o f p r o t r u d i n g d o m a i n d e t e r m i n a n t s absent i n T N V . A l t h o u g h w e h a v e p r o v i d e d b i o c h e m i c a l e v i d e n c e that Olpidium m a y c o n t a i n receptors for C N V  a n d T N V w e d o not yet k n o w the nature o f this receptor. T r e a t m e n t o f  z o o s p o r e s w i t h p h o s p h o l i p a s e C d i d not e l i m i n a t e b i n d i n g w h i l e treatment w i t h t r y p s i n d i d s i g n i f i c a n t l y r e d u c e b i n d i n g (K. K a k a n i a n d D. R o c h o n , u n p u b l i s h e d o b s e r v a t i o n s ) . T h e s e results suggest that p r o t e i n s m a y b e i n v o l v e d i n p a r t i c l e attachment.  112  6 C H A P T E R SIX: USE  OFFITC-LECTINS TOIDENTIFY SUGARS ON THE S U R F A C E O F O. BORNOVANUS  A N D O.  BRASSICAE  ZOOSPORES  6.1 Introduction Olpidium spp. b e l o n g t o the C h y t r i d i o m y c e t e s , w h i c h are p o s t e r i o r l y u n i f l a g e l l a t e  Chytridiomycota. A prominent cell coat consisting of glycoconjugates, produced f r o m Golgi-derived vesicles during zoosporogenesis, has been observed around zoospores o f s o m e c h y t r i d i o m y c e t e s ( D o r w a r d a n d P o w e l l , 1 9 8 3 ; L i et al., 1991; M a n i e r a n d L o u b e s , 1978; M u n n et al., 1981; P o w e l l , 1981; T a y l o r a n d F u l l e r , 1981). It is m o s t l i k e l y that a l l c h y t r i d s h a v e v a r y i n g degrees o f e x t r a c e l l u l a r m a t e r i a l c o a t i n g the p l a s m a m e m b r a n e o f z o o s p o r e s ( P o w e l l , 1994). M e c h a n i s m s f o r c e l l r e s p o n s e to e n v i r o n m e n t a l t r i g g e r s o f t e n i n v o l v e g l y c o p r o t e i n r e c e p t o r s at the c e l l s u r f a c e ( S h a r o n a n d L i s , 1993). D i f f e r e n c e s i n c e l l s u r f a c e s o f z o o s p o r e s c a n h a v e m a j o r f u n c t i o n a l i m p l i c a t i o n s , s u c h as s e l e c t i v e b i n d i n g o f v i r u s e s f o r w h i c h s o m e c h y t r i d i o m y c e t e s s e r v e as v e c t o r s ( r e v i e w e d i n P o w e l l , 1993). S u r f a c e c a r b o h y d r a t e s are i m p o r t a n t i n f u n g a l - p l a n t i n t e r a c t i o n s as a d h e s i v e s t o the p l a n t s u r f a c e ( N i c h o l s o n , 1 9 8 4 ) a n d as d e t e r m i n a n t s o f c e l l - c e l l r e c o g n i t i o n ( C a l l o w , 1987). F l u o r e s c e n t l y l a b e l l e d p l a n t l e c t i n s h a v e b e e n u s e d to d e t e r m i n e the n a t u r e o f s u r f a c e sugars o n f u n g a l c e l l w a l l s ( O ' C o n n e l l , 1 9 9 1 ) a n d the z o o s p o r e p l a s m a m e m b r a n e ( G u i l l o t et al, 1990; B u r r a n d B e a k e s , 1994; B e r b e e a n d K e r w i n , 1993; L e h n e n a n d  113  P o w e l l , 1993). L e c t i n s are p r o t e i n s a n d g l y c o p r o t e i n s w h i c h h a v e a f f i n i t y f o r s p e c i f i c m o n o - a n d o l i g o s a c c h a r i d e s ( R i n i , 1995). T h e T B S V C P s h e l l d o m a i n ( a n d the C N V C P s h e l l b y a n a l o g y ) c o n t a i n s a s t a n d a r d c l a s s I j e l l y r o l l motif, as d o s e v e r a l other i c o s a h e d r a l a n i m a l a n d p l a n t v i r u s C P s as w e l l as the h e m a g g l u t i n i n o f i n f l u e n z a v i r u s ( C h e l v a n a y a g a m et al, 1992). N o n - v i r a l structures w i t h this j e l l y - r o l l m o t i f i n c l u d e the l e c t i n c o n c a n a v a l i n A ( C o n A ) , t u m o r n e c r o s i s f a c t o r ( T N F ) , a n d the l a r g e d o m a i n o f the c a t a b o l i t e g e n e a c t i v a t o r p r o t e i n ( G A P ) ( C h e l v a n a y a g a m et al, 1992). T h e T B S V C P p r o t r u d i n g d o m a i n ( a n d the  CNV  C P p r o t r u d i n g d o m a i n b y a n a l o g y ) i s a u n i q u e e x a m p l e o f a c l a s s LT j e l l y r o l l w h i c h has b e e n h y p o t h e s i z e d to b e a c a p t u r e d c e l l u l a r g e n e ( G i b s o n a n d A r g o s , 1990). S e v e r a l o f the v i r u s e s a n d p r o t e i n s k n o w n to h a v e a c l a s s I j e l l y r o l l are lectins. C o n A has a s p e c i f i c i t y f o r D ( + ) - m a n n o s e . I n f l u e n z a v i r u s h e m a g g l u t i n i n i s the v i r a l s u r f a c e g l y c o p r o t e i n r e s p o n s i b l e f o r attachment to s i a l i c a c i d ( N - a c e t y l n e u r a m i n i c a c i d ) o f o l i g o s a c c h a r i d e s o n h o s t - c e l l s u r f a c e g l y c o p r o t e i n s a n d g l y c o l i p i d s a n d is c o n s i d e r e d a l e c t i n ( W i l e y a n d S k e h e l , 1987; R i n i , 1995). T N F has a l e c t i n - l i k e s p e c i f i c i t y f o r the c a r b o h y d r a t e N , N ' - d i a c e t y l c h i t o b i o s e ( S h e r b l o m et al, 1988). T h e studies d e s c r i b e d i n this c h a p t e r w e r e u n d e r t a k e n to d e t e r m i n e s o m e o f the sugar r e s i d u e s o n the s u r f a c e o f Olpidium z o o s p o r e s . F i v e F I T C - l a b e l l e d l e c t i n s r e p r e s e n t i n g f o u r d i f f e r e n t c l a s s e s o f s u g a r - b i n d i n g s p e c i f i c i t y ( T a b l e 4) w e r e u s e d to p r o b e the s u r f a c e o f O. bornovanus o r O. brassicae z o o s p o r e s . In a d d i t i o n , it was o f interest to d e t e r m i n e i f there are d i f f e r e n t types o f sugars o n z o o s p o r e s o f these t w o s p e c i e s o f Olpidium, s i n c e these f u n g i h a v e d i f f e r e n t s p e c i f i c i t i e s i n t r a n s m i s s i o n o f v i r u s e s a n d v i r u s b i n d i n g in vitro (see C h a p t e r 5).  114  Table 6.1 L e c t i n s u s e d i n b i n d i n g a s s a y s w i t h Olpidium z o o s p o r e s a n d their m a i n b i n d i n g specificities. Lectin  Abbreviation  Binding Specificity and Relative Affinity  Con A  M e t h y l oc-D-mannopyranoside > D(+)M a n n o s e > D(+)-glucose  WGA  N , N ' , N ' - T r i a c e t y l c h i t o t r i o s e > > N , N'Diacetylchitobiose  BPA  N-Acetylgalactosamine > D(+)-Galactose  ECA  N-Acetyllactosamine > a-lactose  TPA  L(-)-Fucose  Mannose Group  Concanavalin A  b  Chitin Group  Wheatgerm agglutinin  0  Galactose/lactose Group Bauhinia purpurea  agglutinin Erythrina corallodendron  agglutinin Fucose Group Tetragonolobus purpurea  agglutinin  "Binding specificity and relative affinity to specific sugars are indicated in the order of greatest to least. from Canavalia ensiformis b  °from Triticum vulgaris  6.2 Results  6.2.1 Concanavalin A-FITC binds to Olpidium zoospores.  C o n c a n a v a l i n A - F I T C f r o m Canavalia ensiformis ( j a c k b e a n ) ( C o n A - F I T C ) , w h i c h r e c o g n i z e s D ( + ) - m a n n o s e ( T a b l e 6.1), b o u n d t o t h e z o o s p o r e s o f b o t h O. bornovanus a n d O. brassicae ( F i g u r e 6.1 B - D a n d F i g u r e 6.2 B ). C o n A - F I T C w a s  a d d e d i n c o n c e n t r a t i o n s o f 1, 16, a n d 120 p.g/ml to z o o s p o r e s u s p e n s i o n s . C o r i A - F I T C b o u n d d e n s e l y a n d u n i f o r m l y o v e r the s u r f a c e o f the z o o s p o r e b o d y a n d f l a g e l l u m o f z o o s p o r e s o f b o t h Olpidium spp. w h e n as l i t t l e as 16 p.g/ml o f C o n A - F I T C w a s a d d e d ( F i g u r e s 6.1 a n d 6.2). W h e n 120 p g / m l C o n A - F I T C w a s a d d e d , a h i g h l y f l u o r e s c e n t  115  3  b a c k g r o u n d w a s observed (not shown). F a i n t fluorescence o f zoospores w a s observed w i t h the a d d i t i o n o f 1 |ig/ml o f C o n A - F I T C to the z o o s p o r e s u s p e n s i o n ( n o t s h o w n ) . T h e a d d i t i o n o f 16 p g / m l o f C o n A - F I T C p r o d u c e d s t r o n g f l u o r e s c e n c e o f a l l o b s e r v a b l e zoospores w i t h m i n i m a l b a c k g r o u n d a n d so these conditions were used i n all further experiments using C o n A - F I T C , unless otherwise specified. T h e presence o f C o n A F I T C i n the O. bornovanus z o o s p o r e s u s p e n s i o n s o m e t i m e s c a u s e d the l o s s o f z o o s p o r e m o t i l i t y a n d the f o r m a t i o n o f l a r g e a g g r e g a t e s o f z o o s p o r e s ( F i g u r e 6.1 D ) , a l t h o u g h i n s o m e e x p e r m e n t s z o o s p o r e s m a i n t a i n e d their m o t i l i t y ( F i g u r e s 6.1 C a n d 6.2 B ) . N o n e o f the f u n g a l structures s h o w e d a u t o f l u o r e s c e n c e ( n o t s h o w n ) . D ( + ) - m a n n o s e , at c o n c e n t r a t i o n s o f 1, 2 0 a n d 1 0 0 m g / m l , w a s a d d e d to 16 p.g/ml C o n A - F I T C f o r 3 0 m i n u t e s i n the d a r k b e f o r e the a d d i t i o n o f z o o s p o r e s to d e t e r m i n e i f the b i n d i n g o f C o n - F I T C to Olpidium z o o s p o r e s w a s s p e c i f i c . T h e a d d i t i o n o f 2 0 m g / m l D ( + ) - m a n n o s e s l i g h t l y r e d u c e d the f l u o r e s c e n c e o f the z o o s p o r e s a n d e l i m i n a t e d the f o r m a t i o n o f a g g r e g a t e s ( n o t s h o w n ) a n d the a d d i t i o n o f 1 0 0 m g / m l D ( + ) - m a n n o s e u s u a l l y e l i m i n a t e d m o s t o f the b i n d i n g o f C o n A - F I T C to the z o o s p o r e s o f b o t h s p e c i e s o f Olpidium ( F i g u r e s 6.1 E a n d F, 6.2 C a n d D ) . W i t h the a d d i t i o n o f 1 0 0 m g / m l o f D ( + ) -  m a n n o s e the z o o s p o r e s m a i n t a i n e d their m o t i l i t y . S o m e t i m e s faint f l u o r e s c e n c e o f O. bornovanus b u t n o t O. brassicae z o o s p o r e s w a s o b s e r v e d w i t h the a d d i t i o n o f 1 0 0 m g / m l  o f the D ( + ) - m a n n o s e c o m p e t i t o r , i n d i c a t i n g that s o m e C o n A - F I T C w a s s t i l l a v a i l a b l e f o r b i n d i n g ( F i g u r e 6.1 F ) .  116  Triticum vulgaris lectin-FITC does not bind to secondary zoospores of Olpidium but does bind to to primary zoospores of Olpidium. 6.2.2  Triticum vulgaris ( w h e a t g e r m ) l e c t i n - F I T C ( W G A - F I T C ) , w h i c h r e c o g n i z e s  c h i t i n ( N , N ' , N ' - t r i a c e t y l c h i t o t r i o s e ) ( T a b l e 4), d i d n o t b i n d t o s e c o n d a r y z o o s p o r e s o f Olpidium spp. b u t d i d b i n d to w h a t a p p e a r t o b e r e s t i n g spores o f O. bornovanus a n d O. brassicae ( F i g u r e 6.3 A - D ; T a b l e 6.2). Z o o s p o r e s r e m a i n e d m o t i l e a n d d i d n o t f l u o r e s c e  w i t h t h e a d d i t i o n o f 125 ixg/ml W G A - F I T C ( F i g u r e 6.3 A a n d B ) . A f t e r t h e a d d i t i o n o f 125 u.g/ml W G A - F I T C , r o o t s w e r e o b s e r v e d w h i c h c o n t a i n e d r o u n d f l u o r e s c e n t b o d i e s w i t h t h i c k w a l l s a n d n o f l a g e l l a w h i c h w e r e d e t e r m i n e d to b e r e s t i n g spores o f O. bornovanus a n d O. brassicae ( F i g u r e 6.3 B a n d 6.3 D ) . C o m p e t i t i o n e x p e r i m e n t s  chitin were not performed.  117  using  F i g u r e 6.1  Binding of Con A - F I T C to 0. bornovanus zoospores ( A ) Photograph of a differential interference contrast (DIC) image of aggregated and free (). bornovanus zoospores after treatment with 16 ug/ml Con A - F I T C (B) Fluorescence image of A showing zoospores treated with C o n A - F I T C (C) Fluorescence image of individual (). bornovanus zoospores after treatment with 16 Ug/ml Con A - F I T C (D) Fluorescence image of a large aggregate of (). bornovanus zoospores after treatment with 16 Ug/ml Con A - F I T C (E) D I C image of 0. bornovanus zoospores after the addition of 16 ug/ml Con A - F I T C pre-incubated with 100 mg/ml D(+)-mannose (F) Fluorescence image of E The photographs in B and F were exposed for equivalent times Images were observed using a Zeiss AxioPhot with D I C and epifluorescence optics Zeiss filter set 487910 (excitation 450 to 490 nm) was used  118  F i g u r e 6.2  B i n d i n g o f C o n A - F I T C to  image o f free  (). brassicae zoospores  ( A ) Photograph o f D I C  (). brassicae zoospores after a d d i t i o n o f 16 p g / m l C o n A - F I T C ( B ) ( C ) D I C i m a g e o f O. brassicae zoospores after a d d i t i o n o f 16  F l u o r e s c e n c e image o f A  u g / m l C o n A - F I T C pre-incubated w i t h 100 m g / m l D(+)-mannose image o f C  (D) Fluorescence  T h e photographs in B and D were exposed for equivalent times  6 1 for details o f m i c r o s c o p y  119  See F i g u r e  F i g u r e 6.3 B i n d i n g o f W G A - F I T C t o O. bornovanus a n d (). brassicae r e s t i n g s p o r e s . ( A ) P h o t o g r a p h o f D I C i m a g e o f 0. bornovanus z o o s p o r e s a n d r e s t i n g s p o r e s i n c u c u m b e r r o o t s a f t e r t h e a d d i t i o n o f 125 u g / m l W G A - F I T C . ( B ) F l u o r e s c e n c e i m a g e o f A . ( C ) P h o t o g r a p h o f D I C i m a g e o f l e t t u c e r o o t c o n t a i n i n g O. brassicae r e s t i n g s p o r e s a f t e r a d d i t i o n o f 1 2 5 p g / m l W G A - F I T C . D. F l u o r e s c e n c e i m a g e o f C . S e e F i g u r e 6.1 f o r d e t a i l s o f m i c r o s c o p y .  F i g u r e 6.4 B i n d i n g o f B P A - F I T C t o 0. brassicae z o o s p o r e s . ( A ) P h o t o g r a p h o f a D I C i m a g e o f (). brassicae z o o s p o r e s a f t e r a d d i t i o n o f 125 |Jg/ml B P A - F I T C . ( B ) F l u o r e s c e n c e i m a g e o f A . ( C ) P h o t o g r a p h o f D I C i m a g e o f z o o s p o r e s o f (). brassicae a f t e r a d d i t i o n o f 125 p g / m l B P A - F I T C p r e - i n c u b a t e d w i t h 100 m g / m l D ( + ) g a l a c t o s e . ( D ) F l u o r e s c e n c e i m a g e o f C . ( E ) P h o t o g r a p h o f D I C i m a g e o f 0. bornovanus z o o s p o r e s a f t e r a d d i t i o n o f 125 ( i g / m l B P A - F I T C . ( F ) F l u o r e s c e n t i m a g e o f E . T h e p h o t o g r a p h s i n B, D, a n d F w e r e e x p o s e d f o r e q u i v a l e n t t i m e s . S e e F i g u r e 6.1 f o r d e t a i l s o f m i c r o s c o p y . 121  F i g u r e 6.5 ECA-FITC  bornovanus or (). brassicae zoospores ( A ) bornovanus zoospores after the a d d i t i o n o f 125 (lg/ml  E C A - F I T C does not b i n d to 0.  P h o t o g r a p h o f D I C i m a g e o f 0.  ( B ) Fluorescence image o f A  ( C ) P h o t o g r a p h o f D I C i m a g e o f ().  brassicae zoospores after a d d i t i o n o f 125 u g / m l E C A - F I T C C  See F i g u r e 6 1 for details o f m i c r o s c o p y  122  ( D ) F l u o r e s c e n c e image o f  F i g u r e 6.6 Binding of TPA-FITC to (). bornovanus and (). brassicae zoospores (A) Photograph of a DIC image of free I), brassicae zoospores after addition of 125 ug/ml TPA-FITC (B) Fluorescence image of A (C) Photograph of fluorescent image of free and aggregated (). bornovanus secondary zoospores after addition of 125 ug/ml TPAFITC (DIC image not available) See Figure 6 1 for details of microscoy  123  Table 6.2  B i n d i n g o f F I T C - l e c t i n c o n j u g a t e s t o Olpidium z o o s p o r e s in vitro . a  O. bornovanus  Lectin  Resting spores  O. brassicae  Zoospores  Resting spores  Con A  -  +  -  WGA  +  -  +  Zoospores  +  BPA  +  ECA TPA  .  . +  . -  . +  "Fluorescent labelling is shown as follows: (+) labelled, (-) unlabelled.  6.2.3 Bauhinia purpurea lectin-FITC binds to O. brassicae but not O. bornovanus zoospores.  Bauhinia purpurea ( c a m e l s f o o t tree) l e c t i n - F I T C ( B P A - F I T C ) , w h i c h b i n d s to  D ( + ) - g a l a c t o s e ( T a b l e 6.1), b o u n d u n i f o r m l y o v e r t h e s u r f a c e o f w a s h e d O. brassicae b u t n o t t o w a s h e d O. bornovanus z o o s p o r e s w h e n u s e d at a c o n c e n t r a t i o n o f 125 p.g/ml ( F i g u r e 6.4 A , B, E , a n d F; T a b l e 6.2). B P A - F I T C b i n d i n g d i d n o t alter O. brassicae z o o s p o r e m o t i l i t y o r c a u s e a g g r e g a t i o n o f z o o s p o r e s . D ( + ) - g a l a c t o s e at 1 0 0 m g / m l w a s a d d e d t o 125 u.g/ml B P A - F I T C a n d a l l o w e d t o i n c u b a t e f o r 3 0 m i n i n t h e d a r k b e f o r e t h e a d d i t i o n o f O. brassicae z o o s p o r e s t o d e t e r m i n e i f the b i n d i n g o f B P A - F I T C w a s s p e c i f i c . A l l O. brassicae z o o s p o r e s r e m a i n e d intact a n d m o t i l e after treatment w i t h t h e l e c t i n s u g a r s o l u t i o n . N o b i n d i n g o f B P A - F I T C w a s o b s e r v e d t o O. brassicae z o o s p o r e s after the p r e - i n c u b a t i o n o f l e c t i n w i t h D ( + ) - g a l a c t o s e ( F i g u r e 6.4 C a n d D ) .  124  6.2.4 Erythrina corallodendron lectin-FITC does not bind Olpidium zoospores.  E r y t h r i n a c o r a l l o d e n d r o n ( c o r a l tree) l e c t i n - F I T C ( E C A - F I T C ) , w h i c h b i n d s Gal a c t o s e ( T a b l e 6.1), d i d n o t b i n d t o either O. bornovanus o r O. brassicae z o o s p o r e s ( F i g u r e 6.5 A - D ; T a b l e 6.2). Z o o s p o r e s w e r e either w a s h e d o r u n w a s h e d w i t h n o b i n d i n g o b s e r v e d w i t h as m u c h as 8 0 u g / m l o f E C A - F I T C . Z o o s p o r e s r e m a i n e d m o t i l e a n d d i d not aggregate i n the presence o f E C A - F I T C .  6.2.5 Tetragonolobus purpureas lectin-FITC binds to Olpidium zoospores. Tetragonolobus purpureas ( w i n g e d p e a ) l e c t i n - F I T C ( T P A - F I T C ) , w h i c h  r e c o g n i z e s L ( - ) - f u c o s e ( T a b l e 6.1), b o u n d t o z o o s p o r e s o f O. bornovanus a n d O. brassicae ( F i g u r e 6.6 A - C ; T a b l e 6.2). B i n d i n g d i d n o t o c c u r u n l e s s t h e z o o s p o r e s w e r e  w a s h e d b e f o r e t h e a d d i t i o n o f the lectin. F l u o r e s c e n c e w a s u n i f o r m o v e r t h e z o o s p o r e b o d y a n d f l a g e l l u m ( F i g u r e 6.6 B a n d D ) . T h e a d d i t i o n o f T P A - F I T C t o O. bornovanus z o o s p o r e s c a u s e d t h e m t o l o s e their m o t i l i t y a n d t o a g g r e g a t e ( F i g u r e 6.6 C ) . T h e m a j o r i t y o f O. bornovanus z o o s p o r e s f o r m e d these l a r g e f l u o r e s c e n t aggregates, m a k i n g i t difficult to locate individual zoospores, although the occasional free fluorescent zoospore c o u l d b e o b s e r v e d ( F i g u r e 6.6 C ) . I n t h e l a r g e a g g r e g a t e s o f z o o s p o r e s , s o m e o f the o u t e r z o o s p o r e s w e r e n o t f l u o r e s c i n g as b r i g h t l y as t h e c e n t r a l l y l o c a t e d z o o s p o r e s ( F i g u r e 6.6 C). T h e individual zoospores observed were fluorescing u n i f o r m l y over the b o d y a n d f l a g e l l u m ( F i g u r e 6.6 B a n d 6.6 C ) . O. brassicae z o o s p o r e s l o s t m o t i l i t y w i t h t h e addition o f T P A - F I T C but n o fluorescent aggregates o f zoospores were observed (Figure 6.6 B ) . C o m p e t i t i o n b e t w e e n L ( - ) - f u c o s e a n d T P A - F I T C f o r b i n d i n g t o Olpidium zoospores was not performed.  125  6.2.6 D i s c u s s i o n T h e p r e s e n t s t u d y w a s u n d e r t a k e n to assess the nature o f sugars o n the s u r f a c e o f O. bornovanus a n d O. brassicae z o o s p o r e s . F i v e F I T C - l a b e l l e d l e c t i n s w i t h d i f f e r e n t  c a r b o h y d r a t e b i n d i n g affinities w e r e u s e d i n c o n j u n c t i o n w i t h e p i f l u o r e s c e n c e m i c r o s c o p y . T h e results o f these b i n d i n g s t u d i e s s u g g e s t that the s u r f a c e o f b o t h O. bornovanus a n d O. brassicae z o o s p o r e s c o n t a i n D ( + ) - m a n n o s e and/or D ( + ) - g l u c o s e a n d  L ( - ) - f u c o s e - c o n t a i n i n g o l i g o s a c c h a r i d e s . D ( + ) - g a l a c t o s e a p p e a r s to b e o n the s u r f a c e o f O. brassicae s e c o n d a r y z o o s p o r e s b u t n o t o n the s u r f a c e o f O. bornovanus z o o s p o r e s . C h i t i n c o v e r s the s u r f a c e s o f r e s t i n g s p o r e s o f b o t h Olpidium s p e c i e s . W a s h i n g o f the z o o s p o r e s u s p e n s i o n b e f o r e the a d d i t i o n o f F I T C - l a b e l l e d l e c t i n s ( e x c e p t f o r C o n A - F I T C ) f a c i l i t a t e d b i n d i n g . B i n d i n g o f C o n A - F I T C to Olpidium z o o s p o r e s o c c u r r e d to s i m i l a r l e v e l s w i t h o r w i t h o u t p r e v i o u s w a s h i n g o f the z o o s p o r e s . Initially, w i t h o u t p r e v i o u s w a s h i n g o f the z o o s p o r e s , o n l y d e b r i s w a s o b s e r v e d f l u o r e s c i n g w i t h the a d d i t i o n o f a l l l e c t i n s ( e x c e p t f o r C o n A - F I T C ) . A f t e r i n c l u d i n g the i n i t i a l z o o s p o r e - w a s h i n g step, s t r o n g f l u o r e s c e n c e w a s o b s e r v e d f o r T P A - F I T C b i n d i n g o f O. bornovanus z o o s p o r e s a n d s t r o n g f l u o r e s c e n c e f o r T P A - F I T C a n d B P A - F I T C b i n d i n g o f O. brassicae z o o s p o r e s . W i t h the a d d i t i o n o f this i n i t i a l w a s h i n g step, b a c k g r o u n d f l u o r e s c e n c e w a s r e d u c e d . It is p o s s i b l e that w a s h i n g r e m o v e d s u b s t a n c e s f r o m the z o o s p o r e s o l u t i o n w h i c h s e q u e s t e r e d the F I T C - l e c t i n s , m a k i n g t h e m u n a v a i l a b l e f o r b i n d i n g to the z o o s p o r e surface. T h e s e i n h i b i t o r s m a y h a v e s l o u g h e d o f f o f the z o o s p o r e s u r f a c e o r t h e y m a y h a v e b e e n r e l e a s e d f r o m the m a t u r e t h a l l u s c o n c u r r e n t l y w i t h z o o s p o r e r e l e a s e f r o m r o o t cells. It i s p o s s i b l e that the i n h i b i t o r s w e r e c a r b o h y d r a t e s or t h e y c o u l d a l s o b e n o n - s p e c i f i c i n h i b i t o r s o f u n k n o w n b i o c h e m i c a l o r i g i n . 126  T w o F I T C - l e c t i n s b o u n d to z o o s p o r e s o f b o t h s p e c i e s o f Olpidium, C o n A - F I T C , and T P A - F I T C . C o n A - F I T C binds D(+)-mannose, and D(+)-glucose with lower affinity, so it i s l i k e l y that there are m a n n o s e and/or g l u c o s e - c o n t a i n i n g o l i g o s a c c h a r i d e s o n the s u r f a c e s o f z o o s p o r e s o f b o t h O. bornovanus a n d O. brassicae. D ( + ) - m a n n o s e at 100 m g / m l was a b l e to c o m p e t i t i v e l y i n h i b i t the b i n d i n g o f C o n A - F I T C to the z o o s p o r e s o f b o t h Olpidium s p e c i e s , i n d i c a t i n g that C o n A b i n d i n g i s s p e c i f i c . A l t h o u g h D ( + ) - m a n n o s e is a b l e to c o m p e t i t i v e l y i n h i b i t C o n A - F I T C b i n d i n g to Olpidium s e o n d a r y z o o s p o r e s , this d o e s n o t p r o v e that D ( + ) - m a n n o s e is the z o o s p o r e s u g a r w h i c h i s r e c o g n i z e d b y C o n A F I T C . T h e p o s s i b i l i t y that C o n A - F I T C i s b i n d i n g to o t h e r c e l l s u r f a c e c o m p o n e n t s cannot be excluded. T h e b i n d i n g o f T P A - F I T C , w h i c h binds L(-)-fucose with high affinity, to the z o o s p o r e s o f b o t h s p e c i e s o f Olpidium suggests that L ( - ) - f u c o s e is a l s o o n the s u r f a c e o f these f u n g i . It w i l l b e n e c e s s a r y to a d d L ( - ) - f u c o s e to T P A - F I T C p r i o r to z o o s p o r e b i n d i n g to d e t e r m i n e i f this b i n d i n g i s s p e c i f i c . B P A - F I T C d i d n o t b i n d to O. bornovanus z o o s p o r e s b u t d i d b i n d to O. brassicae z o o s p o r e s . B P A r e c o g n i z e s N - a c e t y l g a l a c t o s a m i n e a n d D ( + ) - g a l a c t o s e a n d so it is l i k e l y that o n e o f these sugars i s o n the s u r f a c e o f O. brassicae (but n o t o n O. bornovanus z o o s p o r e s ) . T h e a d d i t i o n o f D ( + ) - g a l a c t o s e to B P A - F I T C p r i o r to a d d i t i o n o f the O. brassicae z o o s p o r e s u s p e n s i o n i n d i c a t e d that b i n d i n g was s p e c i f i c . P r e l i m i n a r y  c o m p e t i t i o n b i n d i n g assays, w i t h the a d d i t i o n o f 100 m g / m l D ( + ) - g a l a c t o s e to T N V f o r 60 m i n b e f o r e the a d d i t i o n o f the O. brassicae z o o s p o r e s u s p e n s i o n i n the b i n d i n g a s s a y s h o w e d a s i g n i f i c a n t r e d u c t i o n i n T N V b i n d i n g to O. brassicae ( M . R o b b i n s , u n p u b l i s h e d o b s e r v a t i o n ) . A l t h o u g h f u r t h e r w o r k is s t i l l r e q u i r e d , these d a t a s u g g e s t that D ( + ) galactose is a component o f a putative zoospore receptor for T N V  127  attachment.  W G A - F I T C d i d n o t b i n d t o z o o s p o r e s o f O. bornovanus o r O. brassicae b u t d i d b i n d t o w h a t appears t o b e r e s t i n g spores o f b o t h s p e c i e s o f Olpidium w i t h i n t h e roots. T h e b i n d i n g o f W G A , w h i c h b i n d s c h i t i n ( N , N ' , N ' - t r i a c e t y l c h i t o t r i o s e a n d N , N'd i a c e t y l c h i t o b i o s e ) , i n d i c a t e s that there i s c h i t i n o n t h e s u r f a c e o f Olpidium r e s t i n g spores. C e l l w a l l s o f f u n g i u s u a l l y c o n t a i n m i c r o f i b r i l l a r c h i t i n ( S e l i t r e n n i k o f f et  al.,  1980). T h e r e s t i n g spores o f Olpidium are t h e f u n g a l structures w h i c h m u s t s u r v i v e i n s o i l i n t h e a b s e n c e o f a p p r o p r i a t e c o n d i t i o n s f o r g r o w t h . O n e r o l e o f the c e l l w a l l i s p r o t e c t i o n o f the f u n g u s f r o m o t h e r m i c r o b e s s u c h as b a c t e r i a ( P o w e l l , 1994). E C A - F I T C d i d n o t b i n d t o z o o s p o r e s o f O. bornovanus o r O. brassicae. E C A F I T C b i n d s t o N - a c e t y l l a c t o s a m i n e a n d oc-lactose, i n d i c a t i n g that n e i t h e r o f these sugars are o n t h e s u r f a c e s o f Olpidium z o o s p o r e s . A d i f f e r e n t i a l e x p r e s s i o n o f sugars b e t w e e n O. bornovanus a n d O. brassicae m a y h e l p t o e x p l a i n t h e d i f f e r e n t i a l abilities t o b i n d a n d t r a n s m i t s e v e r a l s o i l - t r a n s m i t t e d viruses. Studies o f zoospores o f Chytridiomycete r u m e n fungi using lectin probes i n d i c a t e d that t h e l e c t i n b i n d i n g pattern f o r d i f f e r e n t i s o l a t e s o f the s a m e g e n u s v a r i e d ( G u i l l o t et al., 1990). T h e s e results s u g g e s t e d that c e l l coats o f r u m e n f u n g a l z o o s p o r e s v a r y i n c o m p o s i t i o n , p e r h a p s e n a b l i n g t h e m t o adapt to d i f f e r e n c e s i n t h e r u m e n e n v i r o n m e n t ( P o w e l l , 1994). G l y c o p r o t e i n s at t h e c e l l s u r f a c e o f o t h e r c h y t r i d z o o s p o r e s b i n d l e c t i n s a n d m a y s e r v e as p o t e n t i a l sites t r i g g e r i n g e n c y s t m e n t o r i n r e c o g n i t i o n o f e n v i r o n m e n t a l c u e s ( D a l l e y a n d S o n n e b o r n , 1982; J e n a n d H a u g , 1979). T h i s w o r k h a s s h o w n that sugars a r e l o c a t e d o n t h e s u r f a c e s o f O. bornovanus a n d O. brassicae z o o s p o r e s , i n c l u d i n g D ( + ) - m a n n o s e and/or D ( + ) - g l u c o s e  and L(-)-fucose.  T h e s e sugars a r e l i k e l y present as o l i g o s a c c h a r i d e s c o v a l e n t l y l i n k e d t o p r o t e i n s 128  ( g l y c o p r o t e i n s ) o r l i p i d s ( g l y c o l i p i d s ) . In the future, e x a m i n a t i o n o f the b i n d i n g o f o t h e r F I T C - l e c t i n s to Olpidium z o o s p o r e s m a y p r o v i d e f u r t h e r i n f o r m a t i o n a b o u t sugars l o c a t e d o n the s u r f a c e o f these f u n g i . T h e i d e n t i f i c a t i o n o f a d i f f e r e n t i a l e x p r e s s i o n o f sugars b e t w e e n O. bornovanus a n d O. brassicae, w h e r e N - a c e t y l g a l a c t o s a m i n e and/or D ( + ) g a l a c t o s e a p p e a r to b e l o c a t e d o n the s u r f a c e o f O. brassicae b u t not o n O. bornovanus, is i n t e r e s t i n g i n l i g h t o f the d i f f e r e n t i a l b i n d i n g o f these f u n g i to v i r u s e s w h i c h t h e y transmit. It i s h o p e d that the r e s e a r c h d e s c r i b e d i n this c h a p t e r w i l l c o n t r i b u t e to the i d e n t i f i c a t i o n o f a s p e c i f i c r e c e c p t o r site f o r v i r u s e s o n the s u r f a c e o f Olpidium z o o s p o r e s .  129  7 C H A P T E R  G E N E R A L  S E V E N :  D I S C U S S I O N  In this t h e s i s the i n t e r a c t i o n b e t w e e n a p l a n t virus, C N V , a n d its f u n g a l v e c t o r , O. bornovanus, was e x a m i n e d at the m o l e c u l a r l e v e l to d e t e r m i n e i f there are s p e c i f i c  s e q u e n c e s and/or structures a s s o c i a t e d w i t h v i r u s p a r t i c l e s and/or f u n g a l z o o s p o r e s m e d i a t i n g t r a n s m i s s i o n . A t the start o f this thesis, the first g o a l was to d e t e r m i n e i f s p e c i f i c s e q u e n c e s and/or structures o f the C N V C P w e r e i n v o l v e d i n t r a n s m i s s i o n s p e c i f i c i t y . P r e v i o u s w o r k i n o u r l a b o r a t o r y s h o w e d that the C N V C P i s r e s p o n s i b l e f o r the s p e c i f i c i t y o f C N V t r a n s m i s s i o n b y z o o s p o r e s o f O. bornovanus ( M c L e a n et al., 1994). V i r u s e s w i t h m u t a t i o n s i n their C P g e n e s w i t h a l t e r e d t r a n s m i s s i b i l i t y w e r e r e q u i r e d to f u r t h e r d e l i n e a t e r e g i o n s o f the C N V C P i n v o l v e d i n t r a n s m i s s i o n s p e c i f i c i t y . N o C N V v a r i a n t s h a d yet b e e n i d e n t i f i e d w i t h a r e d u c e d a b i l i t y to b e t r a n s m i t t e d b y O. bornovanus. T o i d e n t i f y s u c h t r a n s m i s s i o n mutants, a p o p u l a t i o n o f C N V  which had  b e e n m e c h a n i c a l l y p a s s a g e d f o r 13 g e n e r a t i o n s i n the a b s e n c e o f its f u n g a l v e c t o r ( R o c h o n , 1991) was i n o c u l a t e d o n t o a l o c a l l e s i o n h o s t a n d i n d i v i d u a l l o c a l l e s i o n s e x a m i n e d f o r their a b i l i t y to b e t r a n s m i t t e d b y z o o s p o r e s o f O. bornovanus ( C h a p t e r 3). Several natural variants o f C N V were identified which were inefficiently transmitted by O. bornovanus ( 0 - 2 0 % o f w i l d - t y p e  CNV).  T h e C N V t r a n s m i s s i o n m u t a n t s w e r e c h a r a c t e r i z e d at the m o l e c u l a r l e v e l to assess the b a s i s f o r their r e d u c e d t r a n s m i s s i b i l i t y . A r e d u c t i o n i n t r a n s m i s s i o n c o u l d b e the result o f p r o b l e m s i n a n y o f s e v e r a l aspects o f the v i r a l l i f e - c y c l e i n c l u d i n g r e d u c e d r e p l i c a t i o n , l o w e r p a r t i c l e stability, o r i n e f f i c i e n t a t t a c h m e n t to p u t a t i v e r e c e p t o r 130  m o l e c u l e s a s s o c i a t e d w i t h the vector. It w a s h y p o t h e s i z e d that s o m e v i r a l v a r i a n t s w o u l d b e i d e n t i f i e d w h i c h h a d l o s t t h e i r a b i l i t y to attach to the f u n g a l vector. T h e s e attachmentd e f i c i e n t v a r i a n t s c o u l d b e a n a l y z e d to d e t e r m i n e s e q u e n c e s and/or structures d i f f e r i n g f r o m w i l d - t y p e i n the h o p e s o f i d e n t i f y i n g v i r a l attachment sites o n the p a r t i c l e surface. It w a s f o u n d that s o m e C N V m u t a n t s r e p l i c a t e d p o o r l y as a r e s u l t o f the p r e s e n c e o f D I R N A s a n d others w e r e m i s s i n g the C P c o d i n g r e g i o n f r o m t h e i r g R N A ( C h a p t e r 3). O t h e r m u t a n t s l i k e l y h a d m u t a t i o n s w i t h i n t h e i r C P g e n e s as these v i r u s e s d i d not p r o d u c e C P d e s p i t e c o n t a i n i n g w i l d - t y p e l e n g t h g R N A . O n e m u t a n t , L L 5 , h a d stable p a r t i c l e s a n d r e p l i c a t e d to w i l d - t y p e l e v e l s i n plants. It w a s h y p o t h e s i z e d that L L 5 h a d o n e o r m o r e m u t a t i o n s i n its C P p r e v e n t i n g v i r i o n s f r o m a t t a c h i n g to the z o o s p o r e surface. O n e p r o b l e m w i t h this n e g a t i v e s e l e c t i o n strategy f o r o b t a i n i n g v i r a l m u t a n t s i s that reasons f o r the p h e n o t y p e other t h a n those w h i c h are s o u g h t m u s t b e e l i m i n a t e d b y c a r e f u l e x a m i n a t i o n o f s e v e r a l c h a r a c t e r i s t i c s o f the m u t a n t . L L 5 w a s f u r t h e r c h a r a c t e r i z e d b y c l o n i n g a n d s e q u e n c i n g o f its C P g e n e t o d e t e r m i n e i f there w e r e d i f f e r e n c e s b e t w e e n its C P a n d that o f w i l d - t y p e C N V . T h e C P g e n e o f L L 5 c o n t a i n e d t w o s i n g l e n u c l e o t i d e s u b s t i t u t i o n s ; one, i n the s h e l l d o m a i n w h i c h p r o d u c e d a n a m i n o a c i d s u b s t i t u t i o n o f G l u to L y s , a n d a s e c o n d i n the a r m w h i c h c h a n g e d a P h e t o C y s ( C h a p t e r 3). T h e L L 5 C P g e n e w a s c l o n e d i n t o the C N V c D N A c l o n e a n d i t w a s d e t e r m i n e d that the L L 5 C P i s r e s p o n s i b l e f o r r e d u c e d t r a n s m i s s i o n . S i t e - d i r e c t e d m u t a g e n e s i s to p r o d u c e the t w o n u c l e o t i d e s u b s t i t u t i o n s i n d i v i d u a l l y i n separate C N V c D N A c l o n e s i n d i c a t e d that i t w a s the G l u to L y s c h a n g e i n the L L 5 C P s h e l l d o m a i n w h i c h w a s r e s p o n s i b l e f o r its r e d u c e d t r a n s m i s s i b i l i t y ( C h a p t e r 3). Interestingly, the L L 5 G l u to L y s c h a n g e i s i m m e d i a t e l y adjacent to a n a s p a r t i c a c i d  131  r e s i d u e k n o w n to m e d i a t e s u b u n i t - s u b u n i t c o n t a c t s i n the v i r u s p a r t i c l e b y b i n d i n g C a . + +  It is p o s s i b l e that the G l u to L y s m u t a t i o n i n the L L 5 s h e l l d o m a i n c a u s e d a d e s t a b i l i z a t i o n o f the p a r t i c l e so that the A s p r e s i d u e s c o u l d n o t b i n d C a , t h e r e b y + +  c a u s i n g s w e l l i n g o f the p a r t i c l e a n d a r e d u c e d a b i l i t y to b i n d to the z o o s p o r e s u r f a c e d u e to a l o s s o f the v i r a l a t t a c h m e n t site. A l t e r n a t i v e l y , the c h a r g e c h a n g e r e s u l t a n t f r o m the G l u to L y s c h a n g e c o u l d h a v e e l i m i n a t e d the a b i l i t y o f a p u t a t i v e r e c e p t o r m o l e c u l e o n the z o o s p o r e s u r f a c e to b i n d to the v i r a l a t t a c h m e n t site. A s l o w e r m i g r a t i o n was  observed  f o r L L 5 particles, w h i c h w o u l d r e s u l t f r o m a c h a r g e c h a n g e a n d c o u l d a l s o b e a f f e c t e d b y s w e l l i n g ( C h a p t e r 3). In the future, e x p e r i m e n t s to e x a m i n e the s e d i m e n t a t i o n c o e f f i c i e n t s o f C N V a n d L L 5 s h o u l d b e a b l e to d e t e r m i n e i f p a r t i c l e s h a v e an a l t e r e d conformation. T h e m e t h o d o l o g y d e v e l o p e d f o r the c h a r a c t e r i z a t i o n o f L L 5 w i l l b e u s e f u l i n the f u t u r e to e x a m i n e m o r e l o c a l l e s i o n s f r o m t h i s p a s s a g e d p o p u l a t i o n o f C N V to f u r t h e r i d e n t i f y r e g i o n s o f the C N V C P i n v o l v e d i n t r a n s m i s s i o n a n d a l s o to d e t e r m i n e the f r e q u e n c y o f the types o f m u t a n t s i d e n t i f i e d i n this study. S u c h s t u d i e s are i n f a c t u n d e r w a y ( K . K a k a n i a n d D. R o c h o n ) a n d s i x a d d i t i o n a l C N V m u t a n t s h a v e b e e n i d e n t i f i e d a n d c h a r a c t e r i z e d . A l s o , o t h e r f u n g u s - t r a n s m i t t e d v i r u s e s , s u c h as M N S V , C L S V , o r T N V , c o u l d b e p a s s a g e d m e c h a n i c a l l y i n the a b s e n c e o f t h e i r Olpidium v e c t o r . T h e l o c a l l e s i o n a s s a y c o u l d b e u s e d to i d e n t i f y v a r i a n t s f r o m these p o p u l a t i o n s o f v i r u s w h i c h h a v e a r e d u c e d a b i l i t y to b e t r a n s m i t t e d to d e t e r m i n e i f s i m i l a r o r d i f f e r e n t r e g i o n s o n the c a p s i d s o f v i r u s e s other t h a n C N V are i n v o l v e d i n the s p e c i f i c i t y o f t r a n s m i s s i o n . T o test the h y p o t h e s i s that L L 5 h a d r e d u c e d t r a n s m i s s i o n d u e to a r e d u c e d a b i l i t y to b i n d to O. bornovanus z o o s p o r e s a b i n d i n g a s s a y was d e v e l o p e d ( C h a p t e r 3). U s i n g  132  this b i n d i n g assay it w a s d e t e r m i n e d that L L 5 v i r i o n s b o u n d to 5 0 % the l e v e l s o f w i l d type C N V , i n d i c a t i n g that at least part o f its r e d u c t i o n i n t r a n s m i s s i o n i s d u e to a r e d u c e d a b i l i t y to b i n d to O. bornovanus z o o s p o r e s . T h e n o n - t r a n s m i s s i b l e b u t c l o s e l y r e l a t e d T B S V - C h d i d n o t b i n d to O. bornovanus, i n d i c a t i n g that the b i n d i n g assay i s c o n s i s t e n t w i t h the b i o l o g y o f these v i r u s - f u n g u s i n t e r a c t i o n s . T h e b i n d i n g assay c o u l d b e u s e d i n future a n a l y s e s o f l o c a l l e s i o n s to d e t e r m i n e w h i c h p o t e n t i a l C N V m u t a n t s s h o w r e d u c e d b i n d i n g to O. bornovanus, t h e r e b y e l i m i n a t i n g m u t a n t s w i t h d e f e c t s i n o t h e r areas o f the infection cycle. T h e a m i n o a c i d s e q u e n c e o f the C L S V C P w a s a l i g n e d w i t h o t h e r O. bornovanustransmitted virues ( C N V , M N S V , and R C N M V ) and nine small spherical viruses not t r a n s m i t t e d b y O. bornovanus to d e t e r m i n e i f r e g i o n s o f s i m i l a r a m i n o a c i d s e q u e n c e c o u l d b e i d e n t i f i e d i n c o m m o n b e t w e e n the O. boraovanw.s'-transmitted  viruses w h i c h  w e r e n ' t p r e s e n t i n the n o n - t r a n s m i t t e d v i r u s e s ( C h a p t e r 4). S e v e r a l r e g i o n s o f C P s e q u e n c e i d e n t i t y w e r e i d e n t i f i e d w h i c h are c a n d i d a t e s f o r m u t a t i o n a l a n a l y s i s as p o t e n t i a l v i r a l attachment sites to p u t a t i v e r e c e p t o r m o l e c u l e s o n the z o o s p o r e surface. A d d i t i o n a l l y , p h y l o g e n e t i c a n a l y s i s b a s e d o n these a l i g n m e n t s s u g g e s t e d that the C P s o f the f o u r O. bornovanus-transmitted v i r u s e s are e v o l u t i o n a r y c l o s e l y r e l a t e d to o n e another d e s p i t e t h e i r b e i n g a s s i g n e d to d i s t i n c t t a x o n o m i c groups. T h e b i n d i n g assay d e v e l o p e d i n C h a p t e r 3 to c h a r a c t e r i z e L L 5 w a s u s e d i n C h a p t e r 5 to d e t e r m i n e i f a p u t a t i v e r e c e p t o r f o r C N V e x i s t s o n the s u r f a c e o f O. bornovanus z o o s p o r e s .  O n e i n t e r e s t i n g o b s e r v a t i o n w a s that b i n d i n g w a s p H - d e p e n d e n t ,  w i t h m a x i m u m b i n d i n g o c c u r r i n g at p H 7.2 to 7.6. B i n d i n g w a s a l s o f o u n d to i n c r e a s e w i t h time, w i t h 5 0 % o f the b i n d i n g o c c u r r i n g w i t h i n the first 10 m i n a n d e q u i l i b r i u m  133  b e i n g r e a c h e d after 6 0 m i n . T h e s p e c i f i c i t y o f v i r u s t r a n s m i s s i o n b y Olpidium spp. w a s r e f l e c t e d i n o u r b i n d i n g assay data, w h e r e C N V , M N S V , a n d C L S V , w h i c h are t r a n s m i t t e d b y O. bornovanus z o o s p o r e s , b o u n d O. bornovanus to h i g h e r l e v e l s t h a n T N V , w h i c h is not t r a n s m i t t e d b y O. bornovanus. T N V , w h i c h is t r a n s m i t t e d b y O. brassicae z o o s p o r e s , b o u n d to O. brassicae to h i g h e r l e v e l s t h a n C N V , M N S V , o r C L S V .  B i n d i n g o f C N V to O. bornovanus a n d T N V to O. brassicae w e r e f o u n d to b e saturable, w i t h a p p r o x i m a t e l y 1.75 X 1 0 C N V v i r i o n s b o u n d p e r z o o s p o r e a n d 1.25 X 1 0 T N V 4  4  v i r i o n s b o u n d p e r z o o s p o r e . C N V w a s f o u n d to c o m p e t e w i t h M N S V a n d T N V f o r b i n d i n g to O. bornovanus b u t d i d not c o m p e t e w i t h C L S V . T a k e n together, these r e s u l t s i n d i c a t e that s p e c i f i c m o l e c u l e s e x i s t o n the s u r f a c e o f Olpidium z o o s p o r e s w h i c h facilitate v i r u s b i n d i n g a n d t r a n s m i s s i o n . F u r t h e r e x p e r i m e n t s u s i n g g l y c o s i d a s e a n d p r o t e a s e treatment o f z o o s p o r e s p r i o r to b i n d i n g m a y h e l p to d e t e r m i n e the nature o f these putative receptor molecules. T o c o m p l e m e n t o t h e r s t u d i e s u n d e r w a y i n m y l a b o r a t o r y o n the nature o f p u t a t i v e r e c e p t o r s o n the s u r f a c e o f Olpidium z o o s p o r e s , F I T C - l e c t i n s w e r e a d d e d to z o o s p o r e s i n the b i n d i n g a s s a y to d e t e r m i n e the nature o f sugars o n the s u r f a c e o f these z o o s p o r e s . T h e b i n d i n g o f C o n A - F i T C to O. bornovanus a n d O. brassicae i n d i c a t e d that D ( + ) m a n n o s e and/or D ( + ) - g l u c o s e are p o s s i b l y c o m p o n e n t s o f the s u r f a c e s o f z o o s p o r e s o f b o t h o f these f u n g i . T P A - F I T C a l s o b o u n d to z o o s p o r e s o f b o t h f u n g i , i n d i c a t i n g that L ( )-fucose is p r o b a b l y a l s o o n the s u r f a c e s o f b o t h s p e c i e s o f Olpidium. E C A - F I T C d i d n o t b i n d to z o o s p o r e s o f e i t h e r fungus, s u g g e s t i n g that a - l a c t o s e i s n o t p r e s e n t o n s u r f a c e s o f either O. bornovanus o r O. brassicae. Interestingly, B P A - F I T C , w h i c h b i n d s D ( + ) g a l a c t o s e , b o u n d to z o o s p o r e s o f O. brassicae b u t n o t to O. bornovanus, i n d i c a t i n g that  134  D ( + ) - g a l a c t o s e i s o n l y l o c a t e d o n the s u r f a c e o f O. brassicae z o o s p o r e s .  The  i d e n t i f i c a t i o n o f a d i f f e r e n t i a l e x p r e s s i o n o f sugars b e t w e e n O. bornovanus a n d O. brassicae i n d i c a t e s that this s u g a r ( D ( + ) - g a l a c t o s e ) c o u l d b e part o f a c a n d i d a t e v i r a l  r e c e p t o r o n the z o o s p o r e surface. M o r e F I T C - l e c t i n s s h o u l d b e e x a m i n e d f o r b i n d i n g to Olpidium z o o s p o r e s i n p a r a l l e l to o t h e r m e a n s o f e x a m i n a t i o n o f c a n d i d a t e m o l e c u l e s f o r  their r o l e i n f a c i l i t a t i n g t r a n s m i s s i o n o f p l a n t v i r u s e s . T h e m a j o r s i g n i f i c a n c e o f the w o r k i n this thesis i s that s e v e r a l m e t h o d o l o g i e s w e r e d e v e l o p e d i n c l u d i n g the i s o l a t i o n a n d e x a m i n a t i o n o f l o c a l l e s i o n m u t a n t s o f  CNV  a n d the d e v e l o p m e n t o f a b i n d i n g a s s a y b e t w e e n C N V a n d O. bornovanus z o o s p o r e s . T h e s e m e t h o d o l o g i e s w e r e u s e d to c h a r a c t e r i z e o n e C N V t r a n s m i s s i o n m u t a n t , L L 5 , w i t h a m u t a t i o n i n the C N V C P s h e l l d o m a i n w h i c h m a y c o n t a i n a v i r a l a t t a c h m e n t site to a p u t a t i v e r e c e p t o r m o l e c u l e o n the z o o s p o r e surface. A m i n o a c i d a l i g n m e n t s u s i n g the C P s o f f o u r O. bornovanus-transmitted v i r u s e s i n d i c a t e d n e w s e q u e n c e s w h i c h s h o u l d b e m u t a g e n i z e d to d e t e r m i n e their r o l e i n the s p e c i f i c i t y o f t r a n s m i s s i o n , to f u r t h e r d e l i n e a t e the v i r a l a t t a c h m e n t site. U s i n g the b i n d i n g assay, the s p e c i f i c i t y , saturability, a n d c o m p e t i t i o n o f v i r u s b i n d i n g to Olpidium z o o s p o r e s i n d i c a t e d that a s p e c i f i c r e c e p t o r m o l e c u l e e x i s t s o n the s u r f a c e o f z o o s p o r e s o f these f u n g i . P r e l i m i n a r y w o r k to d e t e r m i n e the nature o f sugars o n the s u r f a c e o f Olpidium z o o s p o r e s u s i n g the b i n d i n g o f F I T C - l e c t i n s m a y a i d i n c h a r a c t e r i z a t i o n o f the p u t a t i v e receptor. In the n e x t p h a s e o f this research, o t h e r b i o c h e m i c a l t e c h n i q u e s s h o u l d b e u t i l i z e d to d e t e r m i n e the nature o f the p u t a t i v e r e c e p t o r o n the Olpidium z o o s p o r e surface. T h e use o f c a n d i d a t e sugars to c o m p e t e w i t h v i r u s b i n d i n g to z o o s p o r e s i n the b i n d i n g a s s a y a n d a l s o to z o o s p o r e extracts o n m e m b r a n e s ( s u c h as the use o f D ( + ) - g a l a c t o s e to  135  c o m p e t e w i t h T N V b i n d i n g to O. brassicae z o o s p o r e s ) m a y be u s e f u l i n d e t e r m i n i n g i f c a n d i d a t e sugars i d e n t i f i e d v i a F I T C - l e c t i n b i n d i n g are i n d e e d i n v o l v e d i n v i r u s b i n d i n g . G e n e t i c t e c h n i q u e s s u c h as the p r o d u c t i o n o f c D N A l i b r a r i e s f o r O. bornovanus a n d O. brassicae a n d the u t i l i z a t i o n o f s u b t r a c t i v e h y b r i d i z a t i o n m a y a i d i n the i s o l a t i o n o f  • c a n d i d a t e g e n e s w h i c h are i n v o l v e d i n v i r u s b i n d i n g a n d the s p e c i f i c i t y o f t r a n s m i s s i o n . 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