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Puccinia graminis f.sp. tritici, race c17 : physiology of uredospore germination and germtube differentiation Hopkinson, Sarah J. 1988

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PUCCINIA GRAMINIS f . s p . TRITICI, RACE C17: PHYSIOLOGY OF UREDOSPORE GERMINATION AND GERMTUBE DIFFERENTIATION By S a r a h J . Hopkinson B.Sc,  The U n i v e r s i t y o f V i c t o r i a , 1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department  of Plant Science)  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © Sarah Jane Hopkinson,  1988  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  or  publication of this thesis  her  P\c\rVv  DE-6 (2/88)  hnw  \Q  requirements  for  an  advanced  I agree that the Library shall make it  purposes  may  representatives.  It  be is  granted  by the head  understood  that  of  my  copying  or  for financial gain shall not be allowed without my written  *"Sr.\cy,r\c v>.  The University of British Columbia Vancouver, Canada  Date  the  I further agree that permission for extensive  permission.  Department of  of  ABSTRACT  G e r m i n a t i n g u r e d o s p o r e s o f r a c e C17  of Puccinia  graminis  f . s p . t r i t i c i form c h a r a c t e r i s t i c i n f e c t i o n s t r u c t u r e s ( a p p r e s s o r i u m , i n f e c t i o n peg, r e s p o n s e t o a 1.5 germination  a t 19°  h h e a t shock a t 29° C.  The  i n f e c t i o n s t r u c t u r e s was a l c o h o l and,  v e s i c l e , i n f e c t i o n hypha) i n C administered  2 h after  p r o p o r t i o n of s p o r e l i n g s forming  augmented by n u t r i e n t s , n - n o n y l  an a p p r o p r i a t e l y t i m e d h e a t shock.  The  heat  shock t e m p e r a t u r e r e q u i r e d t o i n d u c e maximum d i f f e r e n t i a t i o n had a v e r y p r e c i s e optimum w h i c h v a r i e d s l i g h t l y f o r each spore l o t .  V a r i a t i o n s one degree above o r below t h i s optimum  r e d u c e d t h e p e r c e n t d i f f e r e n t i a t i o n by g r e a t e r t h a n 40%.  The  p r e s e n c e o f an i n h i b i t o r o f p r o t e i n s y n t h e s i s , p u r o m y c i n , i n the germination  medium: (1) p r e v e n t e d  uredosporeling  d i f f e r e n t i a t i o n b u t had no e f f e c t on g e r m i n a t i o n ,  (2)  s i g n i f i c a n t l y reduced t h e p r o p o r t i o n o f germtubes f o r m i n g a p p r e s s o r i a , and germtube n u c l e i .  (3) i n most c a s e s p r e v e n t e d t h e d i v i s i o n o f I t was  concluded t h a t e s s e n t i a l  d i f f e r e n t i a t i o n - s p e c i f i c p r o t e i n s are synthesized onset of germination,  throughout the formation  and t o t h e c o m p l e t i o n o f d i f f e r e n t i a t i o n .  of  from t h e appressoria  These r e s u l t s were  c o n s i s t e n t w i t h t h e observed e f f e c t s o f h e a t shock on t h e of p r o t e i n h y d r o l y s i s .  During germination  t h e r e was  a  h y d r o l y s i s o f p r o t e i n l e a d i n g t o an i n c r e a s e i n s i z e o f endogenous p o o l o f f r e e amino a c i d s and t o an  increased  rate  net the  leakage of amino acids to the germination medium.  Heat shock  e f f e c t i v e l y reduced the amount of endogenous free amino acids and the extent to which amino acids were l o s t to the medium. I t was  concluded that i n heat shocked sporelings protein  synthesis was  increased r e l a t i v e to protein hydrolysis by  comparison with the r e l a t i v e rates of these two processes i n germinating was  (non-shocked) uredosporelings.  Moreover, there  no net protein synthesis during the formation of  structures induced by heat shock. the germination medium was shocked sporelings.  infection  The loss of amino acids to  selective, p a r t i c u l a r l y i n heat  iv TABLE OF CONTENTS Page Number Abstract  i i  L i s t of Tables  viii  L i s t of Figures  x  Acknowledgements  xiii  1.  Introduction  1  2.  L i t e r a t u r e Review  6  2.1  2.2  2.3  U r e d o s p o r e G e r m i n a t i o n and Morphogenesis 2.1.1  G e r m i n a t i o n I n h i b i t o r s and S t i m u l a n t s  ....  6  2.1.2  Thigmodifferentiation  7  2.1.3  Thermodifferentiation  8  2.1.4  Chemodifferentiation  9  2.1.5  Mechanisms o f D i f f e r e n t i a t i o n  9  2.1.6  Role of I n f e c t i o n Structures  10  C y t o l o g i c a l Events 2.2.1  N u c l e a r Changes  11  2.2.2  P r o t e i n Metabolism  13  N u t r i t i o n a l Requirements o f R u s t F u n g i 2.3.1  P h y s i o l o g y o f t h e H o s t - P a r a s i t e Complex .  2.3.2  A x e n i c C u l t u r e and M e t a b o l i s m  17  N u t r i t i o n a l Requirements  20  Synthetic Capacity  21  Sulphur Metabolism  22  M e t a b o l i t e Leakage  22  Endogenous F r e e Amino A c i d s  24  E x p e r i m e n t a l Methods  26  3.1  P r o d u c t i o n and C o l l e c t i o n o f Spores  26  3.2  Spore G e r m i n a t i o n  27  3.3  Spore G e r m i n a t i o n w i t h t h e I n d u c t i o n t o D i f f e r e n t i a t e 27  3.4  C r i t e r i a f o r t h e Assessment o f S p o r e l i n g  Development 27  3.5  S t a i n i n g and Counts  30  3.6  Temperature Range T r i a l s  30  3.7  E s s e n t i a l P r o t e i n S y n t h e s i s : Puromycin  33  3.8  N u c l e a r S t a i n i n g : DAPI  35  3.9  Amino A c i d A n a l y s i s  37  3.9.1  H i g h Performance L i q u i d Chromatography  ..  37  3.9.2  Reagents  39  3.9.3  Instrument  40  3.9.4  Sample C o l l e c t i o n  40  3.9.5  Sample Clean-up  43  3.9.6  HPLC o f Amino A c i d s as P h e n y l t h i o c a r b a m o y l Derivatives B u f f e r System  45  Pre-Column D e r i v a t i z a t i o n  45  Sample P r e p a r a t i o n  46  Chromatography  46  VI  Results  50  4.1  Uredospore  G e r m i n a t i o n and D i f f e r e n t i a t i o n  50  4.2  Temperature Range T r i a l s  4.3  I n f l u e n c e o f N u t r i e n t s on S p o r e l i n g D i f f e r e n t i a t i o n  53  60 4.4  Essential Protein Synthesis  62  4.5  C y t o l o g y o f U r e d o s p o r e l i n g Development 4.5.1  Nuclear Staining  67  4.5.2  Nuclear Behaviour d u r i n g Germination  and  Differentiation 4.6  68  Amino A c i d A n a l y s i s  75  4.6.1  Experimental Plan  75  4.6.2  Complications  78  4.6.3  Exogenous F r e e Amino A c i d s : R e s t i n g Spore Wash 81  4.6.4  Endogenous F r e e Amino A c i d s : E x t r a c t s o f Unwashed R e s t i n g Uredospores  4.6.5  83  E f f e c t o f Heat Shock on t h e F r e e Amino A c i d s i n Germinated Uredospores A f t e r Germination  and i n t h e  Leachate 85  V l l  Discussion 5.1  89  Temperature Requirements  f o r the D i f f e r e n t i a t i o n of  P. g r a m i n i s t r i t i c i U r e d o s p o r e l i n g s 5.2  The I n f l u e n c e o f N u t r i e n t s on  89  Uredospore  Differentiation  90  5.3  The T i m i n g o f E s s e n t i a l P r o t e i n S y n t h e s i s  92  5.4  N u c l e a r B e h a v i o u r Accompanying G e r m i n a t i o n and  5.5  Differentiation  95  5.4.1  97  S t a i n i n g w i t h DAPI  The E f f e c t o f Heat Shock on t h e Amounts and K i n d s o f F r e e Amino A c i d s i n G e r m i n a t i n g Uredospores and Leachates  their 99  Summary  107  Literature Cited  109  V l l l  LIST OF TABLES Page Number I.  The c o m p o s i t i o n and p r e p a r a t i o n  o f t h e Ca-K-P0  4  buffer  and t h e v o l a t i l e g e r m i n a t i o n s t i m u l a n t , n - n o n y l a l c o h o l 28 II.  The c o m p o s i t i o n and p r e p a r a t i o n  of the d i f f e r e n t i a t i o n  medium, MPG III.  29  L a c t o p h e n o l - T r y p a n b l u e ; a mounting and s t a i n i n g medium f o r semipermanent mounts o f f u n g i  IV.  Timing o f e s s e n t i a l p r o t e i n synthesis:  31 Experimental  design  34  V.  A d e s c r i p t i o n of the f i x a t i o n schedules t e s t e d  36  VI.  Pump method; t h e b u f f e r g r a d i e n t system and f l o w rate  VII.  48  A summary o f e x p e r i m e n t s i n v e s t i g a t i n g t h e i n f l u e n c e o f t h e components o f MPG, n-nonyl a l c o h o l , and h e a t shock on t h e p e r c e n t a g e o f g e r m l i n g s d e v e l o p i n g i n f e c t i o n structures  61  V I I I . The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin, on t h e development o f i n f e c t i o n s t r u c t u r e from g e r m i n a t i n g uredospores  63  ix IX.  The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin, on t h e development o f i n f e c t i o n s t r u c t u r e from g e r m i n a t i n g uredospores.  E x p r e s s e d as a mean p e r c e n t o f t h e  preceding structure X.  66  PITC-amino a c i d peaks i d e n t i f i e d by number and r e t e n t i o n time  XI.  77  The f r e e amino a c i d c o m p o s i t i o n o f t h e b u f f e r wash from ungerminated u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i  XII.  ...  82  Endogenous f r e e amino a c i d l e v e l i n unwashed r e s t i n g u r e d o s p o r e s , and i n non-shocked u r e d o s p o r e l i n g s a f t e r 8 h and 20 h  84  X I I I . The f r e e amino a c i d c o n t e n t o f unwashed r e s t i n g u r e d o s p o r e s , and i n h e a t shocked u r e d o s p o r e l i n g s a f t e r 8 h and 20 h XIV.  86  The f r e e amino a c i d c o m p o s i t i o n o f l e a c h a t e from h e a t shocked and non-shocked u r e d o s p o r e l i n g s a f t e r an 8 h and 20 h i n c u b a t i o n p e r i o d  XV.  87  The d i s t r i b u t i o n o f t h e f r e e amino a c i d s a s s o c i a t e d w i t h r e s t i n g s p o r e s , non-shocked and h e a t uredosporelings  shocked 100  X  LIST OF FIGURES Page Number 1.  G e r m i n a t i o n and d i f f e r e n t i a t i o n o f t h e wheat r u s t pathogen, P. g r a m i n i s t r i t i c i , on t h e c e r e a l h o s t . .  2.  The c h e m i c a l s t r u c t u r e o f DAPI ( 4 ' , 6 - d i a m i d i n o - 2 phenyl indole)  3.  3  38  HPLC amino a c i d a n a l y s i s system c o n f i g u r a t i o n f o r RPa n a l y s i s o f amino a c i d s w i t h post-column u l t r a v i o l e t detection  4.  Sample p r e p a r a t i o n scheme f o r c r u d e amino a c i d u s i n g Sep-Pak C  5.  41  1 8  cartridges  44  R e a c t i o n scheme o f amino compound by pre-column d e r i v a t i z a t i o n w i t h PITC  6.  samples  .  47  The p e r c e n t g e r m i n a t i o n o f u r e d o s p o r e s on Ca-K b u f f e r i n b o t h t h e p r e s e n c e and t h e absence o f n - n o n y l alcohol  51  7.  T i m i n g o f u r e d o s p o r e l i n g morphogenesis  52  8.  Temperature r e q u i r e m e n t s f o r a p p r e s s o r i u m f o r m a t i o n from u r e d o s p o r e germtubes  9.  54  Temperature r e q u i r e m e n t s f o r t h e complete d i f f e r e n t i a t i o n of uredospore germlings  10.  The r e l a t i o n s h i p between h e a t shock t e m p e r a t u r e and t h e t r a n s f o r m e d p e r c e n t o f a p p r e s s o r i a formed  11.  56  58  The r e l a t i o n s h i p between h e a t shock t e m p e r a t u r e and t h e transformed percent of t o t a l differentiation  sporeling 59  XI  12.  The p r e s e n c e o f a p r o t e i n i n h i b i t o r (puromycin)  i n the  g e r m i n a t i o n medium s i g n i f i c a n t l y reduced t h e number o f germtubes f o r m i n g a p p r e s s o r i a 13.  64  F o l l o w i n g 2 h g e r m i n a t i o n two i n t e r p h a s e n u c l e i . m i g r a t e d from t h e u r e d o s p o r e i n t o t h e e l o n g a t i n g germtube...  14.  The germtube o f non-shocked u r e d o s p o r e l i n g s remained b i n u c l e a t e f o r up t o 20 h  15.  69  69  I n some c a s e s t h e n u c l e i w i t h i n 2 0 - h o u r - o l d u r e d o s p o r e l i n g s were e l o n g a t e d i n form and had a "ragged" appearance  16.  69  Four h o u r s f o l l o w i n g g e r m i n a t i o n t h e two n u c l e i m i g r a t e d i n t o t h e newly formed a p p r e s s o r i u m i n i t i a l .  Their  e l o n g a t e d form suggested t h a t t h e s e n u c l e i were i n c o n j u g a t e t e l o p h a s e and p r e p a r i n g t o d i v i d e 17.  The f i r s t round o f m i t o s i s u s u a l l y o c c u r r e d w i t h i n t h e mature a p p r e s s o r i u m 5 t o 8 h a f t e r g e r m i n a t i o n  18.  72  Ten h o u r s f o l l o w i n g g e r m i n a t i o n t h e s i x - n u c l e a t e v e s i c l e was d e l i m i t e d from t h e a p p r e s s o r i u m by a septum....  21.  70  O c c a s i o n a l l y two n u c l e i d i v i d e d w i t h i n t h e appressorium  20.  70  The mature a p p r e s s o r i u m c o n t a i n e d f o u r d a u g h t e r nuclei  19.  70  72  I n most c a s e s a n u c l e a r p a i r d i v i d e d i n t h e mature v e s i c l e to y i e l d a t o t a l of eight nuclei  72  X l l  22.  The o r i e n t a t i o n o f n u c l e a r m i g r a t i o n a l p a t t e r n s was found t o be r e l a t e d t o t h e p o l a r i t y o f t h e vesicle  23.  73  The i n f e c t i o n hypha c o n t a i n e d e i g h t , s i x , o r more commonly, f o u r n u c l e i  24.  73  I n some c a s e s a n u c l e a r p a i r f a i l e d t o m i g r a t e  with  o t h e r n u c l e i and remained i n t h e v e s i c l e 25.  Twenty t h r e e h o u r s f o l l o w i n g g e r m i n a t i o n t h e i n f e c t i o n hypha most o f t e n c o n t a i n e d f o u r expanded n u c l e i . . . .  26.  73  S e p a r a t i o n o f amino a c i d s t a n d a r d by r e v e r s e - p h a s e  73 high-  p r e s s u r e l i q u i d chromatography 27.  76  R e p r e s e n t a t i v e RP-HPLC s e p a r a t i o n s o f amino a c i d s i s o l a t e d from non-shocked 2 0 - h o u r - o l d and t h e l e a c h a t e o f 2 0 - h o u r - o l d  uredosporelings  h e a t shocked  uredosporelings 28.  79  Diagrammatic r e p r e s e n t a t i o n o f t h e n u c l e a r b e h a v i o u r o f d i f f e r e n t i a t i n g u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i , C17  race 96  xiii Acknowledgements  I would l i k e t o t h a n k t h e p e o p l e t o whom I owe my p e r s o n a l s a n i t y and t h e f i n a l c o m p l e t i o n o f t h i s t h e s i s .  A special  t h a n k y o u t o Dr. L i n d a Boasson f o r h e r companionship i n t h e l a b and h e r a l w a y s h e l p f u l  suggestions.  Thankyou t o Dr. M i c h a e l Shaw f o r t h e many days s p e n t r e v i e w i n g t h e r e s u l t s and h i s i n v a l u a b l e a s s i s t a n c e t h r o u g h a l l aspects o f t h i s t h e s i s .  T h i s work was s u p p o r t e d by a g r a n t from t h e N a t u r a l S c i e n c e and E n g i n e e r i n g Research  Committee t o Dr. M i c h e a l Shaw.  1 1. INTRODUCTION  R u s t f u n g i a r e b i o t r o p h i c pathogens on p l a n t s , g r e a t l o s s e s t o many c u l t i v a t e d c r o p s .  The  wheat pathogen, P u c c i n i a g r a m i n i s P e r s .  f.sp. t r i t i c i  b l a c k stem r u s t o f  and E. Henn. r a n k s among t h e more s e r i o u s o f c e r e a l The  stem r u s t i s m a c r o c y c l i c  causing  and h e t e r o e c i o u s ;  Eriks diseases.  t h a t i s , the  l i f e c y c l e e x h i b i t s f i v e d i s t i n c t s p o r e s t a g e s on two The  u r e d i a l s t a g e , w h i c h i s t h e most d e s t r u c t i v e  hosts.  economically,  p e r p e t u a t e s t h e fungus t h r o u g h o u t t h e g r o w i n g season by r e i n f e c t i n g the c e r e a l  host.  R u s t f u n g i t h a t appear m o r p h o l o g i c a l l y  identical  but  i n f e c t d i f f e r e n t h o s t genera a r e c l a s s e d as formae s p e c i a l e s (f.sp.).  W i t h i n each forma s p e c i a l i s t h e r e a r e many  p h y s i o l o g i c a l r a c e s w h i c h a r e p a t h o g e n i c on o n l y c e r t a i n v a r i e t i e s w i t h i n the host species. r a c e C17 study.  ( f o r m e r l y r a c e 56), was The  s u b r a c e 56A  P. g r a m i n i s f . s p .  employed i n t h e  tritici.  current  r a c e i s g e n e t i c a l l y s t a b l e w i t h o n l y one  rare  known.  and  I t i s highly aggressive  on wheat  c a p a b l e o f c a u s i n g huge economic l o s s e s i n N o r t h A m e r i c a . The  s p e c i f i c r e a c t i o n of a host c u l t i v a r t o a s e r i e s of  p h y s i o l o g i c a l r a c e s i s d e t e r m i n e d by complementary genes f o r r e s i s t a n c e i n t h e h o s t and v i r u l e n c e genes i n t h e pathogen (i.e.  by gene-for-gene r e a c t i o n s )  ( F l o r 1971).  I n most c a s e s  i n c o m p a t i b i l i t y ( r e s i s t a n t r e a c t i o n ) r e s u l t s from t h e i n t e r a c t i o n o f a dominant gene f o r r e s i s t a n c e  (R) i n t h e h o s t  2  and a complimentary dominant gene f o r avirulence pathogen.  A l l other reactions  (P) i n the  (R-pp, rrP-, and rrpp) r e s u l t  i n host-pathogen compatibility (susceptible reaction). Infection of a host plant by the rust fungi i s characterized by a series of morphological events (Figure 1). The  uredospore germinates r e a d i l y on water.  The germtube of  wheat stem rust grows across the c u t i c u l a r ridges of the l e a f blade; upon contact with a stomatal pore germtube elongation ceases and d i f f e r e n t i a t i o n begins.  D i f f e r e n t i a t i o n involves  the sequential development of i n f e c t i o n structures: appressorium (app), the i n f e c t i o n peg  the  ( i p ) , the substomatal  v e s i c l e (ssv), and the i n f e c t i o n hypha ( i h ) .  Once  established, the mycelium grows primarily i n an i n t r a c e l l u l a r fashion, obtaining nutrients from host c e l l s through s p e c i a l i z e d structures c a l l e d haustoria.  Nutrients are also  absorbed by i n t e r c e l l u l a r mycelium. The susceptible host i s able to both induce or a s s i s t fungal morphogenesis and provide the balance of nutrients e s s e n t i a l f o r further growth.  The a b i l i t y to culture the  wheat rust pathogen axenically was  f i r s t demonstrated using  an  Australian race of wheat stem rust (Williams et a l . 1966). Research u t i l i z i n g axenic culture can provide regarding  the biology  the rust fungi.  information  (metabolism, n u t r i t i o n , and genetics) of  Unfortunately, serious obstacles such as the  very slow growth rates of these fungi, and t h e i r  unpredictable  n u t r i t i o n a l requirements have l i m i t e d the widespread use  of  3  Fig. 1. Germination and d i f f e r e n t i a t i o n of the rust pathogen, P. graminis t r i t i c i , on the cereal host: The wheat stem rust uredospore (Ur) germinates readily on the leaf surface. Contact with a stomatal pore causes the germtube (gt) to cease l i n e a r growth and begin d i f f e r e n t i a t i o n : an appressorium (app) forms over the stomate, an i n f e c t i o n peg (ip) grows between the guard c e l l s and expands to form the substomatal v e s i c l e (ssv), the v e s i c l e gives r i s e to the infection hypha (ih) •  4 a x e n i c c u l t u r e methods (Maclean 1982).  Furthermore,  a p p a r e n t , a t l e a s t w i t h P. g r a m i n i s t r i t i c i , a b e r r a n t forms a r e encouraged  i t seems  that genetically  t o develop, t h a t s p o r u l a t i o n i s  a t b e s t e r r a t i c and t h a t fewer t h a n h a l f t h e r a c e s t h a t have been t e s t e d have s u r v i v e d under t h e c u l t u r e c o n d i t i o n s employed ( W i l l i a m s 1984). A c c o r d i n g t o W i l l i a m s (1971) d i f f e r e n t i a t i o n i s a n e c e s s a r y p r e l u d e towards o b t a i n i n g g e n e t i c a l l y (haploid, d i k a r y o t i c ) pathogenic c o l o n i e s .  normal  The p r o p o r t i o n o f  g e r m l i n g s t h a t form i n f e c t i o n s t r u c t u r e s i s i n f l u e n c e d by n u t r i e n t s , h e a t shock, t h e t i m e o f i n o c u l u m c o l l e c t i o n , and the g e n e t i c c o n s t i t u t i o n of the spore. differentiation  by  Sporeling  i s most o f t e n i n d u c e d by a t i m e d h e a t  shock  (30°C, 1.5 h) a d m i n i s t e r e d 2 h f o l l o w i n g g e r m i n a t i o n a t  19°C.  I n o r d e r t o e s t a b l i s h a g i v e n r u s t fungus i n c u l t u r e , s p e c i f i c n u t r i t i o n a l r e q u i r e m e n t s must be met.  The medium  must c o n t a i n i n o r g a n i c s a l t s , a s o u r c e o f c a r b o h y d r a t e , reduced n i t r o g e n , and reduced s u l p h u r (e.g. c y s t e i n e ) .  Howes  r e p o r t e d t h a t a wide range o f amino compounds were e x c r e t e d i n t o c u l t u r e f i l t r a t e s o f P. g r a m i n i s t r i t i c i i n c u b a t i o n (see Howes and S c o t t 1973).  a f t e r 9 days  The f u l l e x t e n t o f  amino a c i d l e a k a g e d u r i n g t h e f i r s t 20 h o f g e r m i n a t i o n and d i f f e r e n t i a t i o n was n o t a s s e s s e d .  T h i s s t u d y was d e s i g n e d t o  i n v e s t i g a t e t h e l e a k a g e o f f r e e amino a c i d s from  uredospore  g e r m l i n g s c o n c o m i t a n t w i t h t h e p r o c e s s e s o f g e r m i n a t i o n and differentiation  ( i n d u c e d by h e a t s h o c k ) .  The changes i n t h e  5 endogenous free amino acid pool were also assessed.  The objectives of the present study thus were: 1.  To reexamine the temperature requirement f o r the process of d i f f e r e n t i a t i o n of the wheat stem rust fungus.  2.  To examine the influence of nutrients on the proportion of germlings that form i n f e c t i o n structures.  3.  To determine the timing of essential protein synthesis during d i f f e r e n t i a t i o n .  4.  To investigate nuclear behaviour accompanying germination and d i f f e r e n t i a t i o n of P. graminis t r i t i c i , race C17: nuclear d i v i s i o n , nuclear migration patterns, and nuclear d i s t r i b u t i o n .  5.  To determine the amount and kinds of free amino acids present during the germination of wheat stem rust uredospores (race C17) and the d i f f e r e n t i a t i o n of germ tubes into i n f e c t i o n structures. 5.1  To characterize the free amino acids within nongerminated uredospores and those adhered to the outer spore wall.  5.2  To assess the changes i n the endogenous free amino acid pools of uredospore germlings during germination and d i f f e r e n t i a t i o n .  5.3  To determine the amounts and kinds of amino acids leached into the medium during the process of germination and d i f f e r e n t i a t i o n .  6 2.  LITERATURE REVIEW  2.1  2.1.1  U r e d o s p o r e G e r m i n a t i o n and Morphogenesis  G e r m i n a t i o n I n h i b i t o r s and S t i m u l a n t s U r e d o s p o r e g e r m i n a t i o n and germtube d i f f e r e n t i a t i o n i s  s u b j e c t t o r e g u l a t i n g mechanisms i n v o l v i n g endogenous i n h i b i t o r s and s t i m u l a t o r s .  Germination i n h i b i t o r s are  dormancy a g e n t s w h i c h resemble hormones i n t h e i r m o b i l i t y from s p o r e t o s p o r e , t h e i r r e g u l a t o r y a c t i o n and t h e i r h i g h biological activity  ( A l l e n 1976).  Sporostatic levels of c i s -  cinnamates a r e found i n u r e d o s p o r e s .  The e f f e c t o f t h e  i n h i b i t o r s i s r e a d i l y r e v e r s i b l e , f o r example,  during  h y d r a t i o n , and p r i o r t o g e r m i n a t i o n , t h e a c t i v e c i s - c i n n a m a t e s a r e r e l e a s e d and a r e e i t h e r d i l u t e d t o below a c t i v e l e v e l s o r c o n v e r t e d t o i n a c t i v e t r a n s - i s o m e r s by u l t r a v i o l e t l i g h t (420 nm) ( A l l e n 1972).  The g e r m i n a t i o n i n h i b i t o r o f wheat stem  r u s t i s m e t h y l c i s - f e r u l a t e : a m e t h y l e s t e r o f 3-methoxy-4hydroxycinnamate  (Macko e t a l . 1971).  I t has an E D  5 0  o f 0.2  ng/ml (Macko e t a l . 1972) and i s a c t i v e o n l y d u r i n g g e r m i n a t i o n ( m y c e l i a l growth i s n o t a f f e c t e d [ A l l e n 1 9 7 6 ] ) . The i n h i b i t o r p r e v e n t s t h e d i s s o l u t i o n o f t h e germ p o r e p l u g b u t i t s m o l e c u l a r s i t e o f a c t i o n i s unknown (Hess 1975). Endogenous g e r m i n a t i o n s t i m u l a n t s such as n - n o n y l a l c o h o l (nonanal) ( F r e n c h and Weintraub 1957),  6-methyl-5-hepten-2-one  ( R i n e s e t a l . 1974), coumarins, and p h e n o l s (Van Sumere e t a l .  7 1957)  are not species s p e c i f i c .  Macko and c o - w o r k e r s (1976)  suggest t h a t t h e s t i m u l a n t s f a c i l i t a t e germination by p r o m o t i n g t h e r e l e a s e o f t h e i n h i b i t o r s from t h e s p o r e .  2.1.2  Thigmodifferentiation Uredosporeling  physical stimuli.  d i f f e r e n t i a t i o n may be i n d u c e d i n v i t r o by I n a s e r i e s o f e x p e r i m e n t s e m p l o y i n g bean  r u s t g e r m l i n g s and a r t i f i c i a l membranes D i c k i n s o n  (1949, 1971)  demonstrated t h a t t h e formation o f i n f e c t i o n s t r u c t u r e s r e l i e s on a r e c o g n i t i o n e v e n t t h a t i s c o r r e l a t e d w i t h thigmotropic s t i m u l i .  specific  D i f f e r e n t i a t i o n thus stimulated  ( t h i g m o d i f f e r e n t i a t i o n ) requires f i r m attatchment o f t h e germtube t o t h e i n d u c t i v e s u r f a c e  (Wynn and S t a p l e s 1981).  Recent d a t a s u g g e s t t h a t , i n bean r u s t , p r o t e i n s on t h e h y p h a l s u r f a c e a r e i n v o l v e d i n s u b s t r a t e a d h e s i o n and t h e transmission o f a s i g n a l t o the cytoskeleton t o begin  nuclear  d i v i s i o n ( E p s t e i n e t a l . 1985). According  t o Hoch e t a l . (1987) t h e t o p o g r a p h i c a l s i g n a l  r e q u i r e d t o t r i g g e r maximum c e l l d i f f e r e n t i a t i o n i n Uromyces appendiculatus surface.  i s a 0.5  m h i g h r i d g e on t h e s u b s t r a t e  The e l e v a t i o n o f t h e r i d g e i s c r i t i c a l ,  g r e a t e r t h a n 1.0  m o r l e s s t h a n 0.25  ridges  m are not e f f e c t i v e  s i g n a l s and a r e u n a b l e t o promote maximum d i f f e r e n t i a t i o n .  It  was a l s o r e p o r t e d t h a t t h e growth o f t h e germtubes i s o r i e n t e d by r i d g e s p a c i n g s o f 0.5 t o 6.7  m.  A l t h o u g h c o l l o d i o n membranes c o n t a i n i n g p a r a f f i n o i l  8 induced bean rust germlings to d i f f e r e n t i a t e , they had to no e f f e c t on P. q. t r i t i c i  (Maheshwari et a l .  little  1967b).  Staples and h i s group (1983) found that contact s t i m u l i are responsible f o r the induction and positioning of the wheat stem rust appressorium.  However, further development  of the  stem rust germling requires other environmental factors most l i k e l y of host o r i g i n .  2.1.3  Thermodif f e r e n t i a t i o n Another type of induction was reported f o r P. q. t r i t i c i .  The exposure of germinated uredospores to elevated temperatures of 30 to 31°C f o r 90 min followed by the return to  lower temperatures promoted up to 90% d i f f e r e n t i a t i o n  (Maheshwari et a l .  1967, Dunkle and A l l e n 1971).  The  percentage of germlings that formed i n f e c t i o n structures was influenced by the duration of heat shock, the spore to l i q u i d r a t i o , and the composition of the medium present during the induction period (Dunkle and A l l e n 1971). The induction of d i f f e r e n t i a t i o n by heat shock, as described above was repeated by Wisdom (1977).  The r e s u l t s  proved unsatisfactory, y i e l d i n g d i f f e r e n t i a t i o n percentages of l e s s than 5%.  The e f f e c t of the temperature of the heat shock  was not investigated.  The Ca-K buffer used by Dunkle and  A l l e n (1968) was abandoned i n favour of a medium composed of Maheshwari s-buffer 7  glucose (MPG).  (Maheshwari et a l .  1967), peptone, and  MPG supported up t o 80 percent d i f f e r e n t i a t i o n  9 (Wisdom 1977).  2.1.4  Chemodifferentiation Chemodifferentiation  i s the induction of i n f e c t i o n  structures by a chemical agent.  Chemical s t i m u l i include  uredospore d i s t i l l a t e s such as acrolein  (2-propanal) (Macko et  a l . 1978), the potassium ion (Staples et a l . 1982), c e r t a i n reduced nucleotides  (Staples et a l . 1983a), and v o l a t i l e l e a f  constituents and phenolic compounds leached or metabolized from the guard c e l l s (Grambow and Riedel 1977, Grambow and Grambow 1978).  Grambow  1978,  Synthetic short-chain a l i p h a t i c  compounds with conjugated double bonds (e.g. acrolein) are morphogenetically active whereas saturated a l i p h a t i c aldehydes, ketones and alcohols, as well as phenylacrolein unable to induce d i f f e r e n t i a t i o n (Wolf 1982).  are  The a b i l i t y of  a chemical agent to stimulate d i f f e r e n t i a t i o n opens the p o s s i b i l i t y that the agent i s a normal intermediary  in  contact-triggered d i f f e r e n t i a t i o n (Allen 1976).  2.1.5  Mechanisms of D i f f e r e n t i a t i o n The mechanisms involved i n the regulation of germtube  d i f f e r e n t i a t i o n are unknown.  Allen  (1976) suggests that the  formation of i n f e c t i o n structures i s controlled by the uredospore genome. program.  An appropriate stimulus activates the  The observed i n h i b i t i o n of d i f f e r e n t i a t i o n by  protein and RNA  i n h i b i t o r s suggests that the genetic message  10 i s a c t i v a t e d by d e - r e p r e s s i n g  a r e g i o n o f t h e genome.  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 t h a t the  cytoskeleton  ( m i c r o f i b r i l l a r system) i s r e s p o n s i b l e f o r t h e i n d u c t i o n o f n u c l e a r d i v i s i o n i n bean r u s t g e r m l i n g s (Hoch and 1985,  Hoch e t a l . 1984,  S t a p l e s and Hoch 1982).  s u g g e s t t h a t t h e c y t o s k e l e t o n somehow r e p r e s s e s  Staples These a u t h o r s nuclear  d i v i s i o n u n t i l i t i s s u b j e c t e d t o h e a t shock o r an i n d u c t i v e membrane topography.  2.1.6  The R o l e o f I n f e c t i o n S t r u c t u r e s I n n a t u r e t h e i n f e c t i o n s t r u c t u r e s p l a y a number o f  important  roles:  I n f e c t i o n s t r u c t u r e s must form b e f o r e  c o l o n i z i n g hyphae can grow ( D i c k i n s o n 1949, According  Chakravati  t o Wisdom (1977), d i f f e r e n t i a t e d g e r m l i n g s o f  leaf1966). the  wheat stem r u s t fungus i n f e c t exposed h o s t m e s o p h y l l ; u n d i f f e r e n t i a t e d s p o r e l i n g s a r e u n a b l e t o cause i n f e c t i o n . I n f e c t i o n s t r u c t u r e s s e r v e t o anchor t h e fungus a t t h e stomatal s i t e thereby  enabling penetration.  They  represent  t h e s i t e o f t h e t r a n s i t i o n from germtube t o h y p h a l growth. This hypothesis and DNA  i s s u p p o r t e d by t h e a c t i v a t i o n o f p r o t e i n ,  s y n t h e s i s d u r i n g d i f f e r e n t i a t i o n (Dunkle e t a l .  M e i h l e 1972).  And  RNA  1969,  l a s t l y t h e y e n a b l e t h e fungus t o complete  t h e t r a n s i t i o n w i t h t h e r a p i d i t y demanded by a c h a n g i n g (perhaps t o a l e s s c o n d u c i v e ) environment. found t h a t d i k a r y o t i c hyphae b r a n c h i n g  Grambow (1977)  d i r e c t l y from germtubes  a r e p r o d u c e d a t a h i g h r a t e b u t n o t b e f o r e t h e t h i r d day a f t e r  seeding.  Dikaryotic hyphae a r i s e from a complete set of  i n f e c t i o n structures very soon a f t e r germination. According t o Williams (1971) the formation of i n f e c t i o n structures i s an e s s e n t i a l prelude to the establishment and maintenance of g e n e t i c a l l y normal pathogenic colonies.  The  t r a n s i t i o n ( p a r a s i t i c to saprophytic growth), i f appropriately stimulated, occurs with or without the induction of d i f f e r e n t i a t e d structures.  However, i n f e c t i o n structures  appear t o control nuclear behaviour and as a consequence increase the p r o b a b i l i t y of giving r i s e to hyphae which are both normal and stable f o r the dikaryophase. Although heat shock generally appears to stimulate c e l l d i f f e r e n t i a t i o n and improve saprophytic growth of most rust fungi, Bushnell (1976) claims that heat shock does not promote growth of P. graminis f.sp. t r i t i c i , race 17.  Kuck and  Reisner (1985) report that d i f f e r e n t i a t i o n has a negative e f f e c t on the i n v i t r o sporulation of P. graminis t r i t i c i . race 32.  2.2  2.2.1  C y t o l o g i c a l Events  Nuclear Changes The morphological changes leading to the formation of  i n f e c t i o n structures are accompanied by a series of p r e c i s e l y timed nuclear events. haploid.  The r e s t i n g uredospore i s d i k a r y o t i c  Following germination, the two n u c l e i migrate with  12 the cytoplasm into the developing germtube (Heath and Heath 1978).  Given the stimulus t o d i f f e r e n t i a t e the nuclei may  d i v i d e p r i o r t o (Maheshwari et a l . 1967b), or following (Grambow and Muller 1978) the development of the appressorium. Nuclear d i v i s i o n occurs only a f t e r the l i n e a r growth of the germtube i s arrested and i s r a r e l y observed i n nondifferentiated germlings  (Maheshwari et a l . 1967, Grambow  and Muller 1978, Wisdom 1977). Dickinson (1949) reported that two or three rounds of m i t o t i c d i v i s i o n occur during the formation of i n f e c t i o n structures i n P. graminis and P. t r i t i c i n a .  The appressorium  may contain four t o eight nuclei which migrate into the developing v e s i c l e .  A second d i v i s i o n i n the v e s i c l e y i e l d s  eight n u c l e i i n t o t a l .  According to Wisdom (1977) the v e s i c l e  n u c l e i may e i t h e r dissolve or coalesce leaving one t o two n u c l e i i n the i n f e c t i o n hypha. the mature appressorium  A l l e n (1923) also noted that  of P. g. t r i t i c i contains four n u c l e i  and that the i n f e c t i o n hypha contains two.  The i n f e c t i o n  structures induced i n v i t r o are morphologically and c y t o l o g i c a l l y s i m i l a r to those induced on the host plant (Maheshwari et a l . 1967b). Staples e t a l . (1984b) reported that the synthesis of nuclear DNA i n U. fabae sporelings coincides with the onset of mitosis i n response to the stimulus f o r the i n i t i a t i o n of differentiation.  The r e p l i c a t i o n of DNA begins between the  second and fourth hour following germination.  I t i s not known  13 how  external s t i m u l i activate nuclear DNA  and Staples 1974).  polymerase (Yaniv  In the absence of the stimulus to  d i f f e r e n t i a t e the uredosporeling n u c l e i are i n Gl ( i . e . period preceding DNA  r e p l i c a t i o n ) (Staples et a l . 1984b).  Mitochondrial DNA  synthesis occurs during germination but i s  not detected following the appearance of the appressoria (Staples 1974). Uredospore n u c l e i are known to e x i s t i n two p r i n c i p a l but d i s s i m i l a r forms.  Between d i v i s i o n s the interphase nucleus i s  described as "expanded".  The expanded nucleus i s composed of  two parts, the "ectosphere",  containing a l l the chromatin and  the "endosphere", containing the nucleolus.  As the process of  d i v i s i o n begins, the nucleus extrudes the endosphere i n t o the cytoplasm, contracts and rounds up to form the "unexpanded" or d i v i d i n g nucleus (Savile 1939).  Craigie (1959) and Wisdom  (1977) observed both expanded and unexpanded n u c l e i i n P. h e l i a n t h i and P. q. t r i t i c i , respectively.  2.2.2  Protein Metabolism The changes i n proteins and nucleic acids accompanying  germination  of uredospores and d i f f e r e n t i a t i o n of the germtube  into structures has inspired much research Dunkle et a l . 1969, Shaw et a l . 1985,  (Staples  1968,  Kim et a l . 1982a, Huang and Staples  1982,  Wanner et a l . 1985).  Rust uredospores germinate without the appreciable increase i n protein that occurs i n most saprophytes, such as  14 Fusarium (Cochrane et a l . 1971) and Aspergillus niqer (Staples et a l . 1962).  Even so, f u l l y functional ribosomes are present  i n bean and wheat rust uredospores and t h e i r capacity increases three-fold during spore hydration, p r i o r t o germination (Staples e t a l . 1968).  Data from studies using  metabolic i n h i b i t o r s of RNA (actinomycin-D) and protein (puromycin) synthesis indicate that new protein synthesis i s essential for differentiation.  The presence of actinomycin-D  during the heat shock prevents the i n i t i a t i o n of i n f e c t i o n structures.  The presence of puromycin allows germination and  appressorial formation to occur but prevents further i n f e c t i o n structure development  (Dunkle et a l . 1969).  The protein  required f o r germination appears to be stored within the dormant uredospore.  The mRNA responsible f o r d i r e c t i n g the  complete development  of i n f e c t i o n structures i s synthesized at  the time of heat shock.  Essential protein synthesis i s then  programmed by the new mRNA during elaboration of the i n f e c t i o n structures. Although puromycin and actinomycin-D f a i l e d t o prevent germination i t would be erroneous t o conclude that RNA and protein synthesis does not occur during the early stages of development. Uredospores contain a complete system f o r protein synthesis (Yaniv and Staples 1974).  Germination studies on  the incorporation of [ C ] - l a b e l l e d amino acids, -glucose, and 14  - v a l e r i c a c i d into protein of wheat stem rust (Reisener 1967) and bean rust (Staples and Bedigan 1967), and the appearance  15 of new isozymes of glucose,dehydrogenase, cytochrome oxidase, and acid phosphatase  (Staples and Stahmann 1964) are  i n d i c a t i v e of protein synthesis.  Although there i s an  apparent synthesis of protein, germination i s not by net protein synthesis;  accompanied  t o t a l free and bound amino acids  remain unchanged or decrease (Staples et a l . 1962). Heat shock promotes the synthesis of new types of RNA  and  protein (Kim et a l . 1982a, Huang and Staples 1982, Shaw et a l . 1985).  Although the heat shock response has been reported f o r  a wide range of organisms i t s functional s i g n i f i c a n c e i s unknown.  Heat shock proteins (HSP) may serve to protect the  c e l l against environmental stress (Ashburner 1982). al.  Shaw et  (1985) have i d e n t i f i e d seven HSP i n Melampsora l i n i  uredospore germlings. Kim et a l . (1982a) reported on changes i n detergentsoluble polypeptides from uredospores of P. cf. t r i t i c i . Dormant uredospores were found t o contain more than 270 d i s t i n c t polypeptides, several of which varied among physiologic races (Howes et a l . 1982).  A f t e r 2 and 6 hours of  germination the concentrations of at l e a s t f i v e polypeptides decreased considerably.  At 2 hours, a transient concentration  increase was noted f o r two polypeptides, 4 hours l a t e r these concentrations had decreased again to that found i n dormant uredospores.  The t o t a l protein extracted from six-hour-old  germlings was 86 percent of the t o t a l protein extracted from dormant spores.  16 D i f f e r e n t i a t i o n was f i v e polypeptides  accompanied by a decrease i n at l e a s t  and an increase i n four others.  Two  new  polypeptides appeared i n d i f f e r e n t i a t e d sporelings, t h e i r molecular weights were approximately 30.0  and 20.0  kD.  D i f f e r e n t i a t e d sporelings were found to contain only 48 percent of the t o t a l protein extracted from dormant uredospores (Kim et a l . 1982a).  Wanner et a l . (1985) reported  the synthesis of d i f f e r e n t i a t i o n - s p e c i f i c proteins (DSP)  at a  time corresponding to the appearance of the substomatal vesicle. Several p o s s i b i l i t i e s e x i s t f o r the observed decline i n the t o t a l amount of extractable protein and c e r t a i n polypeptides:  Pulse-chase experiments show that  polypeptides  are continually turned over, e x i s t i n g protein i s broken down into i t s substituent amino acids, and the amino acids are e i t h e r u t i l i z e d f o r resynthesis or l o s t into the medium (see Kim et a l . 1982a).  I t i s also possible that proteins bind to  and form an insoluble complex with glucan and mannan residues i n the c e l l wall of the germtube (Kim et a l . 1982a).  Glucan-  protein and mannan-protein complexes have been detected i n yeasts  (Ballou 1974)  and i t i s possible that analogous  complexes occur i n the germtube walls of P. q. t r i t i c i . F i n a l l y , the decline may metabolites  be due to a considerable loss of  and enzymes into the medium during germination and  c e l l wall degeneration. Pfesofsky-Vig and Brambl (1985) report that the  17 appearance of HSP and the depression of general t r a n s l a t i o n a c t i v i t y i s t y p i c a l immediately following a temperature  shift.  The normal pattern of synthesis begins soon a f t e r the c e l l s are returned t o the normal  2.3  2.3.1  temperature.  N u t r i t i o n a l Requirements  of Rust Fungi  Physiology of the Host-Parasite Complex P r i o r to considering the n u t r i t i o n a l requirements of rust  fungi i n v i t r o i t i s necessary to review host-parasite r e l a t i o n s i n susceptible host t i s s u e .  The pustules of rust  fungi act as f o c i f o r the accumulation of host metabolites.  A  compatible i n f e c t i o n increases the rate of t r a n s p i r a t i o n and r e s p i r a t i o n , decreases photosynthetic a c t i v i t y , a l t e r s the d i r e c t i o n of normal phloem transport (Scott 1972)  and  stimulates the leakage of e l e c t r o l y t e s from the host.  These  responses by the host s i g n i f i c a n t l y improve the a v a i l a b i l i t y of nutrients t o the developing fungus. With respect to s t r u c t u r a l and physiological changes associated with rust infected tissues, Bushnell (1984) recognized the juvenile and the a u t o l y t i c host response. During the juvenile response most plant growth hormones increase.  I t i s unclear however whether the observed increase  i s caused by pathogen or host-produced hormones.  There i s  evidence that plant derived hormones do not play an active r o l e i n a compatible host-pathogen r e l a t i o n s h i p (Levin 1985).  18 The increase i n cytokinin a c t i v i t y reported by S z i r a k i et a l . (1976) appeared to delay l e a f senescence by maintaining protein synthesis (Stodart 1981).  The free concentration of  indole a c e t i c acid (IAA) i n plants increased two and a h a l f f o l d at the i n f e c t i o n s i t e , 6 to 14 hr a f t e r inoculation with wheat stem rust pathogen (Artemeko et a l . 1980). I t i s l i k e l y that IAA acts with cytokinin to control the metabolic state of the host c e l l . The host nucleus and nucleolus generally enlarge during rust i n f e c t i o n and the synthesis of nucleolar and extranucleolar RNA  synthesis i s enhanced (Manocha and Shaw  1966, Whitney et a l . 1962,  Bhattacharya et a l . 1965).  However, the t o t a l amount of host RNA that the newly synthesized RNA over.  declines which indicates  i s rapidly degraded and turned  Chakravorty and co-workers (1974) found that c a t a b o l i c  RNAse a c t i v i t y peaks at about 6 days at l e v e l s two to f i v e times those of uninfected leaves. The j u v e n i l e response i s not associated with s i g n i f i c a n t q u a l i t a t i v e or quantitative changes i n host proteins.  Host  proteins changed l e s s i n compatible host-parasite combinations than i n incompatible combinations 1972). New  (von Broembsen and Hadwiger  isozymes have been detected on polyacrylamide gels  and i n most cases they appear to be of fungal o r i g i n et a l . 1968,  (Johnson  Staples 1965).  Amino acids, amides, and carbohydrates accumulate rapidly at the infected s i t e .  During the early juvenile host  response  these metabolites  are synthesized l o c a l l y from photosynthates  and ammonia, and are also translocated from distant s i t e s on the plant.  During the l a t e r a u t o l y t i c stage the l o c a l  degradation of protein provides a r i c h source of free amino acids and amides (see Bushnell 1985). The r a t i o of soluble to insoluble nitrogen increased at i n f e c t i o n s of stem rust on Franke 1938). increased  L i t t l e Club wheat (Gassner and  On a fresh weight basis bound amino acids  almost twofold whereas free amino acids increased  f o u r f o l d (Shaw and Colotelo 1961).  Large increases i n the  amounts of free glutamine, Y-aminobutyric a c i d (ABA), threonine and several basic and aromatic acids occurred e a r l y as two days post-inoculation.  as  Several investigators  have reported d i f f e r e n t i a l increases i n the amounts of asparagine, arginine, phenylalanine, v a l i n e (Farkas and K i r a l y 1961,  leucine or isoleucine,  Shaw and Colotelo 1961)  and  tryptophan (Kim and Rohringer 1969). The movement of host metabolites has been followed with radiotracers.  into p a r a s i t i c mycelium Labelled sucrose i s  inverted to glucose and fructose and r e a d i l y absorbed by the rust hypha.  Glucose, glutamate, alanine, glycine, l y s i n e and  arginine are taken up; the f i r s t four compounds are metabolized by the fungus.  Some amino acids (e.g. serine and  alanine) are absorbed from the host by the fungus more r e a d i l y than others  (e.g. glutamine, glutamate and aspartate).  Amino  acids are leached from the uredosporeling during germination  and t h e i r movement i n t o host tissues has been reported 1966,  (Jones  Daly e t a l . 1967). The i n f e c t i o n of the host by a rust pathogen i n v a r i a b l y  causes an accumulation of free glutamine (Shaw and Colotelo 1961).  This amide, which may be derived e i t h e r by an  endergonic reaction from glutamate or from p r o t e o l y s i s i s r e a d i l y translocated throughout the plant.  Glutamine can  serve as a source of bulk nitrogen f o r axenic culture (Maclean 1982)  and as a precursor f o r the synthesis of fungal c h i t i n  (Farkas and K i r a l y 1961).  I t i s l i k e l y that glutamine plays a  central r o l e i n the metabolism of rust infected leaves.  2.3.2  Axenic Culture and Metabolism  N u t r i t i o n a l Requirements The n u t r i t i o n a l requirements of P. q. t r i t i c i i n axenic culture include inorganic s a l t s , a source of carbohydrate, reduced nitrogen and reduced organic sulphur.  Carbohydrate  requirements are r e l a t i v e l y nonspecific (Coffey and Shaw 1972) .  Nitrogen may be supplied as an inorganic ammonium  s a l t , or as one of a wide range of organic compounds, such as an amino a c i d .  Some amino acids are better sources of  nitrogen than others.  In the presence of cysteine the  i n i t i a t i o n of saprophytic growth was poor on alanine, improved on aspartate and greatest on glutamate (Coffey and A l l e n 1973) .  Sulphur must be provided i n reduced form such as  cysteine, cystine, the t r i p e p t i d e glutathione, or methionine.  Wheat stem rust requires a higher concentration of cysteine than methionine f o r optimal growth (Howes and Scott 1973). The amount and the combination of sulphur containing amino acids i s c r i t i c a l and may vary between races of rust (Singh and Sethi 1982).  The nutrient requirements appear l e s s  exacting once the rust fungus becomes established on  axenic  medium.  Synthetic Capacity Germinating stem rust uredospores slowly absorb, metabolize and synthesize a wide range of compounds. Uredospores germinated on a medium containing [ C]-sucrose 14  synthesize at l e a s t eleven  [ C ] - l a b e l l e d amino acids within 14  two hours (Kasting et a l . 1959).  Among the f i r s t amino  compounds to be synthesized were glutamine, glutamate, and aspartate  (Reisener et a l . 1961).  Glucose i s taken up r e a d i l y  and the carbon appears i n endogenous pools of free glucose, amino acids and phosphate esters of trehalose and (Manners et a l . 1982).  glucose  Reisener et a l . (1961) demonstrated  that 42 percent of the label from v a l e r a t e - 1 - [ C ] 14  was  incorporated i n t o the spore as carbohydrate; organic, f a t t y and amino acids; amides and peptides.  Glutamine, glutamate  and ABA had the highest s p e c i f i c a c t i v i t y of the free amino compounds. Rust uredospores possess a l l the enzymes required f o r glucose catabolism and terminal oxidation (Shaw 1964), the  pentose phosphate pathway, c i t r i c acid cycle, and  lipid  metabolism (Caltrider and Gottlieb 1962).  Sulphur Metabolism Most rust fungi studied so f a r are heterotrophic f o r reduced, organic sulphur when grown on chemically defined medium.  Although rust fungi synthesize sulphur amino acids  r e a d i l y from [ S ] - s u l p h i d e 35  only a l i m i t e d synthesis of  l a b e l l e d protein can be detected  (Howes and Scott 1973).  More  than 70 percent of the label incorporated into sulphur amino acids was  l o s t i n t o the medium as cysteine,  glutathione and cysteinylglycine.  S-methylcysteine,  Labeled methionine  however, appeared i n mycelial protein and only n e g l i g i b l e amounts were l o s t to the f i l t r a t e .  P. q. t r i t i c i was  unable  to reduce inorganic sulphate f o r sulphur-amino-acid synthesis* These r e s u l t s suggest that a metabolic block e x i s t s i n the pathway of inorganic sulphur metabolism.  Howes and Scott  (1973) propose that rust fungi are unable to reduce  3'-  phosphoadenosine-5'-phosphosulphate to thiosulphate or thiosulphate to sulphide.  I t i s i n t e r e s t i n g to note that  sulphate reduction occurs i n prokaryotes, most fungi (Scott 1972)  eukaryotic algae,  and a l l higher plants so f a r rested  (Schiff and Hodson 1973).  Metabolite Leakage In axenic culture the germtube and mycelium leak  metabolic intermediates.  Staples and Wynn (1965), and Tulloch  (1962) mention possible losses of free amino acids,  sulphur-  containing compounds and glycine-containing peptides t o the medium but o f f e r no quantitative data.  Jones and Snow (1965)  reported that [ S ] - l a b e l l e d uredospores of P. coronata avenae 35  l o s t a range of amino compounds including ethionine, ABA, methionine sulphoxide and four sulphur-containing during 12 h of germination.  unknowns  The amino acids detected  i n the  culture f i l t r a t e (basal medium) of P. g. t r i t i c i a f t e r nine days were glutamate, glutamine, glycine, alanine, l y s i n e , arginine, serine, leucine and isoleucine,  phenylalanine,  v a l i n e , threonine, asparagine, proline, cysteine, and methionine (Howes and Scott 1972).  A considerable amount of  cysteine and glycine-containing peptides, such as c y s t e i n y l g l y c i n e and  glutathione accumulated i n the medium.  Very l i t t l e methionine and homocysteine was detected and Scott 1973).  (Howes  From these studies i t was evident that the  loss of sulphur-containing  compounds i s s e l e c t i v e and they are  not l o s t as r a p i d l y nor t o the same extent as other free amino acids.  ABA, Y-glutamylglutamate (Howes and Scott 1972),  carbohydrate, protein, such as ribonuclease  (Chakravorty and  Shaw 1974) and the germination i n h i b i t o r , methyl f e r u l a t e i n the c i s or trans form are also leached into the medium. Mutual stimulation during sporeling development i s demonstrated by the p o s i t i v e e f f e c t of increasing inoculum density (Kuhl et a l . 1971), and the success of "nurse culture"  24 techniques, "conditioned agar" (Scott 1976) and coculture experiments  (Hartley and Williams 1971a, 1971b).  A minimal or  unbalanced medium i n axenic culture would r e s u l t i n a net loss of metabolites, thus imposing an excessive drain on metabolism and depleting internal metabolite pools, which i n turn would r e s u l t i n l e s s e r growth rates (Maclean 1982).  A poor  approximation of amino acid balances may also r e s u l t i n detrimental e f f e c t s , such as methionine t o x i c i t y (Howes and Scott 1972) and the s e l e c t i v e leakage of amino acids from the mycelium (Maclean 1982).  Endogenous Free Amino Acids Interest i n the free amino acid content of rust uredospores has developed i n conjunction with studies r e l a t i n g to s e l f - i n h i b i t i o n of germination (Wilson 1958), the p o t e n t i a l of d i f f e r e n t i a t i n g races of rust on the basis of c h a r a c t e r i s t i c amino acid content (McKillican 1960), and the e f f e c t s of storage on assimilative and synthetic c a p a b i l i t e s (Wynn et a l . 1966). McKillican (1960) reported race 56 as unique i n that the dormant uredospore lacked glutamic acid and contained large amounts of aspartic acid and a-alanine.  Free  amino acids represented approximately 0.5 percent of the o r i g i n a l spore weight.  Using the same race Stefayne and  Bromfield (1965) found that the major ninhydrin-positive compounds were glutamine, glutathione, glutamic acid and ammonia.  Approximately 1.2 percent of the spore weight  was  25 composed of free amino acids.  In l a t e r studies by Wynn e t a l .  (1966) i t was found that glutamic acid made up over h a l f of the t o t a l amino acids.  Other major amino acids were alanine,  aspartic acid, serine, and cystine.  Since no attempt was made  to recover asparagine and glutamine i n t a c t , i t was probable that the l e v e l s of aspartic acid and glutamic acid these amides.  Stefayne and Bromfield  included  (1965) noted e a r l i e r  that the uredospores contain seven times the amount of glutamic a c i d as glutamine.  Analyses of the amino a c i d  composition of wheat rust uredospores vary widely due to sampling and analysis procedures. Accordingly,  I have examined the changes i n the amino  acid composition of the free pool within germinating and d i f f e r e n t i a t i n g sporelings of P. crraminis t r i t i c i at 8 and 20 h following imbibition. providing information  The r e s u l t s are preliminary t o  f o r further studies of nitrogen  metabolism and n u t r i t i o n i n the wheat stem rust fungus.  26 3.  EXPERIMENTAL METHODS  3.1  Production and C o l l e c t i o n of Spores  The wheat stem rust uredospores,  (Puccinia graminis Pers.  f.sp. t r i t i c i E r i k s and E. Henn., race C17), employed i n t h i s study were obtained through the courtesy of Dr. D. Samborski (Agricultural Research Station, Winnipeg) and were increased on Tritium aestivum L., c.v. L i t t l e Club. Plants were grown i n a growth chamber with a 16 h photoperiod of 9684 lux and temperatures dark.  of 25°C l i g h t , 18°C  A f t e r eight days of growth the plants were inoculated  by l i g h t l y spreading a water-based paste of t a l c and uredospores  (3:1) over the l e a f blade.  The plants were then  well-misted with d i s t i l l e d water, covered with a p l a s t i c bag and placed i n a dark incubator a t 18°C f o r 24 h before they were returned to the growth chamber.  Within s i x days the  leaves showed signs of "flecking", and s i x days following the s o r i opened.  Five days l a t e r the uredospores were c o l l e c t e d  by gently shaking the infected leaves into a clean t e s t tube. The f r e s h l y harvested spores were used immediately f o r the amino acid analyses.  In a l l other experiments the spores  were stored i n g e l a t i n capsules at 4°C f o r up to, but not exceeding 24 h. percent.  Germination was generally between 90 and 100  27 3.2  Spore Germination  Depending on the experiment, germination was c a r r i e d out on e i t h e r a mixed calcium and potassium phosphate (Ca-K) buffer (Table I) or MPG medium (Table I I ) . To deplete the endogenous s e l f - i n h i b i t o r 8 mg of uredospores were dispersed uniformly over the surface of 3 ml of Ca-K b u f f e r i n an p e t r i dish using an inoculation loop. (5-mm  8-cm  A f t e r 5 min, 5 loops  diameter) of uredospores were transferred to 2 ml of the  germination medium i n the inner chamber of a 50-ml Conway D i f f u s i o n dish. mm . -2  The f i n a l spore dose was approximately  The outer well contained 4 ml of 1.5 x 1 0  alcohol (Table I ) .  -4  300  M n-nonyl  Dishes were covered with a glass plate,  sealed with vaseline and incubated i n the dark at 19°C.  3.3  Spore Germination with the Induction to D i f f e r e n t i a t e  Inoculated dishes were incubated i n the dark at 19®C f o r 2 h, transferred to a hot water bath set at 29.5°C f o r 1.5 h, then returned to 19°C.  Infection structure development was  complete within 20 h.  3.4  C r i t e r i a f o r the Assessment of Sporelincr Development  A spore was considered germinated when the germtube length was equal to, or exceeded the spore diameter.  Terminal  28  Table I : The c o m p o s i t i o n and p r e p a r a t i o n o f t h e Ca-K-P0 b u f f e r and t h e v o l a t i l e g e r m i n a t i o n s t i m u l a n t , n - n o n y l a l c o h o l (nonanol). 4  Ca-K-P0  4  Buffer  1.5 x 1 0 - M N o n a n o l 4  Ca(H P0 )2 x H 0 5 mg d i s s o l v e i n 100 m l H 0 2  4  2  2  KH P0 27 2 mg d i s s o l v e i n 100 m l H 0 add 39 m l t o a b o v e 2  4  Stock s o l u t i o n : d i l u t e 1:4 p r i o r t o u s e N-nonyl a l c o h o l (9.94M)  70 u l  2  K HP0 3 48 mg d i s s o l v e i n 100 m l H 0 add t o a b o v e u n t i l pH 6.8 ( 6 1 m l ) 2  4  2  Total  volume  H 0 2  1000 m l mix w i t h s t i r b a r a t h i g h speed u n t i l alcohol droplets dispersed  200 m l  I n n e r w e l l o f Conway  cell  O u t e r w e l l o f Conway  cell  29  Table I I : The composition and preparation of the d i f f e r e n t i a t i o n medium MPG. Ca(H P0 ) KH P0 K HP0 Peptone D-Glucose 2  2  4  2  x H0  0. 025 g 0.449 g 1.145 g 5 g 30 g  2  4  2  4  volume made t o 1000 ml with g l a s s d i s t i l l e d H 0 2  The KH P0 and Ca(H P0 ) x H 0 were dissolved i n 200 ml d i s t i l l e d water. K HP0 was added u n t i l pH 6.8 was reached. Peptone and glucose were added and the pH was adjusted as necessary with phosphoric acid or KOH. The medium was s t e r i l i z e d by autoclaving. 2  4  2  2  4  2  4  swellings were counted as appressoria only a f t e r a septum d e l i m i t i n g the appressorium from the germtube was c l e a r l y visible.  The i n f e c t i o n peg was recognized as a small  outgrowth from the mature appressorium.  Substomatal v e s i c l e s  were recognized a f t e r the terminal end of the i n f e c t i o n peg had expanded t o a s i z e equal t o h a l f that of the appressorium. As the v e s i c l e s matured they flattened out forming two lobes on e i t h e r side.  An i n f e c t i o n hypha was counted when one lobe  of the v e s i c l e extended beyond the length of the other.  3.5  Staining and Counts  Sporelings were transferred from each dish to a glass s l i d e , stained with trypan blue i n lactophenol (Table I I I ) , covered with a coverslip and sealed with n a i l p o l i s h .  The  s l i d e s were semi-permanent and may be stored f o r up to one year.  Counts of 100 spores were made per s l i d e and the  component i n f e c t i o n structures were i n d i v i d u a l l y assessed. D i f f e r e n t i a t i o n was expressed as a percentage of the t o t a l spores.  3.6  Temperature Range T r i a l s  Temperature requirements f o r appressorium formation and the t o t a l d i f f e r e n t i a t i o n of uredospore germlings was investigated by applying a heat shock of constant temperature  31  T a b l e I I I : L a c t o p h e n o l - T r y p a n B l u e ; a mounting and s t a i n i n g medium f o r semipermanent mounts o f f u n g i ( B o e d i j n , 1965). Phenol Lactic acid Glycerol D i s t i l l e d water Trypan Blue  20 20 40 20 0.5%  g g g ml  32 (ranging from 26 t o 32°C f o r 1.5 h) 2 h following germination at 19°C.  The temperature range was tested over four sub-  experiments, each of which u t i l i z e d a d i f f e r e n t spore l o t . The heat shock applied during the temperature range t r i a l s was administered by placing the Conway dishes on a temperature regulated brass plate.  Warm water (35°C) from a hot water  bath was c i r c u l a t e d through copper tubing under one end of the brass plate while the ambient temperature cooled the other end.  The temperature gradient (25 t o 35°C) thus generated was  allowed to s t a b i l i z e f o r a t least 2 h p r i o r to the experiment. Following 2 h germination at 19°C the inoculated dishes were transferred to the brass plate and set a t various i n t e r v a l s along the temperature gradient.  A f t e r 1.5 h the temperature  of the Ca-K buffer i n each dish was measured by an e l e c t r o n i c thermometer and a thermocouple. to 19°C f o r 14 h.  The dishes were then returned  Observations were made a f t e r a t o t a l of  17.5 h of incubation. Since the germination and d i f f e r e n t i a t i o n capacity of spore batches d i f f e r e d s l i g h t l y , the data within each subexperiment were adjusted t o allow f o r comparison between experiments.  The maximum d i f f e r e n t i a t i o n value within each  sub-experiment was represented as "100"; a l l other values were calculated as a percentage of the maximum.  The optimum  d i f f e r e n t i a t i o n temperature varied between experiments and ranged from 28.2 t o 30.7°C.  Therefore, the optimum  temperatures were assigned a value of "0", a l l remaining  temperatures were calculated as u n i t s deviating from t h i s optimum.  3.7  E s s e n t i a l Protein Synthesis: Puromycin  Uredospores were inoculated onto the Ca-K  buffer and  induced to d i f f e r e n t i a t e under conditions previously described.  A protein synthesis i n h i b i t o r , Puromycin  dihydrochloride (100 ug/ml) (Sigma Co.), was dissolved i n the buffer and added to, or removed from (using a 10-ml  syringe),  the inner well of the Conway dish at the times s p e c i f i e d i n Table IV.  A f t e r each medium change, the sporelings were  washed three times with fresh medium.  E s s e n t i a l protein  synthesis was measured by assessing the extent of uredosporeling d i f f e r e n t i a t i o n a f t e r 15 h. In a l l treatments designed to examine e s s e n t i a l protein synthesis the uredospores were k i l l e d and stained i n lactophenol-trypan blue upon completion of the experiment. Each treatment had three r e p l i c a t e dishes, two sample counts were taken from each r e p l i c a t e , and 100 spores were counted per sample.  The treatments were compared to the control f o r  each morphogenic group by one-factor analysis of variance. The complete experiment was repeated  twice.  34  Table IV: The timing of essential protein synthesis: Experimental design. The uredosporelings were germinated at 19^C f o r 2 h, exposed to a heat shock f o r 1.5 h, then returned to 19°C f o r 12 h.  Treatment  (control) 2 3 4 5 6 7 8 9 10 11  Stages of germination and d i f f e r e n t i a t i o n (h) a t which puromycin i s added t o the medium (x) 0-2  2-3 . 5  3 . 5-6  -  -  -  X  X  -  -  —  —  |  6-8  1 " 1 8  10  10-15  -  -  X  X  X  X X  X X  X X  X  X  X X X  -  X  -  X  X  —  -  X  X  X  -  X  -  —  35 3.8  N u c l e a r S t a i n i n g : DAPI  U r e d o s p o r e s were g e r m i n a t e d on Ca-K b u f f e r f o r 4 h i n t h e d a r k a t 19°C.  The g e r m l i n g s were t r a n s f e r r e d t o a c l e a n  slide  u s i n g an i n o c u l a t i o n l o o p and a l l o w e d t o d r y . A d r o p o f DAPI (4', 6 - d i a m i d i n o - 2 - p h e n y l i n d o l e ) ,  1 ug/ml g l a s s - d i s t i l l e d  w a t e r , was p l a c e d d i r e c t l y on t h e sample, a c o v e r s l i p was a p p l i e d and t h e edges were s e a l e d w i t h n a i l p o l i s h .  The s l i d e  was o b s e r v e d w i t h i n 20 min by f l u o r e s c e n t m i c r o s c o p y . The i n s t r u m e n t used was a Z e i s s u n i v e r s a l m i c r o s c o p e e q u i p p e d w i t h a 100 W mercury lamp t o d e l i v e r e x c i t a t i o n l i g h t by e p i f l u o r e s c e n t mode, a Z e i s s UG1 e x c i t e r f i l t e r w i t h a passband from c a 300 t o 400 mu, BG38 t o absorb l i g h t and p r o t e c t sample from h e a t , a b a r r i e r f i l t e r w i t h a c u t o f f a t 410 nm, and a Z e i s s N e o f l u o r  40 power o b j e c t i v e .  Transmitted  l i g h t and f l u o r e s c e n c e photographs were made w i t h F u j i c o l o r 400 ASA d a y l i g h t f i l m . The r e s u l t s o b t a i n e d  from u n f i x e d m a t e r i a l were  u n a c c e p t a b l e i n t h a t DAPI s t a i n e d t h e germtube w a l l as w e l l as the n u c l e i . to optimize  S e v e r a l f i x a t i o n s c h e d u l e s were t e s t e d i n o r d e r stain specificity  (Table V ) .  A l l f i x e d samples  were p l a c e d on a g l a s s s l i d e and s t a i n e d i n a drop o f 1 ug/ml DAPI. A l l t i s s u e s t h a t were f i x e d i n a l c o h o l - c o n t a i n i n g f i x a t i v e s were washed i n a l c o h o l o f t h e same c o n c e n t r a t i o n a s that present i n the f i x a t i v e .  A f t e r w a s h i n g t h e t i s s u e was  36  Table V: 1.  A description of the f i x a t i o n schedules tested.  Formalin-acetic acid-alcohol (FAA)*  Ethyl alcohol (50%) 90 ml G l a c i a l acetic acid 5 ml Formalin 5 ml The material was fixed f o r 2, 24, and 48 h, then rehydrated through an alcohol series to water. 2.  Chrom-acetic solution (Johansen,  1940)  Aqueous chromic acid (10%) 2.5 ml Aqueous a c e t i c acid (10%) 5.0 ml D i s t i l l e d water to 500 ml The sample was fixed f o r 20 h then washed three times i n d i s t i l l e d water (30 min per wash). 3.  Ethanol/Acetic acid  (3:1)  The material was fixed f o r 2, 24, and 48 h, dried on a s l i d e , immersed i n 200 mM KCL and rehydrated through a graded alcohol s e r i e s . 4.  Formaldehyde: 4% and 36% *  The sample was fixed f o r 20 h i n either a 4% solution or the vapours of 36% formaldehyde, then washed 3 times i n d i s t i l l e d water (30 min per wash). 5.  Glutaraldehyde: 3% * The sample was fixed f o r 1 h then washed three times i n d i s t i l l e d water (30 min per wash).  6.  Ethanol series  The material was dehydrated through an ethanol series (15%, 30%, 40%, 60%, 70%) allowing 5 min between changes. After at least 4 h i n 70% the sample was rehydrated (10 min between washes) back to d i s t i l l e d water. * These f i x a t i v e s were both used as described and i n combination with a surfactant. Two surfactants were assessed i n d i v i d u a l l y , 0.25% T r i t o n X-100 and 0.01% Tween-20.  l e f t f o r 30 min i n each o f t h e f o l l o w i n g : 50%, 30%, 15% e t h y l a l c o h o l , and d i s t i l l e d w a t e r . The method o f c h o i c e was a s i m p l e a l c o h o l d e h y d r a t i o n and subsequent r e h y d r a t i o n o f t h e sample p r i o r t o s t a i n i n g w i t h DAPI.  The n u c l e i were c l e a r l y v i s i b l e and background  f l u o r e s c e n c e was m i n i m i m a l i n a l l b u t t h e v e r y young (0 t o 2 h ) .  germtubes  N u c l e a r f l u o r e s c e n c e improved f o r 20 min and  remained s t a b l e f o r up t o 10 days when s t o r e d i n t h e d a r k a t 4°C.  3.9  The c h e m i c a l  s t r u c t u r e o f DAPI i s shown i n F i g u r e 2.  Amino A c i d A n a l y s i s  3.9.1  H i g h Performance L i q u i d Chromatography  (HPLC)  HPLC s e p a r a t i o n s a r e c a r r i e d o u t i n h i g h r e s o l u t i o n columns packed w i t h 3 t o 25 distribution.  m p a r t i c l e s o f uniform  size  The columns r e q u i r e t h e u s e o f d e d i c a t e d  i n j e c t o r s f o r sample i n t r o d u c t i o n , s e n s i t i v e d e t e c t o r s , and s p e c i a l pumps w h i c h d e l i v e r c o n s t a n t f l o w a g a i n s t h i g h pressure.  HPLC methods may be u s e d f o r t h e s e p a r a t i o n o f a  d i v e r s e a r r a y o f compounds w i t h m o l e c u l a r w e i g h t s r a n g i n g between 50 and 20 m i l l i o n . Two approaches a r e c u r r e n t l y a v a i l a b l e f o r HPLC a n a l y s i s o f amino a c i d s : a n a l y s i s u s i n g r e v e r s e d - p h a s e s e p a r a t i o n o f p r e d e r i v a t i z e d amino a c i d s , and a n a l y s i s u s i n g ion-exchange methods w i t h p o s t - d e r i v a t i z e d amino a c i d s .  38  H  Fig. 2. DAPI (4\6-diamidmo-2-phenylindole) binds specifically to AT residues of double-stranded DNA. The AT-specificity resides with both the guanidine group and the indole ring, which may bind to the purine of adenosine through base stacking (Otto and Tsou, 1985).  V  39 Reverse-Phase (RP) Bonded-Phase Chromatography The stationary phase of a RP column i s non-polar, consisting of s i l i c a gel with covalently bound hydrocarbon chains (lengths ranging from C^ to C 2)• 2  Tire e l u t i o n p r o f i l e  of a sample r e f l e c t s the degree of hydrophobicity inherently unique to each of i t s components.  The compound with the  highest a f f i n i t y f o r the solid-phase emerges l a s t .  Solvents  from high to intermediate p o l a r i t y are used as mobile phases. Thus, water i s the solvent which gives the longest retention. The retention and s e l e c t i v i t y of the column can be adjusted and optimised by the addition of water-miscible organic solvents (e.g. methanol, a c e t o n i t r i l e ) .  Since the conditions  of separation are mild the formation of a r t i f a c t s i s not a problem.  3.9.2  Reagents Type I reagent grade water was prepared by running g l a s s -  d i s t i l l e d water through the M i l l i - Q water p u r i f i c a t i o n system (Millipore Co).  This system combines activated carbon  adsorption, mixed-bed deionization, an organics-scavenging cartridge (Organex-Q), and 0.22  m s t e r i l i z i n g membrane  f i l t r a t i o n (Millipak F i l t e r U n i t ) . Methanol and a c e t o n i t r i l e were HPLC-grade (BDH Co). grade.  Chemical  A l l remaining components of the buffer system were ACS Amino acid standards  (hydrolyzate mix, No. 20088),  t r i f l u o r o a c e t i c acid (TFA), triethylamine (TEA), and the  40 d e r i v a t i z i n g agent phenylisothiocyanate (PITC) (No. 26922) were obtained from the Pierce Chemical Company.  3.9.3 Instrument The equipment used was designed by Waters and consisted of two Model 510 pumps, a Model 710B WISP, a System Interface Module, an 840 Data and Chromatography Control Station, a Temperature Control Module, and a 490 Programmable Multiwavelength Detector (Figure 3). RP was a Waters C  1 8  The stationary phase f o r  Pico-Tag column f o r protein hydrolyzates  (3.9 mm x 15 cm). RP-HPLC was performed a t 41°C. When not i n use the column and pumps were washed with water t o remove s a l t s and then stored i n a c e t o n i t r i l e .  3.9.4  Sample C o l l e c t i o n and Preparation  The procedures f o r amino acid analysis necessitated s t r i c t control of a l l possible sources of contamination. B a c t e r i a l and fungal contamination were monitored by suspending uredospores i n s t e r i l e water and p l a t i n g s e r i a l d i l u t i o n s onto nutrient agar.  Glassware:  Glassware was f i r s t washed i n soap (Alconox) and  water, rinsed with tap water, oven dried and then soaked i n an acid bath (NoChromix, Chemonics Sci.) f o r at l e a s t 1 h. The glass was then rinsed three times with glass d i s t i l l e d water, rinsed once i n M i l l i - Q water and dried a t 60°C f o r several  41  Buffer A HPLC Pump A I RP-Column [ Column Heater  WISP HPLC Pump B  Buffer B  Programmable Multiwavelength Detector  Temp. Control Module Discard Bottle System Interface Module  Data and Chromatography Control Station  Component Pump A  (Waters)  Function  (Model  510)  d e l i v e r y of e l u t i o n b u f f e r A  Pump B (Model  510)  d e l i v e r y of e l u t i o n b u f f e r B  WISP (Model System  710B)  I n t e r f a c e Module  automated  sample  injection  l i n k s seperate components with C o n t r o l S t a t i o n  840 Data and Chromatography Control Station  controls conditions for HPLC, s t o r e s and analyzes data  Amino A c i d A n a l y s i s Column  reverse-phase  Temperature  r e g u l a t i o n of column c o i l temperature  C o n t r o l Module  Programmable Multiwavelength D e t e c t o r (490)  d e t e c t i o n of  separations and  PITC-derivatives  Figure 3. Waters HPLC amino a c i d analysis system configuration f o r RP-analysis of amino acids with post-column u l t r a v i o l e t detection.  42 hours.  Buffer:  Ca-K buffer was prepared with M i l l i - Q water i n a c i d -  washed glassware, s t e r i l i z e d through a 0.2  m Millipore  f i l t r a t i o n u n i t and stored at 4°C u n t i l required.  Spore Germination:  Procedures e a r l i e r described f o r inducing  germination and d i f f e r e n t i a t i o n were c a r r i e d out using autoclaved, acid-washed Conway dishes and s t e r i l e buffers. Ungerminated, non-differentiated, and d i f f e r e n t i a t e d uredospores and t h e i r leachates were analyzed f o r free amino acid content.  The undifferentiated and d i f f e r e n t i a t e d  sporelings were harvested a f t e r 8 and 20 h.  Each treatment  consisted of s i x dishes, which were l a t e r combined t o be analyzed as one sample.  The 20 h experiment was repeated  three times and the 8 h experiment twice.  The extent of  d i f f e r e n t i a t i o n was monitored f o r each treatment using the method described i n Staining and Counts (page 30) .  Wash:  Freshly harvested spores (50 mg) were shaken i n 10 ml  of b u f f e r and 0.01% Tween-20 f o r 5 min, the suspension was centrifuged and the supernatant transferred to an vessel.  acid-washed  The above procedure was repeated three times y i e l d i n g  a wash volume of 30 ml which was then freeze-dried and taken up i n 1 ml of M i l l i - Q water.  The washed spores were ground  and extracted as described e a r l i e r .  43 Spores:  The spores were c o l l e c t e d , rinsed with M i l l i - Q water,  placed i n an acid-washed mortar, ground with acid-washed  sand  and HPLC-grade 80% ethanol, and then transferred t o a centrifuge tube and stored at  20°C f o r 20 h.  The spore  extract was centrifuged at high speed f o r 10 min and the deproteinized supernatent decanted into an acid-washed  vessel.  The ethanol was evaporated t o 1 ml under a stream of nitrogen gas and transferred to a 10-ml t e s t tube.  The dry weight of  50 mg spores was determined by drying the spores f o r 16 h at 100°C.  Leachate:  The leachate, together with the water used to rinse  the spores, was c o l l e c t e d from the 6 dishes with a syringe, f i l t e r e d (0.2  10-ml  m M i l l i p o r e ) into a 150 ml acid washed  pyrex vessel, s h e l l frozen i n l i q u i d N  2  and freeze-dried.  The  residue was taken up i n 2 ml of M i l l i - Q water and transferred to a 10-ml t e s t tube.  3.9.5  Sample Clean-up A C  1 8  Sep-Pak cartridge (designed by Waters) was used to  remove l i p i d , pigment, residual protein, and other hydrophobic materials from a l l samples. given i n Figure 4.  The sample preparation scheme i s  Following clean-up a l l samples,  including  a standard amino acid mixture, were dried down i n a SpeedVac concentrator (Savant Instruments) without heat.  44  1.  Activate a Sep-Pak C methanol.  2.  Wash with 2-10 ml of 0.1% t r i f l u o r o a c e t i c a c i d (TFA) i n M i l l i - Q water.  3.  Wash with 10 ml of 0.1% TFA i n water and methanol (80:20).  4.  Mix 1 ml sample with 2 ml 0.1% TFA i n water and methanol (70:30).  5.  Pass the sample through the Sep-Pak.  6.  Discard the f i r s t 1 ml of. e f f l u e n t and c o l l e c t the next 2 ml f r a c t i o n which contains a l l the amino acids.  1 8  cartridge with 2-10-ml volumes of  Figure 4. Sample preparation scheme f o r crude amino a c i d samples using Waters Sep-Pak C cartridges. 1 8  3.9.6  HPLC of Amino Acids as Phenylthiocarbamoyl  Derivatives  Buffer System Eluent A:  Sodium acetate trihydrate, 19.0 gm (140 mM)  dissolved i n 1000 ml of M i l l i - Q water and 5% TEA. adjusted t o 6.4 with g l a c i a l a c e t i c acid.  was  The pH was  The s o l u t i o n was  vacuum f i l t e r e d and combined with 25 ml a c e t o n i t r i l e .  Eluent B:  Contained 60% a c e t o n i t r i l e i n M i l l i - Q water.  Degassed by sonication f o r 5 min.  Pre-column Derivatization Derivatization Solution:  An ethanol, TEA and M i l l i - Q water  (7:1:1) solution was combined with 10% PITC.  Sample Diluent:  A phosphate buffer was prepared (710 mg  Na HP0 per 1000 ml t i t r a t e d t o pH 7.4 with 10% phosphoric 2  4  acid), combined with 50 ml a c e t o n i t r i l e , and f i l t e r e d .  Procedure: with 20  The dried sample and standard were derivatized  1 of the d e r i v a t i z i n g solution f o r 30 min at room  temperature.  The solvents were removed under vacuum with the  SpeedVac centrifuge.  The sample and the standards were  reconstituted with 25 and 500 respectively.  1 of sample diluent,  The amino acids were derivatized i n order t o  improve the detection s e n s i t i v i t y as well as overcome the inherent p o l a r i t y of the free amino acids.  PITC-amino acids  have a broad UV spectrum with a maximum absorbance near 269 nm. PITC allows the quantitative d e r i v a t i z a t i o n of both primary and secondary amino acids.  The reaction scheme f o r  amino acids by pre-column d e r i v a t i z a t i o n with PITC i s outlined i n Figure 5.  Sample Preparation P r i o r t o being loaded on the column each sample was f i l t e r e d (MSI, Cameo dispensable nylon f i l t e r s , 3 mm membrane, 0.45  m pore size) and c o l l e c t e d into 0.5 ml Eppendorf micro  t e s t tubes by centrifugation f o r 5 min i n a microcentrifuge (Eppendorf Model 5413).  The tubes were then placed on a  spring i n 1.5 ml glass v i a l s (Pierce Chemical Co.), covered with a membrane, sealed with a cap, and placed on the autosampler tray.  The pump table was programmed t o generate  the appropriate buffer gradient system and flow-rate (Table VI) .  Chromatography Four  1 of standard were injected per run (200 pM), the  f i r s t i n j e c t i o n generated the "junk" chromatograph  and was  discarded, the second i n j e c t i o n produced the chromatograph used t o c a l i b r a t e the column and subsequent runs. i n j e c t i o n volume was 8  The sample  1 and the i n j e c t i o n - t o - i n j e c t i o n cycle  time was 30 min. A f t e r four i n j e c t i o n s and separations of  47  <fO> = = / - N \  /  ^  N  C  H  S  -  L  PH 10,30 min  + NH-C-C-0 R  '  .  11  +  phenylisothiocyanate (in excess)  y  1  4.  S  '  >  amino compound  H  H O  <(0/"N - C - N - C - C - O^  v  temperature  >  R  aqueous acid  /  \  w  A .  N  O  R  phenylthiocarbamyl amino acids  Fig. 5. Reaction scheme of amino compound by pre-column derivatization with PITC.  48  Table VT: Pump method; the buffer gradient system and flowrate designed t o e f f e c t i v e l y contribute to the separation of amino compounds within a p h y s i o l o g i c a l sample. Time I n i t i a l conditions 10. 00 10.50 11. 50 12 . 00 12.50 20. 00 20.50 30.00  Flow  Curve  %A  %B  1.0 1.0 1.0 1.0 1.5 1.5 1.5 1.0 0.1  *  95 55 0 0 0 100 100 100 100  5 45 100 100 100 0 0 0 0  5 6 6 6 6 6 6 11  49 sample the column was  recalibrated with the standard.  The r e l a t i o n s h i p between a s p e c i f i c amino a c i d and a known concentration was factor (RF).  calculated i n terms of a response  The RF i s an adjustment f o r compounds that do  not respond equally i n the detector concentration). of peaks.  (RF= peak area/amino acid  The sample chromatograms contained  a number  Most of the retention times corresponded with those  of i d e n t i f i e d amino acids, the remaining peaks were c l a s s i f i e d as "unknown".  The RF values determined during the c a l i b r a t i o n  run were used to c a l i b r a t e the amount of a s p e c i f i c amino acid within the sample.  Standard solutions of glutamine and  glutathione were prepared, and 200 pM of the standard injected onto the column.  was  Glutamine formed a peak between  serine and glycine and glutathione eluted with ammonia. Three controls were run:  The Ca-K  buffer contained  large sloping peak (eluting at approximately 1 to 2.2 The baseline was  otherwise f l a t with n e g l i g i b l e peaks.  a  min). Acid  washed sand ground with 80% ethanol generated a chromatogram with a f l a t baseline interrupted by two sharp peaks. f i r s t peak eluted at 9.65  min,  shortly a f t e r leucine, and  second followed phenylalanine at 11.1 min. d e r i v a t i v e contained  The  The blank  no i n t e r f e r i n g amino compounds.  the  50 4.  RESULTS  4.1  Uredospore G e r m i n a t i o n and D i f f e r e n t i a t i o n "In V i t r o "  In t h e presence o f 1.5  x 10  - 4  M n-nonyl a l c o h o l ,  100% o f t h e uredospores germinated w i t h i n 2 h s e e d i n g on C a - K b u f f e r  (Fig.  90  to  following  6).  C o n t r a r y t o e a r l i e r s t u d i e s i n which 95% d i f f e r e n t i a t i o n was o b t a i n e d  (Maheshwari e t a l . ,  t h e p r o p o r t i o n o f germtubes structures infection shock  1967;  Dunkle e t a l . .  forming complete  infection  on Ca-K b u f f e r d i d not exceed 60%.  The t i m i n g o f  s t r u c t u r e formation i n response t o a 1.5  (30.5°C)  1969),  on Ca-K b u f f e r i s shown i n F i g . 7.  h heat Germtube  growth was a r r e s t e d s h o r t l y a f t e r exposure t o t h e  elevated  temperature.  3.5 h and  The hyphal t i p began t o s w e l l  after  w i t h i n 5 h t h e appressorium was mature ( i . e .  a cross wall  d e l i m i t e d t h e appressorium from the germtube).  The  infection  peg formed between 5 and 6 h and a t 8 h began t o extend produce the substomatal v e s i c l e .  The development  v e s i c l e was u s u a l l y completed w i t h i n 10 h by t h e a septum between i t and the appressorium.  of  A f t e r 10 h the the  The cytoplasm w i t h i n t h e germtube and t h e  appressorium began t o c l e a r a t 8 and 9 h , Infection  the  formation of  i n f e c t i o n hypha began t o develop on t h e d i s t a l end o f vesicle.  to  respectively.  s t r u c t u r e f o r m a t i o n was complete w i t h i n 15 t o 20 h .  I n i t i a l l y t h e i n d u c t i o n o f d i f f e r e n t i a t i o n by h e a t shock  51  Time (hr) Fig. 6. The percent germination of uredospores on Ca-K buffer in both the presence ( • ) and absence ( O ) of 1.5 x 10 -4 M n-nonyl alcohol.  52  F i g . 7. Timing of sporeling morphogenesis. (A) A sporeling 1 h following germination. (B) A sporeling 3.5 h following germination, the appressorium i n i t i a l has formed. (C) A f t e r 5 h a septum was v i s i b l e and the appressorium was considered mature. (D) Development of the i n f e c t i o n peg was complete 6 h following germination. (E) After 9 h the cytoplasm began to c l e a r within the germtube. (F) The substomatal v e s i c l e began to expand a f t e r 8 h. (G) The cytoplasm migrated from the appressorium into the developing substomatal v e s i c l e and a second septum become apparent (10 h). (H) The i n f e c t i o n hypha was apparent a f t e r 15 h. A d i f f e r e n t i a t e d sporeling a f t e r 20 h. (592 X).  53 as described by Maheshwari e t a l . (1967) yielded poor r e s u l t s (< 5% d i f f e r e n t i a t i o n ) . mm  2  By using a spore density of 300 per  and c o n t r o l l i n g the temperature of the heat shock by  placing the Conway dishes i n a water bath, i t was possible t o a t t a i n up t o 60% d i f f e r e n t i a t i o n .  A l l further attempts t o  improve the percent d i f f e r e n t i a t i o n ; such as, changing the timing and duration of the heat shock were unsuccessful.  4.2  Temperature Range T r i a l s  The temperature required t o induce the maximum proportion of germtubes t o d i f f e r e n t i a t e into i n f e c t i o n structures was found t o l i e within  a  narrow range.  The optimum temperature  appeared t o be c h a r a c t e r i s t i c of a p a r t i c u l a r spore l o t and ranged from 28° t o 30°C.  Incubation a t lower than 27.5°C or  higher than 30.5°C considerably reduced the amount of appressorium formation (Fig. 8) and t o t a l d i f f e r e n t i a t i o n (Fig. 9). The upper and lower temperature l i m i t s f o r a l l spore l o t s tested were estimated as 30.5® and 27.5°C, respectively.  Heat treatment was completely i n e f f e c t i v e when  the temperature was raised 1° above the upper l i m i t or dropped 2° below the lower l i m i t (Figs. 8 and 9). Although the incubation temperature where the maximum t o t a l d i f f e r e n t i a t i o n was achieved varied between spore l o t s , the s e n s i t i v i t y of the sporelings t o temperature changes was similar  (Figures 10 and 11).  Variations 1° above or below the  54  F i g . 8. Temperature requirements f o r appressorium formation from uredospore germtubes. The spores were germinated a t 19°C (2 h ) . then placed a t constant temperatures (ranging from 26 to 33°C) f o r 1.5 h, and replaced t o 19°C. The optimum temperature (><) i s the temperature at which the maximum t o t a l d i f f e r e n t i a t i o n was attained f o r each spore l o t .  55 80 i  <  >  60 40  Fig. 8A. Spores collected July 22,1986. Optimal differentiation temperature (><): 29.3 C .  •  d  •  20  •  0  •  <  60  •  40  Fig. 8B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C.  •  •  20  •  •  0  60  Fig. 8C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C.  >  40  •  20 • 0 <  60  •  Fig. 8D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 ° C .  •  40  • •  20  - 2 - 1  0 + 1  •  •  +2  +3  Variation Below (-) and Above (+) Optimal Temperature (°C)  56  F i g . 9. Temperature requirements f o r the complete d i f f e r e n t i a t i o n of P. graminis t r i t i c i uredospore germlings. The upper and lower l i m i t s f o r t o t a l d i f f e r e n t i a t i o n were estimated to be 27.5 and 30.5°C, respectively. Germination conditions were described i n F i g . 8.  57 60  •  40  Fig. 9A. Spores collected July 22,1986. Optimal differentiation temperature (> <): 29.3 °C.  •  20  I  I  Fig. 9B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C.  n 40-  I J  20  p  I1 o  0  Pi  zn  Fig. 9C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C.  40  <u 20 •  •  PH  •  •  40  Fig. 9D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 °C.  20  • • -1  0  +1  +2  +3  Variation Below (-) and Above (+) Optimal Temperature ( C)  58  100 -,  80 •  60  < 40  20 • i  -1  +1  •  •  • •  •  +2  Variation Below (-) and Above (+) Optimal Temperature (°C)  Fig. 10. The relationship between heat shock temperature and the transformed percent of appressoria formed (TPA).  i  +3  59  Variation Below (-) and Above (+) Optimal Temperature (°C)  Fig. 11. The relationship between heat shock temperature and the transformed percent of total sporeling differentiation (TPD).  60 optimum temperature resulted i n greater than 40% reduction i n the number of sporelings forming i n f e c t i o n structures.  The  formation of appressoria was l e s s s e n s i t i v e to deviations from the optimum temperature  (Fig. 10) than the formation of a  complete set of i n f e c t i o n structures (Fig. 11).  4.3  The Influence of Nutrients on Sporeling D i f f e r e n t i a t i o n  Preliminary experiments were undertaken to investigate the e f f e c t of nutrients (peptone and glucose), a germination stimulant (n-nonyl alcohol), and a heat shock (30°C, 1.5 h), on the percentage of sporelings developing i n f e c t i o n structures (Table VII).  MPG medium (peptone, glucose, and Ca-  K b u f f e r ) , i n conjunction with both a heat shock and n-nonyl alcohol, supported up to 77% d i f f e r e n t i a t i o n .  When the heat  shock was not administered, less than 11% of the sporelings differentiated.  The absence of both, n-nonyl alcohol and the  heat shock resulted i n the growth of long, matted germtubes that f a i l e d to form i n f e c t i o n structures. Although a l l three components of MPG  influenced sporeling  d i f f e r e n t i a t i o n i t was evident that t h e i r roles were not of equal importance.  MPG was broken down t o i t s constituent  parts and each component was tested f o r i t s a b i l i t y to promote differentiation.  The buffer alone supported 66%  d i f f e r e n t i a t i o n , peptone i n d i s t i l l e d water supported 14%, and glucose i n d i s t i l l e d water resulted i n l e s s than 5% sporeling  61  Table VII: A summary of experiments investigating the influence of the components of MPG, n-nonyl alcohol, and a 29.5°C (1.5 h) heat shock on the percent of germtubes forming appressoria (PAS) and the percent of sporelings forming a complete set of i n f e c t i o n structures (PDS). The presence or absence of a treatment i s indicated by '+' or respectively. MPG peptone g l u c o s e Ca-K + + + + + + +  * -  + + + + + +  -  + +  •  + + + +  -  + + + +  n-nonyl alcohol  heat shock  + +  + +  + + + + + + +  + + + + + + +  -  -  *supplemented with tyrosine (49 mg/l), cysteine tryptophan (15 mg/l).  PAS  PDS  89 27 55 0 54 71 84 36 83 5 40 (17 mg/l), and  77 11 50 0 44 32 39 14 66 5 32  62 differentiation.  The combination of peptone and glucose  increased d i f f e r e n t i a t i o n to 32%. uredosporelings (77%) was  The maximum amount of  forming a complete set of i n f e c t i o n  achieved on the complete MPG  medium.  structures  These r e s u l t s  show c l e a r l y that the response to nutrients i s non-additive. Glucose  i n d i s t i l l e d water promoted the growth of long  germtubes which f a i l e d to d i f f e r e n t i a t e .  The addition of  cysteine, tyrosine and tryptophan to glucose and Ca-K  buffer  had no s i g n i f i c a n t e f f e c t on d i f f e r e n t i a t i o n .  4.4  E s s e n t i a l Protein  Synthesis  Sporelings exposed to an i n h i b i t o r of protein synthesis before, during, and a f t e r the inductive period  provide  evidence f o r the nature and timing of protein synthesis during differentiation.  I t was  found i n the present study that the  presence of puromycin (100 ppm)  had a s i g n i f i c a n t e f f e c t on  appressorium formation and completely i n h i b i t e d a l l further development (Table VIII).  In most cases where the heat  treatment arrested l i n e a r growth the germtube produced an apical swelling, which a f t e r 20 h f a i l e d to form a cross wall (Fig. 12a).  The appressorium was  e a r l i e r defined by  Staples  et al.(1983c) as a swelling of the hyphal t i p that i s separated  from the germtube by a septum.  In the absence of  puromycin the appressoria were oval to roundish.  In the  63  Table VIII: The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin (100 ppm), on t h e development o f i n f e c t i o n s t r u c t u r e s from g e r m l i n g s o f P. g r a m i n i s t r i t i c i . To induce t h e d i f f e r e n t i a t i o n o f i n f e c t i o n s t r u c t u r e s a heat shock a t 3 0 ° C f o r 1.5 h was a d m i n i s t e r e d 2 h a f t e r t h e s t a r t o f germination at 1 9 ° C . S p o r e l i n g development  Treatment  Appressoria a* b#  Infection peg  Vesicle a b  (%) Infection hypha  69  100  61  100  53  100  40  100  X  78  114  72  117  60  114  27  67  XX  73  107  69  112  50  96  1  3  XXX  72  105  58  94  17  31  0  0  —xxxx  75  109  54  88  0  0  0  0  xxxxxx  17  2  3  0  0  0  0  - x —  78  114  71  115  49  93  21  53  — x -  76  111  67  109  43  81  12  30  —-x-  78  113  72  118  46  88  11  27  —xx—  72  105  57  93  27  52  3  7  xx-  73  106  55  90  27  51  2  5  (control)  25  Puromycin was p r e s e n t (x) and absent from (-) t h e g e r m i n a t i o n medium (Ca-K b u f f e r ) . ( ~) c o r r e s p o n d s t o t i m e i n t e r v a l s ; 0-2 h , 2-3.5 h , 3.5-6 h , 6-8 h , 8-10 h , and 10-15 h . *development i s expressed as a mean p e r c e n t o f t h e t o t a l spores counted. ^development i s expressed as a percent of the c o n t r o l , underlined values are s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l (95% c o n f i d e n c e - F i s h e r L S D ) .  64  Fig. 12. The presence of a protein i n h i b i t o r (puromycin) i n the germination medium s i g n i f i c a n t l y reduced the number of germtubes forming appressoria. (A) The appressoria were predominantly i r r e g u l a r i n form (bright f i e l d illumination, 592x). (B) Generally the nuclei within the germtube d i d not divide but remained expanded from 0 to 15 h (fluorescent micrograph, 1040x). (C) In cases where a septum was formed the nuclei divided t o give r i s e to four daughter-nuclei. The p a i r i n g of daughter nuclei was not observed (double illumination with U.V. and highly attenuated v i s i b l e l i g h t , 1040x).  65 presence of the i n h i b i t o r however, t h e i r shape was predominantly i r r e g u l a r (Fig. 12a). The r e s u l t s found by Dunkle e t a l . were confirmed (with the exception of appressorium formation) by a series of preliminary experiments.  To further c l a r i f y the timing of  protein synthesis and i t s e f f e c t on the proportion of germtubes forming i n f e c t i o n structures the sporelings were exposed to puromycin at s p e c i f i c times following heat treatment.  Complete d i f f e r e n t i a t i o n was prevented i n those  t r i a l s where puromycin was present from immediately a f t e r heat treatment to the completion of the experiment  (20 h).  During  t h i s time frame the development of the appressorium and the i n f e c t i o n peg were unaffected but a l l further d i f f e r e n t i a t i o n was i n h i b i t e d  (Tables VIII and IX).  Although the sporelings  appeared t o recover from the e f f e c t s of the i n h i b i t o r shortly a f t e r the removal of puromycin from the germination medium, the development of the i n f e c t i o n hypha was s i g n i f i c a n t l y impaired i n a l l cases. Although the development of v e s i c l e s d i d not occur before 10 h, t h e i r formation appeared t o be dependent on proteins synthesized 3.5 to 8 h following germination.  The formation  of the i n f e c t i o n hypha was dependent on the development of the v e s i c l e ; the l a t t e r always preceded the former.  The  proportion of v e s i c l e s developing i n f e c t i o n hyphae was considerably reduced when puromycin was present at 3.5 t o 8, 6 to 10, or 6 to 15 h and completely i n h i b i t e d when the  66  Table IX: The e f f e c t of a protein i n h i b i t o r , puromycin (100 ppm), on the development of i n f e c t i o n structures from germlings of P. graminis t r i t i c i . Development i s expressed as a mean percent of the preceding structure. Sporeling Treatment  I n f e c t i o n peg a* b*  development (%) a  I n f e c t i o n hypha a b  Vesicle b  90  100  86  100  76  100  X  92  103  84  98  44  59  XX  94  105  73  85  2  3  XXX  80  90  29  34  0  0  —XXXX  72  80  0  0  0  0  xxxxxx  10  11  0  0  0  0  —x  91  101  69  81  43  57  88  98  64  74  28  37  93  104  64  75  23  31  79  88  48  56  10  11  76  85  49  57  7  10  (control)  —-xXX—  xx-  Puromycin was present (x) and absent from (-) the germination medium (Ca-K b u f f e r ) . ( ) corresponds to time i n t e r v a l s ; 0-2 h, 2-3.5 h, 3.5-6 h, 6-8 h, 8-10 h, and 10-15 h. *development as a mean percent of the preceding structure. ^development as a mean percent of the preceding structure, expressed as a percent of the control, underlined values are s i g n i f i c a n t l y d i f f e r e n t from the control (95% confidence - Fisher LSD)  67  i n h i b i t o r was present at 3.5 t o 15 h (Tables VIII and IX). The nuclear behaviour of sporelings d i f f e r e n t i a t i n g i n the presence of puromycin was interesting.  Following heat  treatment the 2 nuclei migrated from the spore, into the germtube and towards the appressorial i n i t i a l .  In most cases  the n u c l e i remained i n the germtube, had a granular appearance, and were expanded i n form (Fig. 12b). Occasionally the nuclei migrated into the developing appressorium and completed one round of m i t o t i c d i v i s i o n .  The  presence of an i n h i b i t o r of protein synthesis appeared to arrest germtube development at a s p e c i f i c phase of the c e l l cycle.  The expanded form of the n u c l e i suggests that the c e l l  was e i t h e r i n the l a s t part of Gl or entering the S-phase. The four daughter nuclei d i d not move together but remained separate and expanded throughout the observation period (at l e a s t 20 h) (Fig. 12c).  Nuclear elongation p r i o r to m i t o t i c  d i v i s i o n was not observed.  4.5  4.5.1  Cytology of Uredosporeling Development  Nuclear Staining The most suitable technique tested f o r f i x a t i o n was a  graded ethanol series followed by staining with 1 ug/ml DAPI. DAPI reacted r a p i d l y with the sample, the n u c l e i were c l e a r l y v i s i b l e and fluoresced a bright blue.  Most of the germtubes  remained transparent with l i t t l e t o no fluorescence.  68  Germtiibes 1.5 h o l d or younger fluoresced strongly making i t d i f f i c u l t to discern the n u c l e i .  Nuclear fluorescence  improved f o r 20 min and then remained stable f o r up, to 10 days when stored i n the dark at 4°C.  In cases where the i n f e c t i o n  hypha was developing and the n u c l e i had migrated into the hypha a change was observed i n the v e s i c l e .  The v e s i c l e  cytoplasm became speckled, the "granules" fluoresced a bright gold. The addition of a surfactant, such as T r i t o n X-100  or  Tween-20 allowed the spores to be taken into suspension and s i m p l i f i e d washing procedures by centrifuging the sample between changes. However, i n the presence of surfactants the germtube fluoresced strongly rendering the n u c l e i i n v i s i b l e .  4.5.2  Nuclear Behaviour during Germination and  Differentiation For c y t o l o g i c a l studies the uredospores of P. graminis t r i t i c i were germinated on a Ca-K buffer.  The sporelings were  induced to d i f f e r e n t i a t e as described e a r l i e r (page 27). Uredospore n u c l e i were stained with DAPI at various stages of germtube development. The r e s t i n g uredospore was binucleate. Following germination the two nuclei usually migrated i n tandem from the spore into the developing germtube and toward i t s apex (Fig. 13).  These n u c l e i were round to oval i n shape  and represented the expanded or interphase n u c l e i described by Savile (1939).  In the absence of a stimulus to d i f f e r e n t i a t e  69  F i g . 13. F o l l o w i n g 2 h g e r m i n a t i o n a t 19°C t h e two i n t e r p h a s e n u c l e i m i g r a t e d from t h e uredospore i n t o t h e e l o n g a t i n g germtube (Double i l l u m i n a t i o n w i t h U.V. and h i g h l y a t t e n u a t e d v i s i b l e l i g h t , 1040x). F i g . 14. The germtube o f non-shocked u r e d o s p o r e l i n g s remained b i n u c l e a t e f o r up t o 20 h. The n u c l e i u s u a l l y appeared t o remain i n p e r p e t u a l i n t e r p h a s e ( I 0 4 0 x ) . F i g . 15. I n some c a s e s t h e n u c l e i w i t h i n 2 0 - h o u r - o l d u r e d o s p o r e l i n g s were e l o n g a t e d i n form and had a "ragged" appearance ( f l u o r e s c e n t m i c r o g r a p h , 1040x).  Fig. 16. The l i n e a r growth of the germtube was arrested during the 1.5 h heat shock period. Four hours following germination the two nuclei had migrated into the newly formed appressorium i n i t i a l . Their elongated form suggested that these nuclei were i n conjugate telophase and preparing to divide (double illumination with U . V . and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 1040x). Fig. 17. The f i r s t round of mitosis usually occurred within the mature appressorium 5 and 8 h a f t e r germination (1040x). Fig. 18. The mature appressorium contained four daughtern u c l e i . These n u c l e i moved together to form a compact tetrad (1040x.  the germtube remained binucleate and appeared to be i n perpetual interphase; m i t o t i c d i v i s i o n was not observed i n 0to 20-hour-old sporelings (Fig. 14).  In some cases the nuclei  of nondifferentiated 20-hour-old sporelings had a "ragged" appearance and became elongated i n form (Fig. 15). The nuclear behaviour of the d i f f e r e n t i a t i n g sporeling was complex; nuclear d i v i s i o n was predictable and changes i n form and migration patterns were observed regularly.  Although  nuclear d i v i s i o n was r e a d i l y observed the more subtle changes i n nuclear form, which correspond to s p e c i f i c phases of the c e l l cycle could not be followed with certainty. shock the germtube produced a bulbous appressorial into which the two nuclei migrated.  During heat initial  At 4 h the n u c l e i were  elongated to dumbbell i n shape and were situated one behind the other (Fig. 16).  Their appearance suggested that these  n u c l e i were i n conjugate telophase and nearing the completion of mitosis.  Usually, a f t e r 4.5 h a cross wall formed and  delimited the appressorium from the germtube.  The f i r s t round  of mitosis was usually complete within 5 t o 8 h following imbibition (Fig. 17).  The mature appressorium contained four  n u c l e i arranged as a compact tetrad (Fig. 18). At  7 h the four nuclei migrated toward the developing  i n f e c t i o n peg.  Occasionally two n u c l e i divided i n the  appressorium (Fig. 19) but more commonly the second d i v i s i o n occurred as the nuclei moved into the developing substomatal v e s i c l e (7 h to 13 h).  The nuclei migrated i n c l o s e l y  72  Fig. 19. Occasionally two nuclei divided within the appressorium. The s i x nuclei migrated i n tandem towards the developing i n f e c t i o n peg (double i l l u m i n a t i o n with U . V . and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 104Ox). Fig. 20. Ten hours following germination the six-nucleate v e s i c l e was delimited from the appressorium by a septum (1040x). Fig. 21. In most cases a nuclear p a i r divided i n the mature v e s i c l e to y i e l d a t o t a l of eight n u c l e i . These n u c l e i were d i s t r i b u t e d such that three to four nuclei aggregated on either side of the v e s i c l e (1040x).  73  Fig. 22. The orientation of nuclear migrational patterns was found to be related to the p o l a r i t y of the v e s i c l e . Once growth of the i n f e c t i o n hypha was i n i t i a t e d the nuclei moved into an arrowhead formation with the apex directed towards the developing hypha (double illumination with U.V. and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 1040X).  Fig. 23. The i n f e c t i o n hypha contained eight, six, or more commonly four nuclei (1040x). Fig. 24. In some cases a nuclear p a i r f a i l e d to migrate with the other nuclei and remained i n the v e s i c l e (1040x). Fig. 25. Twenty three hours following germination the i n f e c t i o n hypha most often contained four expanded nuclei (1040x).  74 associated p a i r s . A cross wall delimited the six-nucleate v e s i c l e from the appressorium within 10 h (Fig. 20).  In most cases a nuclear  p a i r divided i n the mature v e s i c l e to y i e l d a t o t a l of eight n u c l e i (Fig. 21).  These n u c l e i , which were rounder and  considerably smaller than those observed i n the germtube, are described as d i v i d i n g or unexpanded.  Nuclear d i v i s i o n was  not  always synchronous, f i v e to eight n u c l e i were often seen within the v e s i c l e .  Within 10 to 13 h a f t e r germination s i x  to eight n u c l e i were d i s t r i b u t e d such that three to four n u c l e i aggregated on e i t h e r side of the v e s i c l e (Fig. 21). The i n f e c t i o n hypha of P. graminis t r i t i c i developed as a polar extension from the v e s i c l e . i n f e c t i o n hypha was  Once growth of the  i n i t i a t e d (ca 13 h) a nuclear  pattern became apparent.  migration  The n u c l e i moved towards the  developing hypha i n the formation of an arrowhead (Fig. 22). The o r i e n t a t i o n of t h e i r migration was p o l a r i t y of the v e s i c l e .  always r e l a t i v e to the  The leading nucleus, which often  fluoresced more strongly than the others, was directed towards the developing hypha.  As growth ensued eight, s i x or four  expanded n u c l e i migrated into the i n f e c t i o n hypha (Fig. 23), occasionally a nuclear p a i r remained i n the v e s i c l e (Fig. 24). A f t e r 23 h four or fewer n u c l e i were observed i n the hypha (Fig. 25).  75  4.6  Amino Acids Analysis  The standard generated 18 peaks with c h a r a c t e r i s t i c retention times.  The separation of a PITC-amino acid  standard i s i l l u s t r a t e d i n F i g . 26, Table X. was observed within 12 min.  Good resolution  An additional 18 min was required  to remove solvent peaks and r e e q u i l i b r a t e the column between runs.  Except f o r h i s t i d i n e from arginine, and glutathione  from ammonia, most amino compounds were adequately separated using the RP-column . As i s c h a r a c t e r i s t i c of RPchromatography, the e l u t i o n order was related t o the increasing hydrophobic nature of the solute ( i . e . the more water soluble the compound, the faster i t was eluted).  4.6.1  Experimental Plan The objective was t o determine the e f f e c t of the heat  shock on (A) the endogenous free amino acid pool and (B) the exogenous free amino acids leaching from the germinating uredospore.  The following fractions were therefore analyzed:  (1)  Buffer wash from r e s t i n g uredospores.  (2)  Alcohol extract from unwashed r e s t i n g uredospores.  (3)  Alcohol extract from eight-hour-old uredosporelings: non-shocked and heat shocked.  (4)  Leachates from eight-hour-old uredosporelings: nonshocked and heat shocked.  Retention Time (min) Fig. 26. Separation of amino acid standards (Pierce H). Eluent A: 140 mM sodium acetate, 5% TEA, pH 6.4; eluent B: 60% acetonitrile in water; gradient: 5% B to 45% B in 10 min on curve 5; flow-rate: 1 mVrnin for 12 min, 1.5 ml/min for 8 min: column: Pico-Tag RP-column for protein hydrolyzates; detector: ultraviolet (254 nm) at 0.1 a.u.f.s. For peak identification see Table X.  Table X. PITC-arnino acid peaks identified by number and retention time as separated by RP-HPLC under the conditions shown in figure 26. Number  Amino Acid  Retention Time (min)  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  Aspartic acid Glutamic acid Serine Glycine Histidine / Axginine Threonine Alanine Proline NH Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Lysine  2.44 2.68 4.48 4.69 5.22 5.37 5.51 5.66 6.00 7.06 7.61 8.00 9.12 9.30 9.50 10.27 11.62  18 19 20  Glutamine Homoserine Glutathione  4.53 4.96 6.00  *  3  Unknown peaks  —  78 (5)  Alcohol extract from 20-hour-old uredosporelings: non-shocked and heat shocked.  (6)  Leachates from 20-hour-old uredosporelings: nonshocked and heat shocked.  4.6.2  Complications A number of problems were encountered i n i d e n t i f y i n g and  quantifying the amino compounds i n uredospores and uredospore leachates.  Representative RP-HPLC amino acid p r o f i l e s from  (A) the alcohol extract from non-shocked uredospores, and (B) the leachate from heat shocked uredospores, are shown i n F i g . 27. Two peaks corresponding to glutamine (#18 - see Table X) and NH /glutathione (#9/20) are predominant 3  i n both p r o f i l e s .  The r e s o l u t i o n between peaks i d e n t i f i e d as serine, glycine, and glutamine was poor.  Therefore, since column s e l e c t i v i t y  could not be improved, s t r i c t l y quantitative estimates of the amounts of these amino compounds per mg spores could not be obtained.  Despite the overlap, peaks corresponding to serine,  glycine, and glutamine were i d e n t i f i e d i n most, but not a l l extracts.  The most serious overlap occurred between glycine  and glutamine and i n F i g 27 glycine was completely obscured by glutamine (#18).  In those runs where the separation between  glycine and glutamine was achieved the glycine present was always only about 1/3 the height of the glutamine peak.  The  response factors (RFs) f o r glycine and glutamine were s i m i l a r i n value.  In order to calculate the order of magnitude of the  79  F i g . 27. Representative R P - H P L C separations of amino acids isolated f r o m ( A ) non-heat shocked 20-hour-old uredosporelings and (B) the leachate of 20-hour-old heat shocked uredosporelings. F o r peak identification see Table X .  80 concentration of glutamine estimates based on the area of the glutamine peak have been reduced by 30%.  Such estimates leave  no doubt that glutamine i s predominant among the amino compounds i n uredospores (Fig. 27A). Similar considerations apply to the analysis of the leachate (Fig. 27B), i n which glutamine was also  predominant.  A further complication i n the analysis of the leachate arose because of the presence of a very large u n i d e n t i f i e d peak (X) i n the buffer (Fig. 27B) which usually overlapped aspartate (#1) and glutamate (#2).  This prevented any meaningful  estimates of the l e v e l s of aspartate and glutamate i n the leachate. In a l l chromatograms ammonia and glutathione eluted o f f the column simultaneously.  I t was therefore impossible to  obtain independent estimates of the l e v e l s of NH glutathione.  3  and  The NH /glutathione peak (#9/20) was usually 3  larger than a l l other amino compounds except glutamine (Fig. 27).  Ten amino acids (alanine, proline, tyrosine, valine,  methionine, cysteine, isoleucine, leucine, phenylalanine, and lysine) were i d e n t i f i e d and quantified with confidence. Where possible (see above) the amount of each amino acid was expressed as nM per mg spore dry weight.  There was some  unavoidable loss of heat shocked uredosporelings during t r a n s f e r from the germination medium t o the extraction vessel because the shorter, d i f f e r e n t i a t e d germtubes d i d not i n t e r l o c k to form a mat.  The loss was v i s u a l l y estimated to  81 be not greater than 15%.  Calculations of the amino acid  l e v e l s per mg dry weight of uredospores were based on the dry weight of the o r i g i n a l sample (2.78 mg) and are therefore too low by a maximum of 15%. Two unknown peaks eluted from the column shortly a f t e r the amino acid phenylalanine (#16). by Dwyer et a l .  Based on r e s u l t s reported  (1987) these peaks may be i d e n t i f i e d  t e n t a t i v e l y as tryptophan (Trp) and ornithine (Orn).  These  compounds appeared soon a f t e r germination occurred; they were detected i n the germinating uredospores and i n the leachate from 20-h-old non-shocked uredosporelings.  4.6.3  Exogenous Free Amino Acids: Resting Spore Wash The buffer i n which resting uredospores were washed  contained the e a r l y - e l u t i n g amino acids. associated with the spore wall.  These appeared to be  Although there was some  overlap of aspartic and glutamic acids with the u n i d e n t i f i e d peak (X) found i n the buffer, i t was possible to estimate the amounts of these amino acids by approximating the area of the peak.  An estimate of glutamine was obtained as described  e a r l i e r (page 77).  The r e s u l t s are shown i n Table XI.  The  amino acids washed o f f the spores were, i n order of predominance, glutamine, alanine, glutamate, aspartate, and tyrosine.  An unknown compound with a retention time of  minutes was also present i n the spore wash.  6.66  Since i t was  unknown i t could not be quantified by i t s comparison with a  82  Table XI. Free amino acid composition of the buffer wash from ungerminated uredospores of P. graminis t r i t i c i .  Amino A c i d  nM/mg spore dry w e i g h t  3  Percent  Ala Pro Tyr Val Met Cys He Leu Phe Lys  0.57 ±.07  26.0  0.25 ±.06  11.4  Glm HomoSer Arg/His/Thr Unknown*  0.72 ±.04 ND  + + +  ND  + + +  ND  a  •++ +  Asp Glu  0.27 ±.03 0.38 ±.06  Total  2.19 ±.26  -  —  32.9  —  12 . 3 17.4 100. 0  Each value represents the mean and range f o r two separate experiments. + = trace amounts, ++ = greater than trace amounts (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids).  a  a  83 standard.  Therefore i t was assigned an RF of 4256 which i s an  average value calculated from the RF of 14 d i f f e r e n t amino acids.  In addition, small peaks corresponding t o the  retention times of one or more of arginine, h i s t i d i n e , and threonine were detected (Table XI). which of these were actually present.  I t was impossible t o t e l l Cysteine, homoserine,  and l y s i n e were not detected.  4.6.4  Endogenous Free Amino Acids: Extracts of Unwashed  Resting Uredospores In order of prevalence the amino compounds i n unwashed r e s t i n g uredospores were alanine, the unknown, glutamic acid, and glutamine  followed by smaller amounts of several other  amino acids (Table XII).  Alanine accounted f o r 30% and the  unknown accounted f o r 22% of the t o t a l free amino acids present. (2.4/0.2).  The r a t i o of glutamic to a s p a r t i c acid was 12 Cysteine and l y s i n e were not detected.  Comparison of the appropriate columns i n Tables XI and XII shows that the t o t a l amino acids washed o f f r e s t i n g uredospores (Table XI) account f o r only 16.5% (2.19 x 100 /13.3) of the t o t a l amino acids extracted from unwashed r e s t i n g spores.  84  Table XII. The endogenous free amino acid l e v e l i n unwashed r e s t i n g uredospores (S), and i n non-shocked uredosporelings of P. graminis t r i t i c i a f t e r 8 h (N8S), and 20 h (N20S)  nM/mg spore dry  Amino Acid  S  Ala Pro Tyr Val Met Cys He Leu Phe Lys  4 . 04 0.20 + 0.43 + ND 0.25 0.23 0.29 ND  Glm HomoSer Arg/His Unknown* Orn Trp a  Asp Glu Total Glu:Asp  N8S 47 0  ± 0  ±. 01 ±. 02 ±. 02  2 .23 ± 30 ++ ++ 2 .98 ±. 75 ND ND 0. 2 ± 0 2 .4 + .1 13 .3 10:1  ±1.7  4. 99 2 .20 2 .47 1 .80 2 .54 7 . 09 2 . 16 1 .93 2 .93 1 . 17  weight  Ratio  a  N20S  ±. 71 ±. 29 ±. 04 ±. 43 +. 07 ±. 39 ±. 15 ±. 33 ±. 31 ±. 60  2 . 50 1 . 10 2 . 29 1 .23 2 . 09 3 . 19 1 . 07 1 . 50 3 . 10 1 .05  N8S/S  ±. 30 ±. 13 ±. 64 ±. 17 ±. 42 ±. 77 ±. 17 ±. 54 ±. 58 ±. 02  6.99 +. 84 10 .74 ±1 . 2 ++ ++ ++ ++ 2 .47 ±. 10 2 .95 ±. 79 ++ ++ ++ ++ 2 . 2 ±. 7 10 . 0 ±2 .9 50 .9 5:1  1 . 5 ±. 2 4. 1 ±. 8  ±7 . 6 38 . 4 ±6 . 7 3: 1  N20S/S  1. 2 11. 0 27 .4 2 .9 50. 8 (>1 000) 8. 6 8 .4 10. 1 (>2 00)  0. 6 5. 5 25. 4 2 .7 41. 8 (>6 00) 4 .3 6 .5 10. 7 (>2 00)  3 .1  4 .8  1. 0  1. 2  22 .0 4 .2  15. 0 1. 7  3 .8  2 .9  -  -  Each value represents the mean and range f o r two separate experiments. + = trace amount, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids. a  a  85 4.6.5  E f f e c t of Heat Shock on the Free Amino Acids i n  Germinated Uredospores and i n the Leachate A f t e r Germination Unwashed uredospores were placed on Ca-K buffer a t 0 h at 19° C, heat shocked at 29° C from 2 h t o 3.5 h and harvested at e i t h e r 8 h or 20 h.  Non-shocked controls were subjected to  the same protocol but were maintained at 19° C throughout the germination period. analyzed separately.  Uredosporelings and leachates were The r e s u l t s are presented i n Tables XII,  XIII, and XIV. The data i n Table XII show that the endogenous free amino acid l e v e l i n non-shocked uredospores at 8 h was 3.8-fold, and at 20 h was 2.9-fold that i n unwashed r e s t i n g uredospores. Among the i n d i v i d u a l amino acids, alanine and the unknown compound showed r e l a t i v e l y l i t t l e change on germination but a l l the other amino compounds increased markedly.  Large  r e l a t i v e increases occurred i n cysteine and lysine, which were below the l i m i t of detection i n r e s t i n g uredospores.  The  r a t i o of glutamate t o aspartate decreased sharply on germination. Table XIII shows that, while the endogenous free amino acid l e v e l i n heat shocked uredospores increased approximately 2-fold a t 8 h, there was no further increase at 20 h. the i n d i v i d u a l amino compounds there were decreases 50%) i n alanine and the unknown compound.  Among  (about  Valine was  r e l a t i v e l y unchanged. A l l other amino compounds increased s i g n i f i c a n t l y , p a r t i c u l a r l y cysteine and l y s i n e .  In general  86  Table XIII. Endogenous free amino acid content of unwashed r e s t i n g uredospores (S), and heat shocked uredosporelings of P. graminis t r i t i c i a f t e r 8 h (H8S), and 20 h (H20S).  Amino Acid  nM/mg spore dry w e i g h t S  H8S  4.04 ±.47 0.20 ±.0  Ala Pro Tyr Val Met Cys He Leu Phe Lys  2.23 ±.30  8.69 ±.89  8.38 ±.35  2.98 ±.75 ND ND  1.43 ±.18  1.54 ±.48  0.2 2.4  1.7 ±.4 4.5 ±1.0  +  Asp Glu Total  ++ ++  ±.0 ±.1  ±.03 ±.01 ±.03 ±.13 ±.11 ±.12 ±.05 ±.19 ±.16 ±.11  +'+ ++ ++ +  1.40 0.46 1.20 0.66 0.84 1.45 0.69 1.49 1.44 0.44  H8S/S  ND 0.25 ±.01 0.23 ±.02 0.29 ±.02 ND  +  3  H20S  1.80 0.66 0.93 0.53 0.56 2.89 0.80 1.93 1.41 0.25  0.43 ±.0  Glm HomoSer Arg/His Unknown* Orn Trp  Ratio  3  ±.01 ±.0 ±.16 ±.03 ±.02 ±.03 ±.01 ±.02 ±.11 ±.11  ++ ++  ++ ++  1.0 3.5  ±.1 ±.0  13.3 ±1.7 28.1 ±.16 24.5 ±.76  Adjusted Total  11. l  Glu:Asp  10:1  b  32.3 3 :1  C  28.3  C  4:1  0.5 3. 3 10. 3 1.2 11.2 (>600) 3.2 8.4 4.9 (>60)  H20S/S 0.4 2. 3 13 . 3 1.5 16.8 (>300) 2. 8 6.5 5.0 (>80)  3. 9  3.8  0.5  0.5  17.0 1.9  10. 0 1.5  2.1  1.8  -  -  -  -  E a c h value represents the mean and range f o r two separate experiments. + = trace, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids, ^measured endogenous amino acids minus the amino acids i n the resting spore wash. measured amounts plus 15% t o compensate f o r uredospore losses during transfer. a  3  c  87  Table XIV. The free amino acid composition of leachate emanating from heat shocked (H) and non-shocked (N) uredosporelings of P. graminis t r i t i c i a f t e r an 8 h (H8L and N8L), and 20 h (H20L and N20L) incubation period.  nM/mg spore dry wei g h t  Amino Acid  N8L  Ala Pro Tyr • Val Met Cys He Leu Phe Lys  3  Total: Leachate (L) Wash (W) L -W  2  N2 0L  H2 0L  ±. 21 ±. 17 ±. 14 ±. 01  0.70 ±. 01 2 . 99 ±. 59 + 0.85 ±. 71 + ND + + ND +  4. 52 2 .81 1 .21 1 .91 1 .36 5 . 42 1 . 61 1 . 17 2 . 06 0. 66  2 .40 ± . 20 ++ ++ ND ND ND  2 . 39 ±. 33 ++ ++ ND ND ND  9.93 ±. 00 ++ ++ 3 .31 ±. 35 ++ ++  5. 67 +,30  6.93 ±. 98  35 .97 ±. 02  13 . 79 ±. 95  2 .19 ±. 26  2 .19 ±. 26  2 . 19 ±. 26  2 .19 ±. 26  0.60 1.78 0.29 0. 60 + ND + + + +  Glm HomoSer Arg/His Unknown* Trp Orn  H8L  a  3 .48  4 .74  ±. 07 ± .43 ±. 13 ±. 28 ±. 03 ±. 13 +.03 ±. 03 ±. 05 +.01  33 .78  0.91 ±. 11 2 . 64 ±. 24 0.33 +.08 + + + + + ND ND 9.91 ±1 . 17 ++ ++ ND ND ND  11. 60  E a c h value represents the mean and range f o r two separate experiments. + = trace, ++ greater than a trace, (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids. from Table XI. a  a  a  the  increases i n i n d i v i d u a l amino compounds were smaller than  i n non-shocked  spores.  Moreover the amino a c i d l e v e l i n heat  shocked spores was only 55% (28.1 x 100 /50.9) o f that i n nonshocked spores a t 8 h and 64% (24.5 x 100 /38.4) a t 20 h. I t i s thus c l e a r that heat shock decreases protein hydrolysis i n germinating uredospores. Paraphrasing the well known song, we may ask "Where have a l l the amino acids gone?".  The data i n Table XIV, which  presents the analyses of the leachates, provides the answer. By 8 h non-shocked  and shocked uredospores had l o s t  approximately the same t o t a l amount of amino compounds t o the germination medium, ca 5.7 and 7.0 nM/mg dry weight respectively.  In contrast, by 20 h the non-shocked  uredospores had l o s t nearly 36.0 and the shocked uredospores only ca 13.8 nM/mg spore dry weight respectively.  Both non-  shocked and heat shocked uredosporelings l o s t about equal amounts of glutamine.  At 20 h losses i n alanine, cysteine,  phenylalanine, and the unknown were much greater from the nonshocked sporelings. I t i s important t o note that the leachate from unwashed germinating uredospores presumably includes the amino compounds which could be washed off. the r e s t i n g uredospores (Table XI).  Differences i n the t o t a l amounts of amino  compounds found i n the leachates and i n the spore wash are therefore also shown i n Table XIV.  89 5.  5.1  DISCUSSION  Temperature Retirement f o r the D i f f e r e n t i a t i o n of P.  graminis t r i t i c i Uredosporelings  Maheshwari et a l . (1967a) demonstrated that greater than 90% of P. graminis t r i t i c i uredosporelings undergo complete d i f f e r e n t i a t i o n on Ca-K buffer i n response to a 1.5 h heat shock of 30° C administered 2 h a f t e r seeding.  Wisdom (1977)  obtained only 5% d i f f e r e n t i a t i o n using the methods described above.  The e f f e c t of the heat shock temperature was not  investigated. The r e s u l t s presented i n t h i s t h e s i s show that :  (1)  D i f f e r e n t i a t i o n has a sharp temperature optimum. Aberrations one degree above or below the optimum heat shock temperature s i g n i f i c a n t l y reduced (by greater than 40%) the amount of t o t a l sporelings forming a complete set of i n f e c t i o n structures.  (2)  The optimum heat shock temperature v a r i e s  s l i g h t l y f o r d i f f e r e n t spore l o t s .  When the temperature of  the heat shock was optimised up to 70% d i f f e r e n t i a t i o n was obtained.  These r e s u l t s indicate that Wisdom's f a i l u r e to  obtain better than 5% d i f f e r e n t i a t i o n on Ca-K buffer was most l i k e l y due t o the use of a sub-optimal heat shock temperature.  90 5.2  The Influence of Nutrients on Uredospore D i f f e r e n t i a t i o n  Since Wisdom (1977) obtained only 5% d i f f e r e n t i a t i o n on Ca-K b u f f e r she abandoned the buffer i n favour of her nutrient-enriched MPG medium.  Using MPG i n conjunction with a  1.5 h heat shock of 30°C, Wisdom obtained up t o 80% d i f f e r e n t i a t ion. Williams (1971) had e a r l i e r shown that nutrients stimulate d i f f e r e n t i a t i o n and that the nutrient stimulus i s more e f f e c t i v e when a heat shock i s applied.  The r e s u l t s  obtained i n the present study support t h i s claim.  Moreover,  t h i s response of the uredosporelings t o nutrients i n the germination medium was found t o be non-additive.  I t was also  found that n-nonyl alcohol not only stimulates germination but also increases the proportion of germtubes forming  infection  structures. Results from preliminary experiments indicated that MPG was superior t o Ca-K buffer as a d i f f e r e n t i a t i o n medium f o r P. orraminis t r i t i c i uredosporelings.  The precise temperature  optimum f o r the heat shock observed with sporelings germinated on Ca-K buffer (page 87) was not observed with sporelings germinated  on MPG.  On MPG, a single given temperature (30° C)  consistently induced the d i f f e r e n t i a t i o n of a high  percentage  of uredosporelings. Ca-K buffer appeared t o be the most e f f e c t i v e component of MPG.  Baker et a l . (1987) reported that C a , but not K+, + 2  91 stimulated the germination of Uromyces phaseoli uredospores. I t i s also possible that calcium plays a key r o l e i n uredosporeling d i f f e r e n t i a t i o n .  One of the many functions of  calcium i n b i o l o g i c a l systems i s the regulation of c e l l membrane permeability to water and ions; low calcium generally increases permeability whereas high calcium decreases i t .  The  potassium phosphates supply the hyphal c e l l with phosphorus. Phosphorus i s required f o r the formation of nucleic acids and phospholipids, as well as a key molecule of energy metabolism, ATP. Peptone i n d i s t i l l e d water was a poor medium f o r differentiation.  In combination with glucose and Ca-K b u f f e r  peptone s i g n i f i c a n t l y increased the number of sporelings that formed i n f e c t i o n structures (up to 77%). contains  Bacto-peptone  a v a r i e t y of simple nitrogenous compounds (including  reduced organic sulphur), ions, minerals, and a n e g l i g i b l e amount of protease and complex nitrogenous compounds (Difco Laboratories 1953). Glucose i s a potential source of energy f o r hyphal growth.  In the presence of glucose (dissolved i n glass-  d i s t i l l e d water) the germtube d i d not appear t o respond to heat shock but continued l i n e a r growth without the formation of i n f e c t i o n structures.  Although glucose alone was  i n e f f e c t i v e as a d i f f e r e n t i a t i o n medium i t played an important r o l e as a constituent of MPG.  According t o Manners and co-  workers (1982) glucose i s r e a d i l y absorbed and metabolized by  92 the rust hyphae.  The carbon appeared i n endogenous pools of  free glucose, amino acids and phosphate esters of trehalose and glucose (Reisener et a l . 1961). Previous studies on the enzymatic c o n s t i t u t i o n of uredospores of the wheat stem rust have indicated the presence of complete enzyme systems f o r the metabolism of amino acids, l i p i d s , and carbohydrates (see Shaw 1964).  The presence of  nutrients i n the medium may provide the uredosporeling with metabolites that a s s i s t with the processes involved i n germtube morphogenesis.  5.3  The Timing of Essential Protein Synthesis  The timing of RNA and protein synthesis during d i f f e r e n t i a t i o n has been described by Dunkle et a l . Wisdom (1977).  (1969) and  I t has been shown that new polypeptides appear  during the d i f f e r e n t i a t i o n of non-shocked bean rust uredosporelings (Huang and Staples 1982), and the d i f f e r e n t i a t i o n of heat shocked wheat stem rust uredosporelings (Kim et a l . 1982a).  Although not  d i f f e r e n t i a t e d , heat shocked f l a x rust uredosporelings synthesized a number of heat shock proteins (Shaw et a l . 1985).  These r e s u l t s have been discussed i n greater d e t a i l i n  the l i t e r a t u r e review (pages 15). The e f f e c t of puromycin on d i f f e r e n t i a t i o n was reinvestigated under the optimum heat shock conditions  93 described e a r l i e r (page 87).  Puromycin i s recognized as an  e f f e c t i v e i n h i b i t o r of protein synthesis.  I t i s a structural  analogue of the aminoacyl adenosine, the amino-acid-bearing end of transfer-RNA.  I t binds r e a d i l y t o the aminoacyl s i t e  of the large ribosomal subunit and forms a complex with the elongating peptide chain.  The peptidyl-puromycin complex  prevents further amino acid incorporation.  The complex  dissociates from the ribosome and chain elongation i s terminated (Gale et a l . 1972). During the present study puromycin was added to, or removed from, the germination medium (Ca-K buffer) at various stages during sporeling development.  The e f f e c t of puromycin  on germtube morphogenesis was dependent on the timing and duration of the puromycin treatment.  The addition of  puromycin to Ca-K buffer at 3.5 h followed by i t s removal at 8 h caused a s i g n i f i c a n t reduction i n the proportion of i n f e c t i o n pegs forming v e s i c l e s .  Although the v e s i c l e s were  v i s i b l e a f t e r 10 h t h e i r formation appeared to be preceded by the synthesis of e s s e n t i a l proteins.  The r e s u l t s suggest that  these proteins are synthesized 2 h t o 6.5 h p r i o r to v e s i c l e formation. When puromycin was present i n the germination medium f o r the e n t i r e incubation period (0 h to 16 h) germtube d i f f e r e n t i a t i o n was v i s i b l y affected:  (1)  The  complete  d i f f e r e n t i a t i o n of uredosporelings was prevented. proportion of germtubes forming appressoria was  (2)  The  significantly  94 reduced. form.  (4)  (3) The appressoria were predominantly i r r e g u l a r i n The formation of a septa between the appressoria  and the germtube usually f a i l e d to occur.  (5)  Nuclear  d i v i s i o n was r a r e l y observed, but i n the rare event of d i v i s i o n the p a i r i n g of daughter-nuclei d i d not occur.  The  capacity of the i n h i b i t e d germlings to form i n f e c t i o n structures appeared to recover p a r t i a l l y when puromycin was removed from the germination medium. These r e s u l t s suggest the existence of d i f f e r e n t i a t i o n s p e c i f i c proteins and support the claim that these proteins are stage-specific (Huang and Staples 1982). I t may be concluded that essential proteins are synthesized throughout the entire process of d i f f e r e n t i a t i o n , including the formation of the appressoria. The presence of puromycin i n the germination medium throughout the incubation period usually prevented the d i v i s i o n of germtube n u c l e i .  Nuclear d i v i s i o n i s generally  preceded by the r e p l i c a t i o n of nuclear DNA.  P r i o r t o the  onset of the r e p l i c a t i o n of DNA certain enzymes such as, thymidine kinase, thymidylate synthetase and others, must increase within i n the c e l l .  The absence of nuclear d i v i s i o n  observed i n t h i s study suggests that these enzymes are synthesized p r i o r to the formation of the appressorium.  In  rare cases the enzymes appear to be present i n s u f f i c i e n t amounts t o enable r e p l i c a t i o n and d i v i s i o n t o occur.  95 5.4  Nuclear Behaviour Accompanying Germination and  Differentiation  The two n u c l e i of nondifferentiated uredosporelings d i d not divide but appeared to remain i n perpetual interphase throughout the e n t i r e observation period of 20 h.  In  d i f f e r e n t i a t i n g uredosporelings nuclear d i v i s i o n was a regular event.  I t s occurrence was c l o s e l y associated with the  timing of i n f e c t i o n structure development.  The c y t o l o g i c a l  events taking place during the f i r s t 24 h of germination and d i f f e r e n t i a t i o n are summarized i n F i g . 28. A c h a r a c t e r i s t i c pattern of nuclear behaviour was observed i n d i f f e r e n t i a t i n g uredosporelings. The protoplasm migrated into the developing i n f e c t i o n structures leaving an empty space i n the proximal regions of the germtube.  Paired  daughter n u c l e i migrated i n tandem with the cytoplasm.  The 4  to 6 n u c l e i observed i n the appressorium migrated into the v e s i c l e as a c l o s e l y associated group.  This was not observed  by Wisdom (1977) who reported that the movement of the 4 n u c l e i from the appressorium into the developing v e s i c l e was not synchronous.  She noted that one p a i r moved into the  v e s i c l e while the other p a i r remained i n the appressorium f o r up to 10 h.  Moreover, Wisdom (1977) d i d not report the  p o t e n t i a l f o r a second nuclear d i v i s i o n i n the appressorium. In the present study 6 n u c l e i were commonly observed moving into the developing i n f e c t i o n peg.  Migration of n u c l e i into  96  Two expanded nuclei migrate down the germtube toward i t s apex. The f i r s t round of mitosis i s generally completed within the mature appressorium. 7 h  0=?  The cytoplasm moves into the i n f e c t i o n peg with the nuclei. A second d i v i s i o n occurs either p r i o r to or during nuclear migration. The mature substomatal v e s i c l e contains six unexpanded nuclei. A t h i r d m i t o t i c d i v i s i o n may occur to y i e l d eight n u c l e i .  13 h  Eight, s i x , or four nuclei enter the i n f e c t i o n hypha, occasionally a nuclear p a i r remains i n the v e s i c l e . Four or fewer nuclei migrate along the infection hypha.  F i g . 28. Diagrammatic representation of the c y t o l o g i c a l events taking place during the d i f f e r e n t i a t i o n of uredosporelings of P. graminis t r i t i c i , race C17. The times given are approximate.  97 the developing i n f e c t i o n structures was characterized by a s l i g h t change i n nuclear shape.  The n u c l e i appeared t o be  "tear drop" i n shape as i f they were "pulled" into the i n f e c t i o n structures.  When a break occurred i n the germtube  the n u c l e i were extruded along with the cytoplasm.  In some  cases, when n u c l e i were migrating into the i n f e c t i o n hypha, the foremost nucleus appeared brighter than the following ones.  Wisdom (1977) described an analogous s i t u a t i o n i n which  a single intensely Feulgen-positive nucleus preceded the other n u c l e i into the i n f e c t i o n hypha.  In the rare event of a  b i p o l a r v e s i c l e forming, two "bright" nuclei could be seen. One nucleus was situated on either side of the v e s i c l e and adjacent t o the i n i t i a l s of the i n f e c t i o n hyphae.  Finally 2  or more n u c l e i appeared to either break down or coalesce within the i n f e c t i o n hypha, so that a f t e r 24 h the hypha contained up to a maximum of four d i s t i n c t n u c l e i (Fig. 28).  5.4.1  Staining with DAPI DAPI (4', 6-diamidino-2-phenylindole) i s a DNA-specific  fluorochrome.  I t binds r e l a t i v e l y s p e c i f i c a l l y to AT residues  of double-stranded DNA and exhibits a much enhanced fluorescence i n the association (Otto and Tsou 1985).  This  fluorescence i s not observed with RNA or protein (Coleman et a l 1981).  Chemically, i t i s suggested that the AT-  s p e c i f i c i t y resides with both the guanidine group and the benzimidazole or indole ring, which may bind t o the purine of  98 adenosine through base stacking (Otto and Tsou 1985). DAPI binds to DNA and fluoresces i n proportion to the amount of DNA present.  The r e l a t i v e DNA content per nucleus  can be read i n s i t u by measuring the i n t e n s i t y of fluorescence with a microspectrofluorometer (Coleman et a l 1981).  The  enhanced i n t e n s i t y of fluorescence observed i n the leading nucleus (preceded the other nuclei into the i n f e c t i o n hypha) may  suggest that t h i s nucleus i s not haploid but contains  additional  DNA.  DAPI reacted rapidly with the sample and within minutes one could e a s i l y assess nuclear state and i d e n t i f y nuclear abnormalities.  The technique appeared to be useful f o r  determining the numbers and positions of n u c l e i and t h e i r d i v i s i o n s i t e s within the i n f e c t i o n structures.  The s t a i n i s  water-soluble and has been used successfully as a v i t a l dye i n studies on red algae and pollen development (Goff and Coleman 1984,  Coleman and Goff 1985).  I t should have further  a p p l i c a t i o n i n assessing the e f f e c t s of a number of parameters (e.g. n u t r i t i o n , fungicides, and host genotype manipulation) on the nuclear behaviour of the rust pathogen.  99 5.5  The E f f e c t of Heat Shock on the Amounts and Kinds of Free  Amino Acids i n Germinated Uredospores and Their Leachates  The r e s u l t s presented i n Tables XI t o XIV show c l e a r l y that heat shock decreases the s i z e of the endogenous pool of free amino acids and the extent to which uredosporelings lose amino acids t o the medium.  The r e s u l t s are summarized i n  Table XV which presents a "balance sheet" showing the s i z e of the endogenous and leachate amino acid pools. Under the conditions employed only endogenous reserves are a v a i l a b l e t o the germinating uredospores.  The amino a c i d  data (Table XV) therefore c l e a r l y show that there i s a net hydrolysis o f protein during germination, leading t o an increase i n the l e v e l of endogenous free amino acids.  These  r e s u l t s are consistent with data obtained from pulse chase experiments  (Kim et a l . 1982a) which demonstrated that the  majority of uredospore proteins are turned over during germination.  The l e v e l of t o t a l amino acids remained  r e l a t i v e l y unchanged i n the heat shocked sporelings from 8 h to 20 h (39.3 t o 42.0 nM/mg), whereas the l e v e l of t o t a l amino acids i n non-shocked sporelings increased from 55.8 nM/mg a t 8 h t o 74.4 nM/mg at 20 h.  The t o t a l free amino acid l e v e l  (endogenous and leachate) associated with non-shocked uredosporelings was thus 1.8-fold (74.4/42.0) higher than the amount of amino acids associated with heat uredosporelings a t 20 h.  shocked  I t i s thus c l e a r that heat shock  100  Table XV. The d i s t r i b u t i o n of the free amino compounds associated with r e s t i n g spores, non-shocked 8- (N8S) and 20h-old (N20S) uredosporelings, and heat shocked 8- (H8S) and 20-h-old (H20S) uredosporelings. The r e s u l t s are expressed as nM amino acid per mg spore dry weight.  S Endogenous Leachate^ Wash Total  11. l  b  2 .19 13.3  N8S  H8S  50.1  32 . 3  . 5.67  6 .95  55. 8  C  N20S  H20S  38 . 4  28 . 2  36.0  13.8  39.3  74 . 4  42.0  ^measured endogenous amino acids minus the amino acids i n the r e s t i n g spore wash. Measured amounts plus 15%, see Table XII. ^includes amino acids i n resting spore wash (2.19 nM/mg).  C  101 d e c r e a s e s t h e amount o f p r o t e i n h y d r o l y s i s i n  germinating  uredospores. The  r e s u l t s show f u r t h e r t h a t t h e r e i s no n e t p r o t e i n  s y n t h e s i s during the formation of i n f e c t i o n s t r u c t u r e s induced by h e a t shock.  Nevertheless,  protein synthesis i s increased  r e l a t i v e t o p r o t e i n h y d r o l y s i s by comparison w i t h t h e  relative  r a t e s o f t h e s e two p r o c e s s e s i n n o n d i f f e r e n t i a t i n g (nonshocked) u r e d o s p o r e l i n g s .  These r e s u l t s and t h e  conclusions  drawn from them a r e c o n s i s t e n t w i t h t h e e f f e c t o f puromycin on differentiation.  The  i n h i b i t i o n of p r o t e i n synthesis  by  p u r o m y c i n i n h i b i t s d i f f e r e n t i a t i o n b u t does n o t appear t o d e c r e a s e t h e l i n e a r growth o f n o n d i f f e r e n t i a t e d (non-shocked) uredosporelings.  The  r e s u l t s are a l s o c o n s i s t e n t with  the  o b s e r v e d e f f e c t s o f h e a t shock on t h e i n c o r p o r a t i o n o f [  3 5  S]-  m e t h i o n i n e i n t o newly s y n t h e s i z e d h e a t shock p r o t e i n s i n germinating  f l a x r u s t uredosporelings  r e p o r t e d by Shaw e t a l .  (1985). A h i g h p r o p o r t i o n o f t h e t o t a l amino a c i d p o o l was t o t h e medium d u r i n g g e r m i n a t i o n , uredosporelings.  The  lost  p a r t i c u l a r l y i n non-shocked  extent of t h i s leakage i n d i c a t e s t h a t  t h e l o s t amino a c i d s must o r i g i n a t e from t h e h y d r o l y s i s o f protein.  D a l y and h i s c o l l e a g u e s  (1967) a l s o found t h a t i n  non-shocked u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i a  considerable  p o r t i o n o f t h e f r e e amino a c i d s a r i s i n g from p r o t e i n degradation  a r e l o s t t o t h e medium and t h a t o n l y a s m a l l  p o r t i o n are u t i l i z e d f o r the resynthesis of p r o t e i n s .  The  102 r e s u l t s i n t h i s thesis show that the loss of free amino acids from non-shocked sporelings to the medium i s 2.6 (36.0/13.8) times greater than the loss from heat shocked sporelings.  As  spore germination progressed (from 8 h to 20 h) the loss of amino acids from non-shocked uredosporelings  t o the medium  increased and appeared l e s s s e l e c t i v e than the l o s s from heat shocked sporelings. In contrast, heat shock markedly decreased the loss a t 20 h of most of the amino acids l i s t e d on Table XIV. P a r t i c u l a r l y s t r i k i n g decreases occurred i n the losses o f alanine, cysteine, phenylalanine  and an unknown amino acid.  On the other hand heat shock had no s i g n i f i c a n t e f f e c t on the losses o f p r o l i n e and glutamine.  The loss of p r o l i n e and  glutamine i s not s u r p r i s i n g since they are r e a d i l y formed v i a the conversion of many amino acids and metabolites.  Proline  commonly functions as a long-distance transport compound f o r carbon.  Proline i s an ideal translocation molecule by v i r t u e  of i t s high metabolic c a p a b i l i t i e s (eg. t o form glutamate, aketoglutarate, and pyruvate v i a succinate)  ( M i f l i n 1977).  Glutamine, on the other hand i s the p r i n c i p l e nitrogen donor f o r many biosynthetic reactions, and i t s formation  is a  d e t o x i f i c a t i o n mechanism f o r the removal of ammonia. Individual amino acids were l o s t t o the medium at d i f f e r e n t rates and i n d i f f e r e n t amounts (Table XV). For example, non-shocked sporelings l o s t 0.6 nM/mg alanine i n 8 h and 4.5 nM/mg i n 20 h (ie. the loss at 20 h was 7.5 times the  103 loss a t 8 h).  Non-shocked sporelings also l o s t 2.4 nM/mg  glutamine a t 8 h and 9.9 nM/mg a t 20 h ( i e . the loss a t 20 h was 4.2 times the loss a t 8 h).  On the other hand the l o s s of  alanine from heat shocked sporelings a t 20 h was 1.14 times the loss a t 8 h and the loss of glutamine a t 20 h was 4.15 times the loss a t 8 h.  Comparing alanine and glutamine we see  that they were each l o s t a t d i f f e r e n t rates i n non-shocked and heat shocked sporelings.  Comparing non-shocked and heat  shocked sporelings we see that alanine was l o s t a t d i f f e r e n t rates but glutamine a t the same rate i n the two sets of sporelings.  Similar comparisons can be made f o r the other  amino acids from the data i n Table XV.  I t follows that i f  amino acids are l o s t from uredosporelings v i a a membrane defect, the defect does not a f f e c t the l o s s of a l l amino acids to the same extent.  Therefore, the loss o f amino acids from  the uredosporeling t o the medium i s considered t o be selective.  In summary, the composition of each amino acid  pool (exogenous and leachate pools) i s unique.  The percent  composition of each amino acid t o the t o t a l amino acids i n each pool i s : (1) d i f f e r e n t , (2) a l t e r s as germination progresses through 8 h t o 20 h, and (3) i s a l t e r e d by heat shock. The question which next a r i s e s i s the mechanism of the e f f e c t of the heat shock i n s e l e c t i v e l y decreasing the loss of amino acids.  While heat shock promotes the synthesis of  c e r t a i n highly conserved heat shock proteins and depresses the  104 synthesis of other proteins (Shaw et a l . 1985), the mechanisms involved are unknown.  exact  With respect to the leakage  of amino acids into the medium i t i s possible that heat shock d i r e c t l y a f f e c t s on the i n t e g r i t y and hence the permeability of the c e l l membranes.  A l t e r n a t i v e l y i t may  transport processes across the membranes.  a l t e r amino-acid  The data i n t h i s  t h e s i s do not provide any d i r e c t evidence on which to base an answer to these questions. Irrespective of the e f f e c t of heat shock, the growth of the germtube i s accompanied by the loss of amino acids to the medium.  I t i s possible that exogenous amino acids a r i s e from  e i t h e r the mechanical rupture or enzymatic degradation of the germtube c e l l w a l l .  Although germtube l y s i s was  not observed  i t i s l i k e l y that some degradation of the hyphal wall occurs during germination. facilitate metabolites  The break down of the germtube wall would  the loss of amino acids as well as other and enzymes.  cell  In addition, i t i s possible that the  loss of amino acids to the medium i s the r e s u l t of a s t r u c t u r a l or chemical defect i n the c e l l membrane. membrane defect may  A  lead to the loss of s e l e c t i v e permeability  and/or an a l t e r a t i o n i n amino acid transport systems. Scott and Maclean (1969) have suggested that the rust fungi resemble mammalian c e l l cultures i n that both c e l l  lines  appear to have a membrane defect allowing the loss of newly synthesized metabolites  into the medium.  The appearance of  free amino acids within the leachate implies the absence of  105 regulation of permeability i n the hyphal membrane (Scott and Maclean 1969).  I f t h i s assumption i s v a l i d i t may  explain the  high uredospore density usually required f o r i n i t i a t i n g normal saprophytic growth.  I t i s well known that radioactive amino  acids fed to non-shocked germinating uredospores are taken up and incorporated i n t o protein (Shaw 1964,  Shaw et a l . 1985).  At high c e l l d e n s i t i e s the leaked nutrient can reenter another c e l l i n close proximity, thus maintaining an e f f e c t i v e i n t e r c e l l u l a r l e v e l by cross feeding between c e l l s .  At low  c e l l d e n s i t i e s a metabolite may be l o s t into the medium at a rate equal to i t s rate of synthesis.  A c r i t i c a l population  density i s able to b u i l d up an e x t r a c e l l u l a r concentration of the metabolite that i s i n equilibrium with the minimum i n t r a c e l l u l a r l e v e l , before the c e l l s die of a s p e c i f i c n u t r i t i o n a l deficiency (Eagle and Piez 1962). Amino acids have important p h y s i o l o g i c a l r o l e s which govern many metabolic processes activity).  (e.g. growth and enzyme  A number of i n t e r e s t i n g avenues of research have  come to t h i s authors attention during t h i s study:  the e f f e c t  of exogenous amino acids on germination and d i f f e r e n t i a t i o n ; the e f f e c t of some amino acids (pathway products)  on  extractable enzyme a c t i v i t i e s ; the e f f e c t of exogenous amino acids on enzyme a c t i v i t y (derepression/repression of genes); and to explore the phenomenon of growth i n h i b i t i o n due to amino a c i d imbalances.  Although research i s being pursued i n  these areas most of the work has focussed on organisms other  106 than the rust fungi. Most of the recent work published  i s concerned with the  timing and the products of protein synthesis; however, the a c t i v a t i o n of protein synthesis during germination and  the  formation of i n f e c t i o n structures has not been well documented.  Given the information  contained i n the present  study i t would be of i n t e r e s t to determine the amount of charged tRNA f o r each amino acid.  To s i g n i f i c a n t l y a f f e c t  protein synthesis the absence of a free amino acid would r e s u l t i n the absence of the aminoacyl-tRNA. Although some growth of several s t r a i n s of wheat stem rust has been obtained axenically, only the Australian 126-ANZ-67, cultured by William's group (1966, 1967) found to grow vigorously.  has been  Analyses of the amino a c i d  composition of the leachate may formulating  race,  provide a useful basis f o r  the amino acid component of media f o r the axenic  culture of stem rust.  Moreover, i t would also be of i n t e r e s t  to extract and p u r i f y the free amino a c i d f r a c t i o n from germinating uredospores.  This f r a c t i o n could be used to  provide the amino acid component of an axenic medium f o r rust culture.  107 6.  SUMMARY  (1)  The percent germination of P. graminis t r i t i c i , race C17, uredospores and the proportion of germtubes forming complete i n f e c t i o n structures was augmented by n-nonyl alcohol.  (2)  A p r e c i s e l y timed heat shock and exogenous nutrients stimulate d i f f e r e n t i a t i o n . This stimulus i s most e f f e c t i v e i n the presence of n-nonyl alcohol.  (3)  The heat shock temperature required to induce maximum d i f f e r e n t i a t i o n has a very precise optimum. Variations one degree above or below t h i s optimum f o r a given spore l o t reduced the percent d i f f e r e n t i a t i o n by greater than 40%.  (4)  Although the optimum temperature of the heat shock varied s l i g h t l y depending on the p a r t i c u l a r spore l o t the s e n s i t i v i t y of sporeling development to temperature changes i s remarkably constant.  (5)  Compared t o Ca-K buffer, MPG was a superior germination and d i f f e r e n t i a t i o n medium.  (6)  Ca-K b u f f e r was the most e f f e c t i v e component of  (7)  Uredosporeling d i f f e r e n t i a t i o n occured as a series of p r e c i s e l y timed morphological, c y t o l o g i c a l , and physiological events.  (8)  DAPI s t a i n i n g i s a simple, rapid, and reproducible way to assess nuclear behaviour i n P. graminis t r i t i c i uredosporelings.  (9)  Nuclear d i v i s i o n was a regular event i n d i f f e r e n t i a t i n g uredospores. I t s occurrence was c l o s e l y associated with the timing of i n f e c t i o n structure development.  MPG.  108 (10)  Nuclear d i v i s i o n was a rare event i n germinating (nonshocked) uredospores.  (11)  E s s e n t i a l , presumably d i f f e r e n t i a t i o n - s p e c i f i c proteins, were synthesized from the onset of germination t o the completion of d i f f e r e n t i a t i o n . These proteins are required f o r the formation of appressoria, v e s i c l e s and, i n f e c t i o n hyphae.  (12)  Spore germination was accompanied by a rapid decrease i n the glutamic:aspartic acid r a t i o (from 20:1 to 3:1).  (13)  There i s a net hydrolysis of protein during germination, leading t o an increase i n s i z e of the endogenous pool of free amino acids and t o an increased leakage of amino acids to the germination medium.  (14)  Relative t o non-shocked uredosporelings, heat shock decreased both the s i z e of the endogenous pool of amino acids and the extent t o which uredosporelings lose amino acids t o the medium.  (15)  There was no net protein synthesis during the formation of i n f e c t i o n structures induced by heat shock.  (16)  A high proportion of the t o t a l amino a c i d pool was l o s t to the medium during germination, p a r t i c u l a r l y i n nonshocked uredosporelings.  (17)  Free cysteine was detected i n the leachate i s o l a t e d from non-shocked sporelings only.  (18)  The loss of amino acids to the germination medium i s s e l e c t i v e , p a r t i c u l a r l y i n heat shocked uredosporelings.  (19). The composition of the amino a c i d pool i n the leachate may be a useful guide i n formulating media f o r the axenic culture of P. graminis t r i t i c i .  109 7.  LITERATURE CITED  A l l e n , R.F. 1923. A c y t o l o g i c a l study of i n f e c t i o n of Baart and Kanred wheats by Puccinia graminis t r i t i c i . J . Agric. Res. 23: 131-152. A l l e n , P.J. 1972. S p e c i f i c i t y of the cis-isomers of i n h i b i t o r s of uredospore germination i n the rust fungi. Natl. Acad. S c i . USA. 69: 3497-3500.  Proc.  . 1976. Control of spore germination and i n f e c t i o n structure formation i n the rust fungi. In Physiological Plant Pathology, eds. R. Heitefuss, P.H. Williams, 51-78. New York: Springer-Verlag. Artemenko, E.N., Umnov, A.M., and Chkanikov, D.I. 1980. Changes i n the l e v e l of indolyl-3-acetic acid and possible paths of i t s regulation i n leaves of wheat infected with stem rust. Sov. Plant Physiol. (Eng. Transl.) 27: 447-451. Ashburner, M. 1982. The e f f e c t s of heat shock and other stress on gene a c t i v i t y : an introduction. In Heat Shock: from Bacteria t o Man, eds. M.J. Schlesinger, M. Ashburner, A. T i s s i e r s , 1-9. New York: Cold Spring Harbour Lab. Press. Baker, C.J., Tomerlin, J.R., Mock, N., Davidson, L., and Melhuish, J . 1987. E f f e c t s of cations on germination of urediniospores of Uromyces phaseoli. Phytopathology. 77: 1556-1560. B e l l , A.A., and Daly, J.M. 1962. Assay and p a r t i a l p u r i f i c a t i o n of s e l f - i n h i b i t o r s of germination from uredospores of the bean rust fungus. Phytopathology 52: 261266. Bhattacharya, P.K., Naylor, J.M., and Shaw, M. 1965. Nucleic acid and protein changes i n the wheat l e a f n u c l e i during rust i n f e c t i o n . Science 150: 1605-1607. Bidwell, R.G.S. 1979. Plant Physiology, pp 192-227. York: Macmillian Pub. Co. Boedijn, K.B. 1965. Trypan Blue as a s t a i n f o r fungi. Tech. 31: 115-116.  New Stain  Bushnell, W.R. 1976. Growth of races 38 and 17, P. graminis t r i t i c i on a r t i f i c i a l media. Can. J . Bot. 54: 1490-1498.  110 Bushnell, W.R. 1984. Structural and physiological a l t e r a t i o n s i n susceptible host t i s s u e . In The Cereal Rusts .1. Origins, S p e c i f i c i t y , Structure, and Physiology, 477-507. New York: Academic Press Inc. Ltd. C a l t r i d e r , P.G., and Gottlieb, D . 1963. Respiratory a c t i v i t y and enzymes f o r glucose catabolism i n fungus spores. Phytopathology 53: 1021-1030. Chakravorti, B.P. 1966. Attempts to a l t e r i n f e c t i o n processes and aggressiveness of Puccinia graminis t r i t i c i . Phytopathology 56: 223-229. Chakravorti, A.K., Shaw, M., and Scrubb, L.A. 1974. Changes in ribonuclease a c t i v i t y during rust i n f e c t i o n . I. Characterization of multiple molecular forms of ribonuclease from f l a x rust grown i n host-free media. Physiol. Plant Path. 4: 313-334. Cochrane, J . C , Rado, T.A., and Cochrane, V.W. 1971. Synthesis of macromolecules and polyribosome formation i n early stages of spore germination i n Fusarium s o l a n i . J . Gen. Microbiol. 65: 45-55. Coffey, M.D., and Shaw, M. 1972. N u t r i t i o n a l studies with axenic cultures of the f l a x rust Melampsora l i n i . Physiol. Plant Path. 2: 37-46. Coffey, M.D., and A l l e n , P.J. 1973. N u t r i t i o n of Melampsora l i n i and Puccinia h e l i a n t h i . Trans. Br. Mycol. Soc. 60: 245260. Coleman, A.W., Maquire, M., and Coleman, J.R. 1981. Mithromycin and DAPI-staining f o r fluorescence microspectrophotometric measurement of DNA i n n u c l e i , p l a s t i d s and v i r u s p a r t i c l e s . J . Histochem. Cytochem. 19: 959-968. Coleman, A.W., and Goff, L.J. 1985. Applications of fluorochromes to pollen biology. I. Mithromycin and 4',6diamidino-2-phenylindole (DAPI) as v i t a l stains and f o r the q u a n t i f i c a t i o n of nuclear DNA. Stain Tech. 60 (3): 145-154. Craigie, J.H. 1959. Nuclear behaviour i n the d i p l o i d i z a t i o n of haploid infections of Puccinia h e l i a n t h i . Can. J . Bot. 37: 843-855. Daly, J.M., Knoche, H.W., and Wiese, M.V. 1967. Carbohydrate and l i p i d metabolism during germination of uredospores of P. graminis t r i t i c i . Plant Physiol. 42: 1633-1642.  Ill De Jong, E.J., Eskes, A.B., Hoogstraten, J.G.J., and Zadoks, J.C. 1987. Temperature requirements f o r germination, germtube growth and appressorium of urediospores of Hemileia v a s t a t r i x . Neth. J . Plant Path. 93: 61-71. Dickinson, S. 1949. Studies i n the physiology of obligate parasitism. I I . The behaviour of the germtubes of c e r t a i n rusts i n contact with various membranes. Ann. Bot. London 13: 219-236. . 1971. Studies i n the physiology of obligate parasitism. IX. The measurement of thigmotrophic stimulus. Phytopathol. Z. 73: 347-358. Difco Laboratories. 1953. Difco Manual 9 Detroit, U.S.A.: Difco Lab. Inc.  t h  Ed., 265.  Dube, H.C., and Bordia, S. 1982. E f f e c t of amino acids on p e c t y l i t i c enzymes produced by Helminthosporium sacchari, the i n c i t a n t of 'eye-spot' disease of sugarcane. Proc. Nat'l. Acad. S c i . India. B. 52(1): 25-28. Dunkle, L.D., Maheshwari, R., and A l l e n , P.J. 1969. Infection structures from rust uredospores: E f f e c t of RNA and protein synthesis i n h i b i t o r s . Science 163: 481-482. Dunkle, L.D., and A l l e n , P.J. 1971. Infection structure d i f f e r e n t i a t i o n by wheat stem rust uredospores i n suspension. Phytopatholgy 61: 649-652. Dwyer, M.E., Merion, M., and Sousa, K.R. 1987. Applications of a p h y s i o l o g i c a l amino acid analysis system f o r the b i o l o g i c a l researcher. J . Analysis and P u r i f i c a t i o n Sept: 4649. Eagle, H. and Piez, Z. 1962. The population-dependent requirement by cultured mammalian c e l l s f o r metabolites which they can synthesize. J . Exp. Med. 116: 29. Epstein, L., L a c e t t i , L., Staples, R.C, Hoch, H.C., and Hoose, W.A. 1985. E x t r a c e l l u l a r proteins and d i f f e r e n t i a t i o n induction by bean rust uredospore germlings. Phytopathology 75(9): 1073-1076. F l o r , H.H. 1971. Current status of the gene-for-gene concept. Ann. Rev. Phytopath. 9: 275-296. Foudin, A.S., and Wynn, W.K. 1972. Growth of P.graminis t r i t i c i on a defined medium. Phytopathology 62: 1032-1040.  112 French, R.C, and Weintraub, R.L. 1957. Pelargonaldehyde as an endogenous germination stimulator of wheat stem rust spores. Arch. Biochem. Biophys. 72: 235-237. Gassner, G., and Franke, W. 1938. Untersuchungen uber den s t i c k s t o f f h a u s h a l t r o s t i n f i z i e r t e r Getreideblatter. E i n Beitrag zum problem der Teleutospoenbildung. Phytopath. Z. 5: 517-570. Goff, L.J.A., and Coleman, A.W. 1984. Elucidation of f e r t i l i z a t i o n and development i n a red algae by quantitative DNA microspectrofluorometry. Devel. B i o l . 102: 173-194. Grambow, H.J. 1977. The influence of v o l a t i l e l e a f constituents on the i n v i t r o d i f f e r e n t i a t i o n and growth of P.graminis t r i t i c i . Z. Pflanzenphysiol. Bd. 85: 361-372. . 1978. The e f f e c t of Nordihydroguajaretic acid on the development of the wheat rust fungus. Z. Pflanzenphysiol. 88: 369-372. Grambow, H.J., and Grambow, G.E. 1978. The involvement of e p i c u t i c u l a r and c e l l wall phenols of the host plant i n the i n v i t r o development of P. graminis t r i t i c i . Z. Pflanzenphysiol. 90: 1-9. Grambow, H.J., and Muller, D. 1978. Nuclear condition, types of hyphal development from d i f f e r e n t i a t i n g and nond i f f e r e n t i a t i n g uredosporelings, and e f f e c t of 3,3'-bisindolylmethane on P. graminis t r i t i c i i n v i t r o . Can. J . Bot. 56: 736-741. Grambow, H.J., and Riedel, S. 1977. The e f f e c t of morphologically active factors from the host and nonhost plants on the i n v i t r o d i f f e r e n t i a t i o n of i n f e c t i o n structures of P. graminis t r i t i c i . Physiol. Plant Path. 11: 213-224. Hartley, M.J. and Williams, P.G. 1971a. Interactions between s t r a i n s of Puccinia graminis f.sp. t r i t i c i i n axenic culture. Trans. Br. Mycol. Soc. 57:129-136. . 1971b. Morphological and c u l t u r a l differences between races of Puccinia graminis f.sp. t r i t i c i i n axenic culture. Trans. Br. Mycol. Soc. 57: 137-144. Heath, I.B., and Heath, M.C 1978. Microtubules and organelle movements i n the rust fungus Uromyces phaseoli var. vignae. Cytobiology 16: 393-411.  113 Hepper, CM. 1986. Growth of hyphae from Glomus spores i n the presence of sulphur-containing compounds. Mycorrhizae: physiology and genetics. SEM, Dijon, 1-5 J u l y 1985. INRA, Paris: 527-530. Hess, S.L., A l l e n , P.J., Nelson, D., and Lester, H. 1975. Mode of action of methyl c i s - f e r u l a t e , the s e l f - i n h i b i t o r of stem rust uredospore germination. Physiol. Plant Path. 5: 107-112. Hoch, H.C, Staples, R.C, and Bourett, T. 1984. C e l l d i f f e r e n t i a t i o n i n Uromyces. Involvement of the cytoskeleton (abstr.). J . C e l l B i o l . 99: 200a. Hoch, H.C, and Staples, R.C. 1985. The microtubule cytoskeleton i n hyphae of Uromyces phaseoli germlings: I t s r e l a t i o n s h i p t o the region of nucleation and t o the F-actin cytoskeleton. Protoplasma 124: 112-122. Hoch, H.C, Staples, R.C, Whitehead, B., Comeau, J . , and Wolf, E.D. 1987. S i g n a l l i n g f o r growth o r i e n t a t i o n and c e l l d i f f e r e n t i a t i o n by surface topography i n Uromyces. Science 235: 1659-1662. Howes, N.K., and Scott, K.J. 1972. Sulphur n u t r i t i o n of P.graminis t r i t i c i i n axenic culture. Can. J . Bot. 50: 11651170. . 1973. Sulphur metabolism of P. graminis t r i t i c i . Gen. M i c r o b i o l . 76: 345-354.  J.  Howes, N.K., Kim, W.K., and Rohringer, R. 1982. Detergentsoluble polypeptides extracted from uredospores of four physiologic races of Puccinia graminis f.sp. t r i t i c i . Physiol. Plant Pathol. 21(3): 361-366. Huang, B.F., and Staples, R.C. 1982. Synthesis of proteins during d i f f e r e n t i a t i o n of the bean rust fungus. Exp. Mycol. 6: 7-14. J a f f e L.F. 1981. The r o l e of i o n i c currents i n e s t a b l i s h i n g developmental pattern. P h i l . Trans. R. Soc. Lond. B. 295: 553-566. Johansen, D.A. 1940. Hill.  Plant Microtechnique.  New York: McGraw-  Johnson, L.B., Brannaman, B.L., and Zscheile, F.P. 1968. Protein and enzyme changes i n wheat leaves following i n f e c t i o n with Puccinia recondita. Phytopathology 58(5): 578-583.  114 Jones, J.P. 1966. Absorption and t r a n s l a t i o n of -"S i n oat plants inoculated with labeled crown rust uredospores. Phytopathology 56: 272-275. Jones, J.P., and Snow, J.P. 1965. Amino acids released during germination of S - l a b e l e d crown rust spores (abstr.). Phytopathology 55: 499. 35  Kasting, R., McGinnis, A.J., and Broadfoot, W.C. 1959. Biosynthesis of some amino acids from sucrose by germinating uredospores of wheat stem rust fungi, race 15B. Nature 184: 1943. Kim, W.K., and Rohringer, R. 1969. Metabolism of aromatic compounds i n healthy and rust-infected primary leaves of wheat. I I I . Studies on the metabolism of tryptophan. Can. J . Bot. 47(9): 1425-1433. Kim, W.K., Howes, N.K., and Rohringer, R. 1982a. Detergentsoluble polypeptides i n the germinated uredospores and d i f f e r e n t i a t e d uredosporelings of wheat stem rust. Can. J . Plant Path. 4: 328-333. Kim, W.K., Rohringer, R., and Chong, J . 1982b. Sugar and amino acid composition of macromolecular constituents released from walls of uredosporelings of P. graminis t r i t i c i . Can. J . Plant Path. 4(4): 317-327. K i r a l y , Z., and Farkas, G. 1962. Biochemical character of resistance i n wheat to black stem rust. Review of Applied Mycology Abstr. 704. Kropft D.L., Caldwell J.H., Gow N.A.R., and Harold F.M. 1984. T r a n s c e l l u l a r ion currents i n the water mold Achyla. Amino acid proton symport as a mechanism of current entry. J . C e l l B i o l . 99: 486-496. Kuck, K.H., and Reisener, H.J. 1985. In v i t r o sporulation of race 32 of Puccinia graminis f.sp. t r i t i c i E r i k s . and Henn. Physiol. Plant Path. 27(3): 259-267. Kuhl, J.L., Maclean, D.J., Scott, K.J., and Williams, P.G. 1971. The axenic culture of Puccinia species from uredospores: experiments on n u t r i t i o n and v a r i a t i o n . Can. J . Bot. 49: 201-209. Levin, I.M. 1985. Dependence of the development rates of wheat stem rust on changing substance transport i n the detached l e a f treated with phytohormones. Mikologiya i Fitopatologiya 19(3): 256-260.  115 Macko, V., Staples, R.C, A l l e n , P.J., and Renwick, J.A.A. 1971. I d e n t i f i c a t i o n of the germination s e l f - i n h i b i t o r from wheat stem rust uredospores. Science 173: 835-836. Macko, V., Staples, R.C, Renwick, J.A.A., and Pirone, J . 1972. Germination s e l f - i n h i b i t o r s of rust uredospores. Physiol. Plant Pathol. 2: 347-355. Macko, V., Staples, R.C, Yaniv, Z., and Granadas, R.R. 1976. S e l f - i n h i b i t o r s of fungal spore germination. In: The Fungal Spore: Form and Function, eds. D.J. Weber, W.M. Hess, 73-100. New York: John Wiley and Sons. Macko, V., Renwick, J.A.A., and R i s s l e r , J.F. 1978. A c r o l e i n induces d i f f e r e n t i a t i o n of i n f e c t i o n structures i n the wheat stem rust fungus. Science 199: 442-443. . 1982. Axenic culture and metabolism of rust fungi. In The Rust Fungus, eds. K.J. Scott and A.K. Chakravorty, 37-81. London: Academic Press. Maheshwari, R., A l l e n , P.J., and Hildebrandt, A.C 1967a. Physical and chemical factors c o n t r o l l i n g the development of i n f e c t i o n structures from uredospore germtubes of rust fungi. Phytopathology 57: 855-862. . 1967b. The cytology of i n f e c t i o n structure development in uredospore germtubes of Uromyces phaseoli var. typica (Pers.) Wint. Can. J . Bot. 45: 447-450. Manners, J.M., Maclean, D.J., and Scott, K.J. 1982. Pathways of glucose a s s i m i l a t i o n i n P. graminis f.sp. t r i t i c i . J . Gen. Microbiol. 128 (11): 2621-2630. Manocha, M.S., and Shaw, M. 1966. The physiology of hostparasite r e l a t i o n s . XVI. Fine structure of the nucleus i n the rust-infected mesophyll c e l l s of wheat. Can. J . Bot. 44(5): 669-673. M c K i l l i c a n , M.E. 1960. A survey of amino a c i d content of uredospores of some races of wheat rust (Puccinia graminis). Can. J . Chem. 38: 244-247. Meihle, B.R., and Lukezic, F.L. 1972. Studies on c o n i d i a l germination and appressorium formation by Colletotrichum t r i f o l i i Bain and Essary. Can. J . Microbiol. 18: 1263-1269. Mendgen, K., Lange, M., and BretSchneider, K. 1985. A quantitative estimation of the surface carbohydrates of i n f e c t i o n structures of rust fungi with enzymes and l e c t i n s . Arch. Microbiol. 140: 307-311. N  116 M i f l i n , B.J., and Lea, P.J. 1977. Amino acid metabolism. Annu. Rev. Plant Physiol. 28: 299-329. Morris, D.R., and Fillingame, R.H. 1974. Regulation of amino a c i d decarboxylation. Ann. Rev. Biochem. 43: 303-325. Otto, F. and Tsou, K.C. 1985. A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA s t a i n s . Stain Tech. 60 (1): 7-11. Plesosky-Vig, N., and Brambl, R. 1985. Heat shock response of Neurospora crassa: Protein synthesis and induced thermotolerance. J . B a c t e r i o l . 162(3): 1083-1091. Rajam, M.V., Weinstein, L.H., and Galston, A.W. 1985. Prevention of plant disease by s p e c i f i c i n h i b i t i o n of fungal polyamine biosynthesis. Proc. Nat'l. Acad. S c i . U.S.A. 82: 6874-6878. Reisener, H.J. 1967. Untersuchungen uber den Aminosaure und Proteinstoffwechsel der Uredosporen des Weizenrostes. Arch. Mikrobiol. 55: 382-397. Reisener, H.J., and Jager, K. 1966. The quantitative importance of some metabolic pathways i n uredospores of Puccinia graminis var. t r i t i c i • Planta 72: 265-283. Reisener, H., McConnell, W.B., and Ledingham, G.A. 1961. Studies on the metabolism of v a l e r a t e - l - C by uredospores of wheat stem rust. Can. J . Biochem. Physiol. 39: 1559-1566. 1 4  Rines, H.W., French, R.C, and Daasch, L.W. 1974. Nonanal and 6-methyl-5-hepten-2-one: Endogenous germination stimulators of uredospores of P. graminis t r i t i c i and other rusts. J . Agr. Food Chem. 22: 96-100. Saunders, S. and Youatt. 1983. Amino acids i n the control of d i f f e r e n t i a t i o n of sporangia i n Allomyces macrogynus. Aust. J. B i o l . S c i . 36: 435-443. Savile, D.B.O. 1939. Nuclear structure and behaviour i n species of the Uredinales. Am. J . Bot. 26: 585-609. S c h i f f , J.A. and Hodson, R.C. 1973. The metabolism of sulphate. Ann. Rev. Plant Physiol. 24: 381-414. Scott, K.J. 1972. fungi. B i o l . Rev.  Obligate parasitism by phytopathogenic 47: 537-572.  Scott, K.J. 1976. Growth of biotrophic parasites i n axenic culture. In Physiological plant pathology, eds. R. Heitefuss, P.H. Williams. New York: Springer-Verlag.  117 Shaw, M. 1963. The p h y s i o l o g y and h o s t - p a r a s i t e the r u s t s . Ann. Rev. Phytopath. 1: 259-294. Shaw, M. 1964. The p h y s i o l o g y o f r u s t P h y t o p a t h . Z . 50: 159-180.  relations  of  uredospores.  Shaw, M . , and C o l o t e l o , N. 1961. The p h y s i o l o g y o f h o s t p a r a s i t e r e l a t i o n s . V I I I . The e f f e c t o f stem r u s t on t h e n i t r o g e n and amino a c i d s i n wheat l e a v e s . Can. J . B o t . 39: 1351-1372. Shaw, M . , Boasson, R . , and Scrubb, L . 1985. E f f e c t o f heat shock on p r o t e i n s y n t h e s i s i n f l a x r u s t u r e d o s p o r l i n g s . Can. J . B o t . 63: 2069-2076. S i n g h , H . and S e t h i , I . 1982. Growth responses o f r a c e o f P . crraminis f. s p . t r i t i c i i n d e f i n e d media. Can. J . M i c r o b i o l . 28: 486-492.  222  S t a p l e s , R . C . 1962. I n i t i a l products of acetate u t i l i z a t i o n by bean r u s t u r e d o s p o r e s . C o n t r i b . Boyce Thompson I n s t . P l a n t Res. 21: 487-497. S t a p l e s , R . C . 1965. A c i d phosphatases from h e a l t h y and r u s t i n f e c t e d P i n t o Bean l e a v e s . I I . E f f e c t o f removing a p i c a l meristem. C o n t r i b . Boyce Thompson I n s t . P l a n t Res. 23(4): 8391. S t a p l e s , R . C . 1968. P r o t e i n s y n t h e s i s by uredospores o f bean r u s t fungus. N e t h . J . P l a n t P a t h . 74: 25-36.  the  S t a p l e s , R . C . 1974. S y n t h e s i s o f DNA d u r i n g d i f f e r e n t i a t i o n o f bean r u s t u r e d o s p o r e s . P h y s i o l . P l a n t P a t h . 4: 415-424. S t a p l e s , R . C , Syamananda, R . , Kao, V . , and B l o c k , R . J . 1962. Comparative b i o c h e m i s t r y o f o b l i g a t e l y p a r a s i t i c and saprophytic fungi. I I . A s s i m i l a t i o n of • C - l a b e l l e d substrates by g e r m i n a t i n g u r e d o s p o r e s . C o n t r i b . Boyce Thompson I n s t . 21: 345-362. L 4  S t a p l e s , R . C . and Stahmann, M . A . 1964. Changes i n p r o t e i n and s e v e r a l enzymes i n s u s c e p t i b l e bean l e a v e s a f t e r i n f e c t i o n by t h e bean r u s t fungus. Phytopathology 54: 760-764. S t a p l e s , R . C and Wynn, W.K. 1965. The p h y s i o l o g y o f uredospores o f the r u s t f u n g i . Botan. Gaz. 31: 537-564. S t a p l e s , R . C . and B e d i n g i a n , D. 1967. P r e p a r a t i o n o f an amino a c i d i n c o r p o r a t i o n system from uredospores o f the bean r u s t fungus. C o n t r i b . Boyce Thompson I n s t . P l a n t Res. 23: 345-347.  118 Staples, R.C., Bedingian, D. and Williams, P.H. 1968. Evidence f o r polysomes i n extracts of bean rust uredospores. Phytopathology 58: 151-154. Staples, R.C, Grambow, H.J. and Hoch, H.C 1982. Potassium ion induces bean rust germlings t o develop i n f e c t i o n structures (abstr. 221). Phytopathology 72: 957. Staples, R.C. and Hoch, H.C. 1982. A possible r o l e f o r microtubules and microfilaments i n the induction of nuclear d i v i s i o n i n bean rust uredospore germlings. Exp. Mycol. 6: 293-302. Staples, R.C, Grambow, H.J., Hoch, H.C. and Wynn, W.K. 1983a. Contact with membrane grooves induces wheat stem rust uredospore germlings t o d i f f e r e n t i a t e appressoria but not v e s i c l e s . Phytopathology 73 (10): 1436-1439. Staples, R.C, Macko, V., Wynn, W.K. and Hoch, H.C. 1983b. Terminology t o describe the d i f f e r e n t i a t i o n response by germlings of fungal spores. Phytopathology 380. Staples, R.C, Gross, D., Tiburzy, R., Hoch, H.C, Hassouna, S., and Webb, W.W. 1984. Changes i n DNA content of n u c l e i i n rust uredospore germlings during the s t a r t of d i f f e r e n t i a t i o n . Exp. Mycol. 8: 245-255. Stefanye, D. and Bromfield, K.R. 1965. Amino a c i d composition of uredospores of P. crraminis t r i t i c i . Can. J . Bot. 43: 695-699. S z i r a k i , I., Barna, B., Waziri, E., and K i r a l y , Z. 1976. E f f e c t of rust i n f e c t i o n on the cytokinin l e v e l of wheat c u l t i v a r s susceptible and r e s i s t a n t t o P. graminis t r i t i c i . Acta Phytopath. Acad. S c i . Hung. 11: 155-160. Tabor, CW. 1981. Mutants of Sacchromvces cerevisiae d e f i c i e n t i n polyamine biosynthesis: studies on the regulation of ODC Medical Biology 59: 272-278. VanSumere, C.F., VanSumere de Preter, C , Vining, L . C and Ledingham G.A. 1957. Coumarins and phenolic acids i n the uredospore of wheat stem rust. Can. J . Microbiol. 3: 847862. Von Broembsen, S.L., and Hadwiger, L.A. 1972. Characterization of disease resistance responses i n c e r t a i n gene-for-gene interactions between f l a x and Melampsora l i n i . Physiol. Plant Path. 2: 207-215  119 Walters, D.R. 1986. The e f f e c t s of a polyamine biosynthesis i n h i b i t o r on i n f e c t i o n of V i c i a faba L. by the fungus Uromyces vicia-fabae (Pers.) Schroet. New Phytol. 104: 613-619. Wanner, R.H., Forster, K., Mendgen, R.C. and Staples, R.C. 1985. Synthesis of d i f f e r e n t i a t i o n - s p e c i f i c proteins i n germlings of the wheat stem rust fungus a f t e r heat shock. Exp. Mycol. 9: 279-283. Whitney, H.S., Shaw, M., and Naylor, J.M. 1962. The physiology of host-parasite r e l a t i o n s . XII. A cytophotometric study of the d i s t r i b u t i o n of DNA and RNA i n rust-infected leaves. Can. J . Bot. 1533-1544. Williams, P.G. 1971. A new perspective of the axenic culture of Puccinia graminis f.sp. t r i t i c i from uredospores. Phytopathology. 61: 994-1002. Williams, P.G. 1984. Obligate parasitism and axenic culture. In The Cereal Rusts, v o l . 1, 399-428. New York: Academic Press Inc. Williams, P.G., Scott, K.J., and Kuhl, J.C. 1966. Vegetative growth of Puccinia graminis f.sp. t r i t i c i i n v i t r o . Phytopathology. 56: 1418-1419. Wilson, E.M. 1958. Aspartic and glutamic acids as s e l f i n h i b i t o r s of uredospore germination. Phytopathology 48: 595600. Wisdom, C. 1977. Morphogenesis of the wheat stem rust uredospore. M.Sc. Thesis. University of B r i t i s h Columbia. Wolf, G. 1982. Physiology and biochemistry of spore germination. In The Rust Fungi, eds. K.J Scott and A.K. Chakravorty, 151-178. New York: Academic Press. Wynn, W.K. , Staples, R.C, Strouse, B. and Gajdusek, C. 1966. Phsiology of uredospores during storage. Contrib. Boyce Thompson Inst. 23: 229-242. Wynn, W.K., and Staples, R.C. 1981. Tropisms of fungi i n host recognition. In: Plant Disease Control: Resistance and S u s c e p t i b i l i t y , eds. R.C. Staples, G.H. Toniessen, 45-69. New York: Wiley. Yaniv, Z. and Staples, R.C. 1974. Ribosomal a c t i v i t y i n urediospores germinating on membranes. Phytopathology 64: 1111-1114.  120 Youatt, J . 1986. Evidence of methionine biosynthesis i n Allomyces macrocrynus. Trans. Br. Mycol. Soc. 86 (4) : 653655. Zadoks, J.C. 1968. Meterological factors involved i n the d i s p e r s a l of cereal rusts. In Proc. Regional t r a i n i n g seminar on Agro-meterology. ed. A.J.W. Borghorst. Wageningen: 179194.  

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