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UBC Theses and Dissertations

Pseudorecombinants of cherry leaf roll virus Haber, Stephen Michael 1979

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9 * PSEUDORECOMBINANTS OF CHERRY LEAF ROLL VIRUS by Stephen Michael Haber B.Sc. (Biochem.), U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department of P l a n t Science) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1979 ©. Stephen Michael Haber, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Plant Science The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e J u l - 27. 1Q7Q ABSTRACT Cherry l e a f r o l l v i r u s , as a nepovirus w i t h a b i p a r t i t e genome, can be g e n e t i c a l l y analysed by comparing the p r o p e r t i e s of d i s t i n c t ' p a r e n t a l ' s t r a i n s and the pseudorecombinant i s o l a t e s generated from them. In the present work, the el d e r b e r r y (E) and rhubarb (R) s t r a i n s were each p u r i f i e d and separated i n t o t h e i r middle (M) and bottom (B) components by sucrose gradient c e n t r i f u g a t i o n f o l l owed by near-e q u i l i b r i u m banding i n cesium c h l o r i d e . RNA was e x t r a c t e d from the the separated components by treatment w i t h a d i s s o c i a t i o n b u f f e r f o llowed by sucrose gradient c e n t r i f u g a t i o n . E x t r a c t e d M-RNA of E - s t r a i n and B-RNA of R - s t r a i n were mixed and i n o c u l a t e d to a s e r i e s of t e s t p l a n t s as were M-RNA of R - s t r a i n and B-RNA of E - s t r a i n . New l o c a l l e s i o n types i n N i c o t i a n a c l e v e l a n d i i induced by these heterologous RNA combinations were passaged three times through l o c a l l e s i o n s on N. c l e v e l a n d i i i n order to generate pure pseudorecombinant stocks. M-RNA determined s e r o l o g i c a l s p e c i f i c i t y , the d i s t r i b u t i o n of v i r u s p a r t i c l e components, systemic symptoms i n N^ . c l e v e l a n d i i and N. tabacum cvs. Samsun and Xanthi as w e l l as the l o c a l and systemic symptoms i n Chenopodium amaranticolor and C. quinoa. B-RNA determined the a b i l i t y to induce systemic symptoms i n Gomphrena globosa and the type of l o c a l l e s i o n i n N. c l e v e l a n d i i . When the procedure used f o r o r i g i n a l l y generating the pseudorecombinants from the p a r e n t a l s t r a i n s was a p p l i e d to the pseudorecombinant i s o l a t e s themselves, i s o l a t e s were obtained i n the p r e d i c t e d manner that were i d e n t i c a l to the o r i g i n a l p a r e n t a l E- and R - s t r a i n s . i i i TABLE OF CONTENTS Page TITLE PAGE ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v LIST OF TABLES v i i i ACKNOWLEDGEMENT i x INTRODUCTION 1 MATERIALS AND METHODS 16 A. Vi r u s e s 16 B. Host P l a n t s 16 C. P u r i f i c a t i o n Methods 17 D. Sucrose Density Gradients 18 E. P r e p a r a t i v e N e a r - E q u i l i b r i u m CsCl Density-Gradient C e n t r i f u g a t i o n 19 F. I s o l a t i o n and A n a l y s i s of Nucleoprotein Components 21 G. Isopycnic Banding i n the Model-E A n a l y t i c a l U l t r a c e n t r i f u g e 21 H. Pr e p a r a t i o n of RNA f o r Polyacrylamide Gel E l e c t r o p h o r e s i s 22 I . P r e p a r a t i o n of RNA f o r Inoculum 23 J . Generation of Pseudorecombinants 24 K. Comparision of P a r e n t a l and Pseudorecombinant I s o l a t e s 25 1. Conponent D i s t r i b u t i o n 25 2. Symptomatology 25 3. Serology 25 L. Regeneration of Parents from Pseudorecombinants 26 RESULTS 28 A. Comparative Symptomatologies of CLRV S t r a i n s 28 B. P u r i f i c a t i o n Methods 28 C. Sucrose Gradient C e n t r i f u g a t i o n and Scanning P r o f i l e s of P a r t i c l e s 32 D. Pr e p a r a t i v e N e a r - E q u i l i b r i u m CsCl Density-Gradient C e n t r i f u g a t i o n 33 E. A n a l y s i s of Nucleoprotein Components by A n a l y t i c a l U l t r a c e n t r i f u g a t i o n 37 F. I s o l a t i o n and A n a l y s i s of Nucleoprotein Components 37 1. Comparative Spectrophotometric A n a l y s i s 40 2. I n f e c t i v i t y of Nucleoprotein Components 40 G. RNA of Separated Components i n Polyacrylamide Gel E l e c t r o p h o r e s i s 44 i v Page H. RNA of Separated Components as Inoculum 44 I. Generation of Pseudorecombinants 47 J . Comparision of P a r e n t a l and Pseudorecombinant I s o l a t e s 50 1. Symptomatology 50 2. P a r t i c l e Component D i s t r i b u t i o n 59 3. Serology 61 K. Regeneration of Parents from Pseudorecombinants 61 1. Symptom Evidence 61 2. P a r t i c l e Component D i s t r i b u t i o n Evidence 63 3. S e r o l o g i c a l Evidence 65 DISCUSSION 67 SUMMARY 75 LITERATURE CITED 76 BIOGRAPHICAL INFORMATION V LIST OF FIGURES Page FIGURE 1. Systemic symptoms of CLRV i n f e c t i o n i n C_. amaranticolor. 30 A. i n o c u l a t e d w i t h e l d e r b e r r y (E) s t r a i n : dwarfing and a p i c a l t u f t i n g a f t e r 60 days; B. i n o c u l a t e d w i t h rhubarb (R) s t r a i n : systemic mottle and p a r t l y f a s c i c u l a t e growth a f t e r 60 days 2. Absorbance scan patterns of sucrose d e n s i t y - g r a d i e n t s 34 showing d i f f e r e n c e s i n p a r t i c l e component d i s t r i b u t i o n of the e l d e r b e r r y and rhubarb s t r a i n s of CLRV p u r i f i e d from N. c l e v e l a n d i i . A. middle (M) and bottom (B) component nucleoproteins of the e l d e r b e r r y s t r a i n ; B. top (T) component RNA-free empty p a r t i c l e s and middle (M) and bottom (B) component nucleoproteins of the rhubarb s t r a i n 3. Absorbance scan patterns of sucrose d e n s i t y - g r a d i e n t s 35 showing p a r t i c l e component d i s t r i b u t i o n s of the e l d e r b e r r y and rhubarb s t r a i n s of CLRV p u r i f i e d from C_. quinoa. A. middle (M) and bottom (B) component nucleoproteins of the e l d e r b e r r y s t r a i n B. top (T) component RNA-free p a r t i c l e s and middle (M) and bottom (B) component nucleoproteins of the rhubarb s t r a i n 4. Absorbance scan patterns showing extent of separation of 36 middle and bottom n u c l e o p r o t e i n components (rhubarb s t r a i n ) . In each scan, the peak at the greatest depth corresponds to the bottom component. A. L i n e a r - l o g sucrose gradient c e n t r i f u g a t i o n ; B. 'Middle' component of 'A' a f t e r n e a r - e q u i l i b r i u m CsCl d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n ; C. 'Bottom' component of 'A' a f t e r n e a r - e q u i l i b r i u m CsCl d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n 5. C_. amaranticolor p l a n t s 45 days a f t e r i n o c u l a t i o n (at the 38 f o u r - l e a f stage) w i t h middle (M), bottom (B) and combined (M + B) n u c l e o p r o t e i n components of the rhubarb s t r a i n of CLRV. The components had been separated by sucrose gradient c e n t r i f u g a t i o n followed by n e a r - e q u i l i b r i u m banding i n CsCl ( p= 1.45 g-cm - 3). S c h l i e r e n p a t t e r n from a n a l y t i c a l u l t r a c e n t r i f u g a t i o n of CLRV-rhubarb s t r a i n i n CsCl d e n s i t y - g r a d i e n t of e q u i l i b r i u m (44,770 rpm; 24 h). Density increases from l e f t to r i g h t . Middle component n u c l e o p r o t e i n appears as sm a l l e r d e n s i t y -band near the centre of the gradient; bottom component nu c l e o p r o t e i n appears as the l a r g e r density-band to the r i g h t of the middle component. Absorbance scan patterns of CLRV nucleoproteins and ex t r a c t e d RNA, uncorrected f o r l i g h t s c a t t e r i n g , from 220-295 nm. A. separated middle component ( ) and bottom component ( ) n u c l e o p r o t e i n s ; B. e x t r a c t e d RNA Polyacrylamide g e l e l e c t r o p h o r e s i s of RNA e x t r a c t e d from separated n u c l e o p r o t e i n components and 'whole-virus' preparations of CLRV. A. RNA e x t r a c t e d from 'pure' middle component (M) nu c l e o p r o t e i n ; B. RNA e x t r a c t e d from a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of CLRV; C. RNA e x t r a c t e d from f u l l y p u r i f i e d CLRV; D. RNA e x t r a c t e d from 'pure' bottom component (B) nu c l e o p r o t e i n Absorbance scan patterns of RNA obtained by sucrose d e n s i t y -gradient c e n t r i f u g a t i o n . The RNA absorbance peaks are the doublets about o n e - t h i r d of the way down the gradient. A. RNA e x t r a c t e d from the e l d e r b e r r y (E) s t r a i n of CLRV; B. RNA e x t r a c t e d from the rhubarb (R) s t r a i n of CLRV Absorbance scan patterns of RNA (of the rhubarb s t r a i n of CLRV) obtained by sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n , showing the monodisperse nature and s l i g h t d i f f e r e n c e s i n sedimentation v e l o c i t y of RNA e x t r a c t e d from separated middle (M-RNA) and bottom (B-RNA) component n u c l e o p r o t e i n . A. RNA e x t r a c t e d from the separated middle component nu c l e o p r o t e i n (M-RNA); B. RNA e x t r a c t e d from a re-mixture of separated middle and bottom component n u c l e o p r o t e i n s ; C. RNA e x t r a c t e d from separated bottom component n u c l e o p r o t e i n (B-RNA); D. RNA e x t r a c t e d from a 'whole-virus' p r e p a r a t i o n v i i Page 11. Comparison of l o c a l - l e s i o n symptom types induced by p a r e n t a l 51 and pseudorecombinant i s o l a t e s 4 days a f t e r i n o c u l a t i o n on N. c l e v e l a n d i i . 12. Comparison on opposite h a l f - l e a v e s of l o c a l - l e s i o n symptom 52 types induced by p a r e n t a l and pseudorecombinant i s o l a t e s i n Xanthi tobacco. 13. Comparisons on opposite h a l f - l e a v e s of l o c a l - l e s i o n symptom 53 types induced by p a r e n t a l and pseudorecombinant i s o l a t e s i n C_. amaranticolor. 14. Comparison of systemic symptoms induced i n Samsun tobacco 55 induced by p a r e n t a l and pseudorecombinant i s o l a t e s . 15. Comparison of systemic symptoms induced i n C_. amaranticolor 57 60 days a f t e r i n o c u l a t i o n w i t h p a r e n t a l and pseudorecombinant i s o l a t e s . 16. Absorbance scan patterns of sucrose d e n s i t y - g r a d i e n t s showing 60 r e l a t i o n s h i p s of p a r t i c l e component d i s t r i b u t i o n s among pa r e n t a l and pseudorecombinant i s o l a t e s p a r t i a l l y p u r i f i e d from C_. quinoa. 17. D o u b l e - d i f f u s i o n aagar g e l serology showing genetic 62 determination of coat p r o t e i n a n t i g e n i c i t y . A. C e n t r a l w e l l contains a 1:100 d i l u t i o n of antiserum (titre=2560) made against p u r i f i e d E - s t r a i n . P e r i p h e r a l w e l l s contain preparations of E R p u r i f i e d E - s t r a i n (-) and R - s t r a i n (-) parents, i i K E R and - - and - - pseudorecombinants at A , = 1.0; R E Zou B. C e n t r a l w e l l contains a 1:50 d i l u t i o n of antiserum (titre=1280) made against p u r i f i e d R - s t r a i n . P e r i p h e r a l w e l l s are as i n F i g . 17A. 18. Comparison of l o c a l - l e s i o n symptoms i n !N. c l e v e l a n d i i 64 demonstrating regeneration of p a r e n t a l from pseudorecombinant i s o l a t e s . 19. Absorbance scan patterns of sucrose d e n s i t y - g r a d i e n t s 66 showing p r e d i c t e d r e l a t i o n s h i p s of p a r i t c l e component d i s t r i b u t i o n s among pseudorecombinant and 'regenerated' p a r e n t a l i s o l a t e s p u r i f i e d from C_. quinoa. v i i i LIST OF TABLES Page TABLE 1. Comparative symptomatologies of the s t r a i n s of CLRV 29 i n l o c a l and systemic i n f e c t i o n s 2. Enhancement of i n f e c t i v i t y by complementation of separated 43 middle (M) and bottom (B) n u c l e o p r o t e i n components, as measured by i n d u c t i o n of l o c a l l e s i o n s on opposite h a l f - l e a v e s of C_. amaranticolor 3. Enhancement of i n f e c t i v i t y by complementation of separated 43 middle (M) and bottom (B) n u c l e o p r o t e i n components, as measured by i n d u c t i o n of l o c a l l e s i o n s on opposite h a l f - l e a v e s of C_. amaranticolor 4. R e l a t i v e i n f e c t i v i t i e s at A.,_ = 0.10 of middle (M) and 49 z o U bottom (B) RNA components of CLRV e l d e r b e r r y (E) and rhubarb (R) s t r a i n s i n o c u l a t e d as separate components and i n homologous and heterologous equiabsorbant combinations ACKNOWLEDGEMENT The author wishes to express h i s s i n c e r e thanks to a l l who have helped make t h i s work p o s s i b l e : to Dr. R.I. Hamilton and Dr. R. Stace-Smith ( A g r i c u l t u r e Canada, Vancouver Research S t a t i o n ) , who as research supe r v i s o r s provided e s s e n t i a l guidance, c r i t i c i s m and encouragement; to Dr. V.C. Runeckles and Dr. R.J. Copeman of the Department of Pl a n t Science, U n i v e r s i t y of B r i t i s h Columbia, f o r t h e i r t i r e l e s s e f f o r t s i n a i d i n g the completion of t h i s t h e s i s and i t s r e l a t e d requirements; and to Dr. M. Weintraub and the u n f a i l i n g l y h e l p f u l s t a f f at the Vancouver Research S t a t i o n f o r t h e i r support and a s s i s t a n c e . 1 INTRODUCTION In terms of genome o r g a n i z a t i o n , v i r u s e s may be assigned to one of three broad c a t e g o r i e s : a) s i n g l e , unsegmented genome, b) segmented • genome w i t h a l l segments contained i n one p a r t i c l e type, and c) segmented genome with segments d i s t r i b u t e d among d i f f e r e n t p a r t i c l e types. A m a j o r i t y of both DNA and RNA v i r u s e s of animals, p l a n t s and b a c t e r i a belong to the f i r s t category. Examples are adeno- and paramyxoviruses of animals (Fenner et al., 1974), caulimo-, tobamo-and luteoviriiBes of p l a n t s (Tremaine, 1977) and phages lambda, T4 and phi-6 of b a c t e r i a (Casjens and King, 1975). Viruses belonging to the second category i n c l u d e the double-stranded RNA animal and pla n t r e o v i r u s e s (Fenner et a l . , 1974; Casjens and King, 1975) and the sin g l e - s t r a n d e d RNA orthomyxoviruses of animals (Casjens and King, 1975). In the case of the orthomyxovirus, i n f l u e n z a A, the a n a l y s i s of the genetic recombination 'rescues' of mutants d e f e c t i v e i n d i f f e r e n t c a p a c i t i e s and genome segments has succeeded i n e s t a b l i s h i n g a complete genetic map (Ritchey ej: a l . , 1976). Almost a l l v i r u s e s of the t h i r d category are pla n t v i r u s e s (Casjens and King, 1975). Among v i r u s e s of t h i s t h i r d category, new i s o l a t e s can be generated by a r t i f i c i a l l y combining complementary genome segments of r e l a t e d i s o l a t e s . I f the separate components are by themselves u n i n f e c t i o u s , but y i e l d a new productive i n f e c t i o n upon heterologous combination, the r e s u l t a n t new i s o l a t e s are termed 'pseudorecombinant' (Gibbs and H a r r i s o n , 1974). 'Pseudorecombination' w i t h v i r a l genome segments i s d i s t i n g u i s h e d from 'recombination' (as occurs w i t h i n f l u e n z a v i r u s ) by the f a c t that the d i f f e r e n t genome segments do not come together i n one p a r t i c l e but are encapsidated i n d i f f e r e n t com-ponent p a r t i c l e s . D e l i b e r a t e pseudorecombination, l i k e recombination w i t h i n f l u e n z a v i r u s , can be employed as a u s e f u l t o o l f o r genetic a n a l y s i s i f the f o l l o w i n g c r i t e r i a are met: a) the v i r a l genomic components can be separated to the extent that each component on i t s own i s not, or only s l i g h t l y , i n f e c t i o u s but combined w i t h the complementary components i s h i g h l y i n f e c t i o u s ; the q u a n t i t a t i v e measure of success i s termed the 'enhancement-of-infectivity' r a t i o , u s u a l l y defined as the number of l o c a l l e s i o n s induced by a d e l i b e r a t e mixture of. the separated components d i v i d e d by the average number of l o c a l l e s i o n s (at equal p a r t i c l e c oncentration on equal l e a f area) induced by each of the separated components alone (Bruening, 1977). Higher r a t i o s i n d i c a t e s u p e r i o r s e p a r a t i o n of components, b) there are r e -l a t e d v i r u s e s or s t r a i n s that are d i s t i n c t from one another i n a number of observable p r o p e r t i e s , and c) the new i n f e c t i o n r e s u l t i n g from pseudorecombination i s s u f f i c i e n t l y productive that p r o p e r t i e s of the pseudorecombinants can be observed and compared w i t h those of the c o n t r i b u t i n g ' p a r e n t a l ' i s o l a t e s . Several v i r u s groups (and as yet ungrouped or monotypic v i r u s e s ) appear on the b a s i s of t h e i r d i v i d e d genomes to be candidates f o r pseudorecombination. A l l p l a n t v i r u s e s i n t h i s category are s i n g l e -stranded RNA v i r u s e s of h e l i c a l , i s o m e t r i c or b a c i i l i f o r m geometry (Tremaine, 1977). In what f o l l o w s , the e s s e n t i a l f i n d i n g s of pseudore-combination s t u d i e s w i t h p l a n t v i r u s e s that possess two s e p a r a t e l y encapsidated RNA species whose combined a c t i v i t y i s r e q u i r e d f o r f u l l i n f e c t i v i t y ( ' f u n c t i o n a l l y b i p a r t i t e ' ) , w i l l be b r i e f l y reviewed. Tobraviruses are h e l i c a l p l a n t v i r u s e s w i t h b i p a r t i t e genomes and are t r a n s m i t t e d by t r i c h o d o r i d nematodes. They can be seen as counter-parts of the i s o m e t r i c nepoviruses, which are tran s m i t t e d by l o n g i d o r i d nematodes (Harrison et a l . , 1971; Shepherd et a l . , 1976) . Tobraviruses were among the f i r s t v i r u s e s to be st u d i e d by pseudorecombination a n a l y s i s . A l l s t u d i e s to date have been performed w i t h the type number, tobacco r a t t l e v i r u s (TRV), and s t r o n g l y i n d i c a t e that the coat p r o t e i n i s determined by the smaller of the two RNA species (Ghabrial and L i s t e r , 1973). Symptomatology appears to be determined by e i t h e r or both RNAs, depending on the p a r t i c u l a r host species (Saenger, 1968; 1969; G h a b r i a l and L i s t e r , 1973). A l l other p l a n t v i r u s e s w i t h d i v i d e d genomes c o n s i s t i n g of two separately encapsidated RNA species are of i s o m e t r i c geometry. The only members i n t h i s category are the monotypic c a r n a t i o n r i n g s p o t v i r u s (CRSV) and the como- arid nepovirus groups (Tremaine, 1977). The genome of CRSV c o n s i s t s of two RNA species of 0.5 and 1.5 m i l l i o n daltons molecular weight. The b i p a r t i t e nature of the genome i s only apparent upon a n a l y s i s of the e x t r a c t e d RNA since the genome i s packaged i n p a r t i c l e s which sediment as a s i n g l e n u c l e o p r o t e i n component (Dodds e t a l . , 1977). From pseudorecombinant genetic a n a l y s i s i t appears that the l a r g e r RNA determines the coat p r o t e i n (Dodds et a l . , 1977). Besides pea enation mosaic v i r u s (PEMV), whose genome i s f u n c t i o n a l l y b i p a r t i t e but contains a t h i r d RNA species which appears not to i n f l u e n c e i n f e c t i v i t y and i s smaller than e i t h e r of the two f u n c t i o n a l RNA species ( H u l l and Lane, 1973), CRSV i s the only other divided-genome plant v i r u s i n which the coat p r o t e i n i s known to be determined by the l a r g e s t f u n c t i o n a l RNA species. 4 As a group, the comoviruses have been favored objects of study by p l a n t v i r o l o g i s t s (Bruening, 1977) and the type member, the y e l l o w i n g s t r a i n of cowpea mosaic v i r u s (GPMV-Sb), was one of the f i r s t s ubjects of pseudorecombinant genetic a n a l y s i s (Bruening, 1969). Since each comovirus capsid contains 60 sub'units of each of two polypeptide species (Wu and Bruening, 1971), the d i s t r i b u t i o n of the genetic determinants between the two (middle and bottom) n u c l e o p r o t e i n component RNAs i s of i n t e r e s t . A l l pseudorecombinant analyses performed to date i n d i c a t e a n t i g e n i c determination of the capsid p r o t e i n by the s m a l l e r , middle component RNA (Kassanis et a l . , 1973; Moore and S c o t t , 1971; Thongmeearkom and Goodman, 1978). Recently, Gopo and F r i s t (1977) have shown the smaller polypeptide i t s e l f to be determined by the smaller RNA. The genetics of symptom determination among comoviruses appear to vary w i t h the host species and the o r i g i n of the v a r i a n t s t r a i n (de Jager and van Kammen, 1970; Wood, 1972; de Jager, 1976; Thongmeearkom and Goodman, 1978; O x e l f e l t and Abdelmoeti, 1978). Nepoviruses, l i k e comoviruses, have a b i p a r t i t e genome d i s t r i b u t e d between two d i f f e r e n t i a l l y sedimenting c l a s s e s of constant-diameter p a r t i c l e s , but they d i f f e r from comoviruses i n c e r t a i n p h y s i c a l and b i o l o g i c a l p r o p e r t i e s . Nepoviruses possess only one c o a t - p r o t e i n polypeptide species (with the p o s s i b l e exception of a few members whose sta t u s as nepoviruses i s s t i l l a matter of debate). They are t r a n s -mitted by the nematode genera Longidorus and Xiphinema and tend to have wide host ranges i n which they cause ringspot and mottle symptoms, ofte n followed by symptomless i n f e c t i o n (Harrison and Murant, 1977a). A l l nepoviruses (Shepherd et a l . , 1976; H a r r i s o n and Murant, 1977a) appear to have two main'RNA s p e c i e s , the heavier one (RNA-1) clo s e to 5 2.4 m i l l i o n d a l t o n s i n a l l members and making up about 42% by w e i g h t of the b o t t o m component n u c l e o p r o t e i n , and the l i g h t e r one (RNA-2) v a r y i n g w i t h the p a r t i c u l a r member of t h e group from 1.3 t o 2.3 m i l l i o n d a l t o n s and thus r a n g i n g from about 27-40% by w e i g h t of t h e m i d d l e component n u c l e o p r o t e i n . Much s m a l l e r s a t e l l i t e RNAs (RNA-3) i n a d d i t i o n t o t h e two l a r g e r s p e c i e s have been r e p o r t e d i n t h r e e n e p o v i r u s e s : t o b a c c o r i n g s p o t v i r u s (TRSV) ( S c h n e i d e r e t a l . , 1972), tomato b l a c k r i n g v i r u s (TBRV) (Murant and Mayo, 1976) and m y r o b a l a n l a t e n t r i n g s p o t v i r u s (MLRV) (Dunez et^ a l _ . , 1976) . R e c e n t l y , i t has been shown t h a t i n a t l e a s t t w o . n e p o v i r u s e s , TRSV and TomRSV, a s m a l l p r o t e i n c o v a l e n t l y l i n k e d ( p o s s i b l y a t t h e 5'-end) t o b o t h main RNA s p e c i e s i s r e q u i r e d f o r i n f e c t i v i t y ( H a r r i s o n and B a r k e r , 1978). U n t i l r e c e n t l y , the n e p o v i r u s c a p s i d was d e s c r i b e d as b e i n g a T = l i c o s o c h e d r a l s t r u c t u r e w i t h 60 p o l y p e p t i d e s u b u n i t s , t h e p o l y p e p t i d e s u b u n i t h a v i n g a m o l e c u l a r w e i g h t o f about 55,000 d a l t o n s ( H a r r i s o n and M u r a n t , 1977a). Some v i r u s e s , s u c h as s t r a w b e r r y l a t e n t r i n g s p o t v i r u s (SLRV), have, i n most r e s p e c t s , p r o p e r t i e s s i m i l a r t o n e p o v i r u s e s , b u t t h e i r c a p s i d s c o n s i s t , l i k e t h o s e o f c o m o v i r u s e s , o f two p o l y p e p t i d e s w i t h a p p a r e n t m o l e c u l a r w e i g h t s of 29,000 and 44,000 d a l t o n s (Mayo et^ a l . , 1974). V e r y r e c e n t a n a l y s e s of the c o a t p r o t e i n of TRSV by Chu and F r a n c k i ( 1 9 7 9 ) , u s i n g S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p r e c e e d e d by d i s s o c i a t i o n i n t h e p r e s e n c e o f SDS, u r e a and 2-mercaptoe-t h a n o l , and t r y p t i c p e p t i d e mapping, have p r o v i d e d s t r o n g e v i d e n c e t h a t the m o l e c u l a r w e i g h t o f t h e p o l y p e p t i d e s u b u n i t o f n e p o v i r u s c a p s i d p r o t e i n s i s about 13,000 d a l t o n s , o n l y o n e - f o u r t h t h e s i z e p r e v i o u s l y d e s c r i b e d . I n the l i g h t o f t h i s i n f o r m a t i o n , a r e a s s e s s m e n t o f s e e m i n g l y t w o - p o l y p e p t i d e v i r u s e s , t e n t a t i v e l y a s s i g n e d t o t h e n e p o v i r u s group, 6 such as SLRV and cherry rasp l e a f v i r u s (CRLV), may r e v e a l that they share w i t h the d e f i n i t i v e nepoviruses a b a s i c 13,000 dal t o n polypeptide subunit. Within the nepovirus group, subgroups based on s e r o l o g i c a l r e l a t i o n s h i p s and the apparent molecular weight of the l i g h t e r RNA-2, have been defined ( Q u a c q u a r e l l i e_t _ a l . , 1976) . The type and E n g l i s h s t r a i n s of raspberry ringspot v i r u s (RRV) form a c l u s t e r of r e l a t e d serotypes as do potato b l a c k r i n g v i r u s (PBRV) and the type and eucharis mottle s t r a i n s of TRSV; the a r a b i s mosaic v i r u s (AraMV) c l u s t e r comprises grapevine f a n l e a f v i r u s (GFV) and AraMV. The RNA-2 of the v i r u s e s of these three serotype c l u s t e r s ranges i n molecular weight from 1.4-1.5 m i l l i o n d a l t o n s . In a d d i t i o n , a p o r t i o n of the nucleoproteins s e d i -menting i n the bottom component c o n s i s t s of p a r t i c l e s which possess two molecules of RNA-2 w h i l e the r e s t c o n t a i n one molecule each of RNA-1. Among nepoviruses that have an RNA-2 of 1.5 - 1.6 m i l l i o n daltons w i t h only one RNA molecule per p a r t i c l e [ p o s s i b l e exception, MLRV (Debos et a l . , 1976)], there are two serotype c l u s t e r s . The TBRV subgroup comprises cocoa n e c r o s i s v i r u s (CNV), grapevine chrome mosaic v i r u s (GCMV), MLRV, and the potato bouquet and beet ringspot s t r a i n s of TBRV, wh i l e a r t i c h o k e I t a l i a n l a t e n t v i r u s (AILV) appears so f a r to have no s e r o l o g i c a l l y c l o s e r e l a t i v e s . F i n a l l y , among the d e f i n i t i v e nepoviruses, tomato ringspot v i r u s (TomRSV), peach r o s e t t e mosaic v i r u s (PRMV) and cherry l e a f r o l l v i r u s (CLRV) have an RNA-2 only s l i g h t l y smaller than RNA-1, a l l o w i n g only one molecule of RNA per p a r t i c l e . The three v i r u s e s are not c l o s e s e r o l o g i c a l r e l a t i v e s but a number of s e r o l o g i c a l l y c l o s e l y r e l a t e d s t r a i n s have been described f o r CLRV (Walkey et^ a l . , 1973) . 7 The f i r s t evidence f o r a f u n c t i o n a l l y b i p a r t i t e genome i n a nepovirus was obtained by Bancroft (1968) who demonstrated a 10 - f o l d enhancement-of-infectivity when middle and bottom n u c l e o p r o t e i n com-ponents of tomato top n e c r o s i s v i r u s (TomTNV), p r e v i o u s l y separated by sucrose gradient c e n t r i f u g a t i o n , were combined i n an inoculum. Jones and Mayo (1972), using s i m i l a r methods w i t h repeated c y c l e s , obtained an enhancement^of-infectivity r a t i o of about 7 w i t h CLRV. Schneider et_ al_. (.1974), working w i t h TomRSV, a l s o employed sucrose d e n s i t y - g r a d i e n t separation of middle and bottom component nucleoproteins and obtained an enhancement-of-infectivity r a t i o of about 4. The r e l a t i v e l y poor r a t i o s obtained were a t t r i b u t e d to poor separations of the two component nu c l e o p r o t e i n s . The middle component nucleo p r o t e i n s had sedimentation c o e f f i c i e n t s only s l i g h t l y lower than those of the bottom component [102S, 115S and 119S r e s p e c t i v e l y f o r TomTNV, CLRV and TomRSV compared to about 127S f o r each bottom component (Harrison and Murant, 1977a)] and sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n does not r e a d i l y r e s o l v e such c l o s e l y sedimenting components. With nepoviruses such as TRSV and RRV, the problem i n o b t a i n i n g high enhancement-of-infectivity r a t i o s i s not mainly due to poor r e -s o l u t i o n of middle and bottom n u c l e o p r o t e i n components but r a t h e r due to the presence of a f u l l genome complement i n bottom component p a r t i c l e s ; some bottom-component p a r t i c l e s , contain.one RNA-1 and others two RNA-2 molecules (Diener and Schneider, 1966; Murant j 2 t a l . , 1972). Murant et a l . (1972), solved t h i s problem by. using RNA components as inoculum. The RNA preparations were heated i n 8M urea to promote denaturation and were w e l l r e s o l v e d by subsequent polyacrylamide g e l e l e c t r o p h o r e s i s . Enhancement-of-infectivity r a t i o s of about 80 8 were obtained w i t h RRV RNA components separated i n t h i s way. The f i r s t s u c c e s s f u l pseudorecombinations w i t h nepoviruses were achieved by H a r r i s o n et a l . (1972) using the s e r o l o g i c a l l y r e l a t e d but d i s t i n c t E n g l i s h and S c o t t i s h s t r a i n s of RRV. The two s t r a i n s a l s o d i f f e r e d i n the systemic symptoms they induced i n Petunia hybrida Vilm. Pseudorecombinants were made from g e l e l e c t r o p h o r e s i s - s e p a r a t e d RNA-1 and -2 using the methods of Murant et^ a l . (1972). Both s e r o l o g i c a l r e l a t i o n s h i p s and systemic symptom expression were i n h e r i t e d through RNA-2. In a l a t e r study (Harrison et a l . , 1974a) the genetics of transmission of the S c o t t i s h s t r a i n by the nematode, Longidorus elongatus deMan were examined. RNA-2 was a l s o found to determine t h i s property. Taken together w i t h the evidence f o r determination of s e r o l o g i c a l pro-p e r t i e s by RNA-2 (Harrison et a l . , 1972), i t c l e a r l y appeared that the smaller RNA contains (as w i t h t o b r a v i r u s e s , the other s p e c i f i c a l l y nematode-transmitted p l a n t v i r u s group) the genetic i n f o r m a t i o n f o r the coat p r o t e i n . This c o n c l u s i o n was confirmed by a more extensive study (Harrison et a l . , 1974a) where pseudorecombinants were generated among four s e r o l o g i c a l l y r e l a t e d s t r a i n s : the E n g l i s h and S c o t t i s h s t r a i n s of e a r l i e r work and the Ll o y d George s t r a i n (capable of i n f e c t i n g the Ll o y d George v a r i e t y of raspberry) and the Dutch s t r a i n , s e r o l o g i c a l l y i d e n t i c a l w i t h the S c o t t i s h s t r a i n but inducing, as the E n g l i s h s t r a i n does, systemic y e l l o w i n g i n P_. hy b r i d a . Assignments of p r o p e r t i e s to pseudorecombinant RNA species were confirmed by backcrossing to generate the p r e d i c t e d p a r e n t a l phenotypes. In a d d i t i o n , RNA-1 was found to determine the a b i l i t y to i n f e c t systemic leaves of Phaseolus v u l g a r i s L. The determination of systemic y e l l o w i n g symptoms i n J?. h y b r i d a , 9 i n d i c a t e d i n e a r l i e r work (Harrison et al_. , 1972) to be c o n t r o l l e d by RNA-2, appeared more complex and was shown to be ass o c i a t e d w i t h u l t r a s t r u c t u r a l changes i n the c h l o r o p l a s t s . The RNA-2 of the E n g l i s h s t r a i n , shown p r e v i o u s l y to determine these symptoms, appeared to be suppressed i n i t s expression of t h i s property when i t was ass o c i a t e d w i t h the RNA-1 of the Lloyd George s t r a i n . Recently, Hanada and Har r i s o n (1977), using s i m i l a r m a t e r i a l s and methods as before (Harrison et a l . , 1972; 1974b), have shown that seed t r a n s m i s s i b i l i t y i n chickweed ( S t e l l a r i a media L.) of RRV and TBRV de-pends g r e a t l y on the t r a n s m i s s i b i l i t y of the s t r a i n c o n t r i b u t i n g the RNA-1. RNA-2, however, a l s o exerts a measurable but smaller i n f l u e n c e , the extent of which depends on the RNA-1 w i t h which i t i s combined. This has been i n t e r p r e t e d as suggesting t h a t , i n n a t u r a l l y - o c c u r i n g s t r a i n s , a s e l e c t i o n f o r c o m p a t i b i l i t y of RNA-1 and -2 w i t h respect to seed transmission frequency has taken p l a c e . A s i m i l a r l i n k i n g of expression of RNA-2 to the type of RNA-1 w i t h which i t a s s o c i a t e s has been observed i n a study of the competetiveness i n a mixed i n f e c t i o n of d i f f e r e n t RRV genotypes (Harrison and Hanada, 1976). D i f f e r e n t pseudorecombinants of RRV w i t h one RNA i n common were compared i n mixed i n f e c t i o n s as to the r a p i d i t y w i t h which they system-i c a l l y i n f e c t e d Chenopodium quinoa W i l l d . p l a n t s . This property, the a b i l i t y to 'dominate', was r e l a t e d to the r a p i d i t y w i t h which systemic symptoms were induced i n p l a n t s s i n g l y i n f e c t e d by each of the pseudore-combinant types. RNA-1 was found to be the major determining f a c t o r , though to the extent that RNA-1 'allowed' the expression of d i f f e r e n c e s i n competetiveness between RNA-2 from d i f f e r e n t s t r a i n s , RNA-2 exerted a 10 minor i n f l u e n c e on the competitiveness of d i f f e r e n t pseudorecombinant types. Though i n d i r e c t , t h i s evidence supports the n o t i o n gained from s t u d i e s by Hanada and H a r r i s o n (1977), that RNA-1 p r i n c i p a l l y determines the r a t e of seed t r a n s m i s s i o n , s i n c e a more r a p i d l y s y s t e m i c a l l y invading v i r u s would seem to have a b e t t e r chance of i n f e c t i n g the seed forming t i s s u e s at an e a r l i e r stage. The only other nepovirus, f o r which pseudorecombination genetic analyses have been reported to any extent, i s TBRV. I t s RNA-2, at 1.5 m i l l i o n d a l t o n s , l i k e that of RRV, i s considerably smaller than i t s 2.5 m i l l i o n d a l t o n RNA-1. In c o n t r a s t to RRV, however, TBRV bottom component p a r t i c l e s contain only one molecule each of RNA-1 (Murant et a l . , 1973; Randies e^ t a l . , 1977). TBRV was f i r s t shown to have a f u n c t i o n a l l y b i p a r t i t e genome by Murant et a l . (1973), who found three RNA species of which only the l a r g e r two c o n t r i b u t e d to i n f e c t i v i t y . Separated' RNA-2 was only s l i g h t l y i n f e c t i o u s , RNA-1 moderately i n f e c t i o u s . Combinations of d i l u t e component i n o c u l a gave enhancement-of-infectivity r a t i o s of over 30. The smallest RNA component, (RNA-3), of 0.5 m i l l i o n daltons apparent molecular weight, was n e i t h e r i n t r i n s i c a l l y i n f e c t i v e nor able to c o n t r i b u t e to the i n f e c t i v i t y of any combination of component RNAs. Instead, i t appeared to i n h i b i t l e s i o n formation when added to f u l l y i n f e c t i o u s mixtures of RNA-1 and -2. This l e s i o n -i n h i b i t i n g a b i l i t y disappeared when the RNA-3 was i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t . I f not included i n the inoculum, i t f a i l e d to be subsequently detected i n the progeny v i r u s , i n d i c a t i n g i t s ' s a t e l l i t e ' nature. Randies et_ a l . (1977) used both nucle o p r o t e i n s separated by sucrose 11 d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n and RNAs separated by polyacrylamide g e l e l e c t r o p h o r e s i s to generate a pseudorecombinant from s e r o l o g i c a l l y d i s t i n c t , s a t e l l i t e - f r e e i s o l a t e s derived from the S c o t t i s h beet ringspot s t r a i n (A) and the German potato bouquet s t r a i n (G-12) of TBRV. As a p r e l i m i n a r y step, they demonstrated that i n both i s o l a t e s middle component (M) nucleoproteins contained only RNA-2, w h i l e bottom component (B) nucleoproteins c a r r i e d only RNA-1. The combination of M n u c l e o p r o t e i n or RNA-2 of TBRV-G12 w i t h r e s p e c t i v e l y B n u c l e o p r o t e i n or RNA-1 of TBRV-A f a i l e d to enhance i n f e c t i v i t y over the a d d i t i v e i n f e c t i v i t y of the separated components, i n d i c a t i n g poor c o m p a t i b i l i t y . The reverse combination, however, of M n u c l e o p r o t e i n or RNA-2 of TBRV-A w i t h r e s p e c t i v e l y B n u c l e o p r o t e i n or RNA-1 of TBRV-G12 showed a l e v e l of i n f e c t i v i t y enhancement about 12-25% of that observed i n homologous component combinations. The pseudorecombinant, which was much l e s s i n f e c t i o u s than e i t h e r parent and which induced smaller l o c a l l e s i o n s on C_. quinoa, was s e r o l o g i c a l l y i d e n t i c a l w i t h the TBRV-A i s o l a t e that had co n t r i b u t e d i t s RNA-2. This r e s u l t , i n d i c a t i n g coat p r o t e i n determination by the smaller RNA, i s i n agreement w i t h that obtained i n pseudorecombination genetic analyses of RRV (Harrison et a l . , 1972; 1974b). H a r r i s o n and Murant (1977b) used the same i s o l a t e s to i n v e s t i g a t e the genetic determination of nematode t r a n s m i s s i b i l i t y of TBRV. TBRV-A i s s p e c i f i c a l l y t r a n s m i t t e d , as i s the S c o t t i s h beet r i n g s p o t s t r a i n from which i t i s derived by L. elongatus, while TBRV-G12, l i k e i t s o r i g i -n a l German potato bouquet source, i s tran s m i t t e d by L_. attenuatus Hooper. As i n the other study (Randies et a l . , 1977), only one of the two pseudorecombinations between these two parent i s o l a t e s was achieved. 12 S p e c i f i c nematode-transmissability was determined by RNA-2, although the pseudorecombinant was transmitted l e s s f r e q u e n t l y by L_. elongatus than the TBRV-A parent which had co n t r i b u t e d the RNA-2. Lesion type i n C. quinoa was a l s o determined by RNA-2, though the pseudorecombinant-induced l e s i o n s took longer to appear than those induced by e i t h e r parent. This was i n t e r p r e t e d as i n d i c a t i n g that the property of speed of l e s i o n appearance r e q u i r e s determinants i n both RNAs. Hanada and H a r r i s o n (1977) used the same parent and pseudore-combinant i s o l a t e s to study seed transmission of TBRV i n chickweed ( S t e l l a r i a media L . ) . As w i t h RRV (Hanada and H a r r i s o n , 1977), seed transmission appeared to be mainly determined by RNA-1. The p o s s i b l e secondary r o l e of RNA-2 (as w i t h RRV) i n seed transmission could not be determined, given the i n a b i l i t y to generate the r e c i p r o c a l pseudore-combinant. In another s e r i e s of experiments i n the same study, RNA-2 rath e r than RNA-1 appeared to i n f l u e n c e seed tra n s m i s s i o n i n Xanthi-nc. tobacco but not i n Samsun NN tobacco. In the absence of the r e c i p r o c a l pseudorecombinant, however, these data are only suggestive. Using the o r i g i n a l S c o t t i s h beet ringspot (S) and German potato bouquet (G) s t r a i n s which harbor a s a t e l l i t e RNA-3 f o r comparison, i t was found (Hanada and H a r r i s o n , 1977) that RNA-3 was recovered upon seed tra n s m i s s i o n but d i d not appear i n any way to a f f e c t the frequency of seed tra n s m i s s i o n i n S^  media. This i s i n c o n t r a s t to RNA-3's e f f e c t on l o c a l - l e s i o n formation; Murant e_t a l . (1973) had p r e v i o u s l y observed that a d d i t i o n of RNA-3 to an i n f e c t i o u s combination of the two l a r g e r RNAs i n h i b i t e d l o c a l - l e s i o n formation. Of the nepoviruses that f a l l i n t o the category of having an RNA-2 only s l i g h t l y smaller than the RNA-1, such as peach r o s e t t e mosaic 13 v i r u s (PRMV) and tomato ri n g s p o t v i r u s (TomRSV), the most promising candidate f o r pseudorecombinant genetic a n a l y s i s appears to be cherry l e a f r o l l v i r u s (CLRV). A number of d i s t i n c t , w e l l - c h a r a c t e r i z e d , s e r o l o g i c a l l y c l o s e l y - r e l a t e d " s t r a i n s are known (Walkey et a l . , 1973). In a d d i t i o n , a number of s u c c e s s f u l p u r i f i c a t i o n p r o t o c o l s have been worked out. While the small d i f f e r e n c e i n sedimentation c o e f f i c i e n t s between the bottom component (127S) and middle component (115S) nu c l e o p r o t e i n does not a l l o w e f f i c i e n t s e paration of the two components by sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n , there i s at l e a s t no concern that the bottom component nucleoprotein. might cont a i n two molecules of RNA-2. Moreover, even w i t h t h i s d i f f i c u l t y , enhancement of i n f e c t i v i t y of combined compared to separated nucleoproteins has been demonstrated (Jones and Mayo, 1972). The a p p l i c a t i o n of techniques f o r b e t t e r separation of the n u c l e o p r o t e i n or RNA could be expected to open the way to s u c e s s f u l pseudorecombination genetic analyses s i n c e the c l o s e l y - r e l a t e d , n a t u r a l l y - o c c u r r i n g s t r a i n s w i t h r e p r o d u c i b l e , d i s t i n c t genetic markers are at hand. Cherry l e a f r o l l v i r u s was f i r s t described by Cropley (1961) , though i t had been r e p o r t e d j e a r l i e r (Posnette and Cropley, 1955; Posnette, 1956) as a g r a f t - t r a n s m i s s i b l e agent causing severe l e a f r o l l i n g i n cherry t r e e s . Cropley (1960), upon t r a n s f e r r i n g i t to N i c o t i a n a and Chenopodium s p e c i e s , found symptom types s i m i l a r to those observed w i t h the s o i l - b o r n e v i r u s e s , a r a b i s mosaic, raspberry r i n g s p o t and tomato black r i n g . The r e a c t i o n s i n some hosts, however, d i d d i s t i n g u i s h cherry l e a f r o l l from these three v i r u s e s (Cadman et a l . , 1960). The l o c a l i z e d d i s t r i b u t i o n of the disease i n orchards suggested 14 s o i l - b o r n e t r a n s m i s s i o n . Attempts to transmit the disease to herbaceous hosts by growing them i n s o i l taken i n s o i l taken from root zones of i n f e c t e d t r e e s , however, were not s u c c e s s f u l (Cropley, 1960). V i r u s p u r i f i e d from tobacco sap appeared, when f i x e d i n f o r m a l i n , as i s o m e t r i c p a r t i c l e s w i t h an average diameter of 32 nm, c a s t i n g shadows s i m i l a r to those of a r a b i s mosaic, raspberry r i n g s p o t and tobacco b l a c k r i n g v i r u s e s (Harrison and Nixon, 1960). The p h y s i c a l p r o p e r t i e s of the v i r u s and i t s symptoms i n i n f e c t e d p l a n t s suggested a s i m i l a r i t y between cherry l e a f r o l l v i r u s and these three v i r u s e s , so s e r o l o g i c a l and c r o s s - p r o t e c t i o n r e l a t i o n s h i p s were examined but none were detected (Cropley, 1960). Jones and Murant (1971), using g e l - d i f f u s i o n s e r o l o g i c a l t e s t s showed that the 'type' cherry l e a f r o l l v i r u s i s o l a t e d and described by Cropley (1960) was s e r o l o g i c a l l y r e l a t e d to 'elm mosaic v i r u s ' (Varney and Moore, 1952), 'golden e l d e r b e r r y v i r u s ' (Hansen, 1967) as w e l l as a rhubarb i s o l a t e of cherry l e a f r o l l v i r u s (Tomlinson and Walkey, 1967). Since the evidence i n d i c a t e d close r e l a t i o n s among the four i s o l a t e s and the Cropley i s o l a t e (1960) had been the most s t u d i e d , i t was suggested that the elm, golden e l d e r b e r r y and rhubarb i s o l a t e s should be considered s t r a i n s of cherry l e a f r o l l v i r u s (CLRV) (Jones and Murant, 1971). Waterworth and Lawson (1973) i s o l a t e d a v i r u s from dogwood (Cornus f l o r i d a L.) which, although i t appeared c o n s i s t e n t l y smaller than the Cropley type s t r a i n of CLRV (25 nm diameter compared to 28 nm), was found to be s e r o l o g i c a l l y r e l a t e d to CLRV type and e l d e r b e r r y s t r a i n s , and d i s t a n t l y r e l a t e d to the elm s t r a i n . Walkey e_t al. (1973) examined the comparative p h y s i c a l and b i o l o g i c a l p r o p e r t i e s o f - t h e s e f i v e s t r a i n s of CLRV. A l l s t r a i n s had 15 coat p r o t e i n w i t h molecular weights of about 55,000 d a l t o n s , s i m i l a r l y sedimenting components of 52S, 114S and 132S and middle and bottom component n u c l e o p r o t e i n RNAs of 2.1 and 2.4 m i l l i o n daltons respec-t i v e l y , but s e r o l o g i c a l data revealed d i f f e r e n t degrees of 'relatedness among the f i v e s t r a i n s . The dogwood and elm s t r a i n s showed a clo s e s e r o l o g i c a l r e l a t i o n s h i p , the cherry s t r a i n was more c l o s e l y r e l a t e d to the dogwood than the elm s t r a i n w h i l e the rhubarb and e l d e r b e r r y s t r a i n s were more c l o s e l y r e l a t e d to each other than to the other three s t r a i n s . Since pseudorecombinants are g e n e r a l l y e a s i e r to prepare from c l o s e l y r a t h e r than d i s t a n t l y r e l a t e d s t r a i n s (Bruening, 1977), the dogwood-elm or elderberry-rhubarb s t r a i n p a i r s would seem to be the best s t a r t i n g m a t e r i a l s f o r pseudorecombination s t u d i e s . The elm s t r a i n , however, has been reported as being more d i f f i c u l t to p u r i f y than the other s t r a i n s ( F u l t on and F u l t o n , 1970). The p r o p e r t i e s most a c c e s s i b l e to examination i n any such pseudo-recombination genetic a n a l y s i s would be s e r o l o g i c a l r e l a t i o n s h i p s and symptomatology i n a range of d i f f e r e n t i a l hosts. The r e s u l t s would be examined to see how they conform to the p a t t e r n e s t a b l i s h e d i n previous genetic analyses w i t h RRV (Harrison e_t aj.. , 1972; 1974a; H a r r i s o n and Hanada, 1976; Hanada and H a r r i s o n , 1977) and TBRV (Randies et a l . , 1977; Hanada and H a r r i s o n , 1977). In a b r i e f report of p r e l i m i n a r y work, Jones (1977) s t a t e d that pseudorecombinants had been made from the e l d e r b e r r y and rhubarb s t r a i n s of CLRV. S e r o l o g i c a l p r o p e r t i e s were i n h e r i t e d through the middle component, w h i l e the a b i l i t y to induce systemic symptoms i n Gomphrena  globosa L. appeared to be~conferred by the bottom component of the rhubarb s t r a i n . 16 MATERIALS AND METHODS Viruses The dogwood (D), cherry (C), e l d e r b e r r y (E) and rhubarb (R) s t r a i n s of CLRV, i d e n t i c a l to those described by Walkey j 2 l t al. (1973) , were obtained i n the form of sealed f r e e z e - d r i e d l e a f samples from Dr. R. Stace-Smith. Apart from i n i t i a l comparisons of symptoms done wi t h a l l four s t r a i n s , most experiments employed the e l d e r b e r r y (E) and rhubarb (R) s t r a i n s , which were maintained by s e r i a l t r a n s f e r s i n N i c o t i a n a c l e v e l a n d i i Gray, host p l a n t s . Host p l a n t s I n o c u l a t i o n s were c a r r i e d out by g r i n d i n g leaves from a source plant ( u s u a l l y IJ. c l e v e l a n d i i ) i n f e c t e d w i t h a given s t r a i n of CLRV i n 0.01M potassium phosphate, pH 7.1 b u f f e r and rubbing the b u f f e r e d sap onto host p l a n t s that had been dusted w i t h carborundum. The f o l l o w i n g host species and c u l t i v a r s were examined f o r t h e i r a b i l i t y to serve as d i f f e r e n t i a l hosts of the four s t r a i n s : N i c o t i a n a c l e v e l a n d i i Gray, Jfl. s y l v e s t r i s L., N. g l u t i n o s a L., N. tabacum L. cvs. Samsun and X a n t h i , Chenopodium amaranticolor Coste & Reyn, €. quinoa W i l l d . , Capsicum  annum L., Phaseolus v u l g a r i s L. cvs. B o u n t i f u l and P i n t o , Vigna  unguiculata E r d l . , Datura stramonium L. and Z i n n i a elegans L. Most l a t e r experiments, i n v o l v i n g only the E- and R - s t r a i n s and the pseudorecombinant i s o l a t e s generated from them, employed the f o l l o w i n g range of d i f f e r e n -t i a l hosts: N. c l e v e l a n d i i , N. tabacum cvs. Samsun and X a n t h i , CJ. amaranticolor, C_. quinoa and Gomphrena globosa L. 1/7 P u r i f i c a t i o n methods The methods of Mayo and Jones (1972) and Walkey et. a l . (1973) were compared. In a d d i t i o n , the f o l l o w i n g method was developed and was the procedure used i n most experiments: A l l procedures were c a r r i e d out at about 4 C. Fresh s y s t e m i c a l l y -i n f e c t e d l e a f t i s s u e of N. c l e v e l a n d i i (harvested 10-14 days a f t e r i n o c u l a t i o n ) or l e a f and stem t i s s u e of C_. quinoa (harvested 6-10 days a f t e r i n o c u l a t i o n ) was homogenized i n two volumes per weight of t i s s u e of c o l d (4C) 0.25M (0.5M f o r C_. quinoa) potassium phosphate pH 6.8 c o n t a i n i n g 0.2% mercaptoethanol and 0.1% sodium d i e t h y l d i t h i o c a r b a m a t e . . The s l u r r y was s t r a i n e d through cheese-cloth, and chloroform and butanol (1:1 v/v) was added to the sap to a concentration of 6% ( v / v ) . The mixture was s t i r r e d f o r about one hour and the e m u l s i f i e d green sap then c e n t r i f u g e d at 8,000 rpm f o r 5 min. ( S o r v a l l GSA r o t o r ) . An amber to yellow supernatant was c o l l e c t e d to which 8% w/v polyethylene g l y c o l (PEG) 6000 ( F i s h e r Carbowax) was added and allowed to f u l l y d i s s o l v e by s t i r r i n g f o r about two hours. A f t e r c e n t r i f u g i n g at 8,000 rpm f o r 20 min. at 4 C ( S o r v a l l GSA r o t o r ) the w h i t i s h p e l l e t was resuspended i n 0.02M sodium phosphate pH 7.0 and allowed to stand f o r at l e a s t one hour, before c e n t r i f u g i n g at 10,000 rpm f o r 10 min ( S o r v a l l SS-34 r o t o r ) to remove remaining crude f l o c c u l e n t host m a t e r i a l . This c l a r i f i e d concentrated sap was then c e n t r i f u g e d at high speed, depending on volume, i n e i t h e r a Beckman No. 30 r o t o r at 27,000 rpm or a Beckman No. 65 r o t o r at 35,000 rpm f o r two to three hours. A f t e r d i s c a r d i n g the supernatant, each p e l l e t was o v e r l a i d w i t h 1.0 mL of 0.02M sodium phosphate pH 7.0 i n No. 30 tubes or 0.5 mL i n No. 65 tubes and allowed 18 to stand f o r one hour before being t r i t u r a t e d i n a ground-glass homogenizer. The t r i t u r a t e was then c e n t r i f u g e d at 8,000 rpm ( S o r v a l l SS-34 r o t o r ) and the supernatant, c o n t a i n i n g the p a r t i a l l y p u r i f i e d v i r u s p r e p a r a t i o n , c o l l e c t e d . To check the y i e l d and b i o l o g i c a l a c t i v i t y of t h i s p r e p a r a t i o n , the o p t i c a l d e n s i t y of a 100-fold d i l u t i o n i n 0.02M sodium phosphate pH 7.0 b u f f e r was measured against b u f f e r alone at 260 and 280 nm i n a G i l f o r d Model 260 spectrophotometer and subsequently i n o c u l a t e d to a s e r i e s of t e s t p l a n t s . This s e r i e s c o n s i s t e d of N_. c l e v e l a n d i i , N. tabacum cvs. Samsun and Xanthi and C_. amaranticolor. In some l a t e r experiments, C_. quinoa and G_. globosa were included i n t h i s s e r i e s . In determining the v i r u s y i e l d , an e x t i n c t i o n c o e f f i c i e n t at 260 nm of 2 10.0 cm /mg was assumed on the b a s i s of the s i m i l a r e x t i n c t i o n c o e f f i -c i e n t reported f o r TomRSV and the bottom component n u c l e o p r o t e i n of TRSV (Stace-Smith, 1970). L i k e CLRV, these are nepovirus p a r t i c l e s c o n t a i n i n g about 40% RNA. The p a r t i a l l y p u r i f i e d p r e p a r a t i o n was stored at 4 C w i t h a t r a c e of c r y s t a l l i n e chloro-butanol added as a p r e s e r v a t i v e , u n t i l needed f o r other experiments. Sucrose d e n s i t y gradients L i n e a r - l o g sucrose gradients were prepared manually f o r Beckman SW 41 c e n t r i f u g a t i o n tubes using the formulae of Brakke and van P e l t (1970) and Jackson e_t a l . (1973) and allowed to form a smooth gradient overnight at 4 C. Sucrose gradients f o r the p u r i f i c a t i o n of v i r u s preparations were made w i t h F i s h e r 'laboratory-grade' sucrose and 0.02M sodium phosphate pH 7.0.buffer. Sucrose gradients f o r the i s o l a t i o n 19 of i n f e c t i o u s RNA were made w i t h r i b o n u c l e a s e - f r e e sucrose (Schwarz-Mann) and 0.02M sodium phosphate pH 7.0/ 0.15M NaCl (PBS) b u f f e r c o n t a i n i n g 5 mg/mL of ben t o n i t e , a ribonuclease i n h i b i t o r (Fraenkel-Cemrat et a l . , 1961). Immediately before c e n t r i f u g a t i o n , the top 0.5-1.0 mL of the gradient was removed and a corresponding volume of pr e p a r a t i o n c a r e f u l l y layered on. A l l r a t e - z o n a l sucrose gradient c e n t r i f u g a t i o n s of p a r t i a l l y p u r i f i e d v i r u s preparations were done at 5 C and 38,000 rpm with the Beckman SW 41 r o t o r . Most runs were of 105 min d u r a t i o n ; runs to achieve b e t t e r component r e s o l u t i o n were of 135 min du r a t i o n . The c e n t r i f u g e d gradients were then scanned at 254 nm w i t h an ISCO UA-4 de n s i t y - g r a d i e n t monitor, and the components corresponding to the absorbance peaks were manually c o l l e c t e d . In some experiments, samples from each component f r a c t i o n were examined i n the e l e c t r o n microscope to monitor the i n t e g r i t y of the p u r i f i e d v i r u s . P r e p a r a t i v e n e a r - e q u i l i b r i u m CsCl d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n E a r l y experiments employed BDH 'Analar' grade but l a t e r experiments made use of BDH 'Technical' grade cesium c h l o r i d e p u r i f i e d by a method adapted from B i r n i e (1978). A CsCl s o l u t i o n i n d i s t i l l e d water (100 g/ 100 mL) was f i r s t f i l t e r e d through Whatman No. 1 f i l t e r paper. A s l u r r y of 10 mL of Dowex 50-mesh 'Chelating Resin' (Sigma) i n d i s t i l l e d water was poured i n t o a long narrow column (about 50 mL capacity) that had been plugged w i t h glass wool and f i t t e d w i t h a T e f l o n stopcock. The f i l t e r e d s o l u t i o n of t e c h n i c a l CsCl was passed through the r e s i n column at the r a t e of about 1 drop per second. The den s i t y of the e l u t e d CsCl s o l u t i o n was determined by p r e c i s e l y weighing 5 mL i n a tared 20 T e f l o n cup and was then made up to the des i r e d stock d e n s i t y of -3 1.45 g-cm and b u f f e r s t r e n g t h of 0.02M sodium phosphate pH 7.0. Pre p a r a t i v e n e a r - e q u i l i b r i u m CsCl d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n was performed i n SW 41 (Beckman) c e n t r i f u g a t i o n tubes w i t h s e l f - f o r m i n g gradients c o n s i s t i n g of 9 mL of the CsCl stock s o l u t i o n on which 3 mL of sample were layered. The samples were u s u a l l y 'separated' sucrose d e n s i t y - g r a d i e n t f r a c t i o n s of e i t h e r the middle or bottom n u c l e o p r o t e i n components. C e n t r i f u g a t i o n was u s u a l l y f o r 18 h at 30,000 rpm (SW 41 ro t o r ) and 5 C. No electromagnetic braking was a p p l i e d at the end of t h i s time, i n order to l i m i t the d i s r u p t i o n of the de n s i t y - g r a d i e n t formed during the run. As w i t h sucrose d e n s i t y - g r a d i e n t s , the c e n t r i f u g e d CsCl gradients were scanned at 254 nm i n an ISCO absorbance monitor with a 100% (w/v) s o l u t i o n of u n p u r i f i e d 'Technical' CsCl s e r v i n g as pump f l u i d . F r a c t i o n s of 0.25 mL each were c o l l e c t e d (ISCO Model 270) from the le a d i n g edge to the apex of the middle component absorbance peak and from the apex to the t r a i l i n g edge of the bottom component peak. The r e s p e c t i v e middle (M) and bottom (B) component f r a c t i o n s were pooled and i n some cases observed under the e l e c t r o n microscope to determine p a r t i c l e i n t e -g r i t y . In some runs, f i v e or s i x f r a c t i o n s from d i f f e r e n t depths of the gradient were c o l l e c t e d and the den s i t y of each of these f r a c t i o n s was determined with an Abbe refTactometer. In e a r l y experiments of t h i s type, the resolved M and B nucleo-p r o t e i n component f r a c t i o n s were d i l u t e d 50-200 f o l d with 0.02M sodium phosphate pH 7.0 b u f f e r . These l e s s p l a s m o l y t i c , d i l u t e preparations of separated n u c l e o p r o t e i n components were i n o c u l a t e d d i r e c t l y to p l a n t s , s e p a r a t e l y , and i n near-equimolar combination, to determine the enhancement-of-infectivity r a t i o and thus the e f f i c i e n c y of component r e s o l u t i o n . I s o l a t i o n and a n a l y s i s bf n u c l e o p r o t e i n components Middle and bottom component nucleoproteins were removed from CsCl by p r e c i p i t a t i o n w i t h PEG (10% w/v) and c e n t r i f u g i n g the suspension at 10,000 rpm f o r 20 min ( S o r v a l l SS-34 r o t o r ) . For l a t e r use, the blu e i s h - w h i t e p e l l e t s were resuspended i n a minimal volume (0.1 or 0.2 mL) of 0.02M sodium phosphate pH 7.0. Absorbance p r o f i l e s of d i l u t e d samples of such n u c l e o p r o t e i n p r e p a r a t i o n s , without c o r r e c t i o n f o r l i g h t s c a t t e r i n g , were obtained from o p t i c a l d e n s i t y measurements at 5 nm i n t e r v a l s from 295-225 nm ( G i l f o r d Model 250 spectrophotometer). I n f e c t i v i t y of the separated and combined n u c l e o p r o t e i n components was determined at A.,. = 0.1, 0.03, and 0.01. Both opposite h a l f -zoU leaves and i n d i v i d u a l p l a n t s of C. amaranticolor, C_. quinoa, and Samsun and Xanthi tobacco were i n o c u l a t e d i n order to measure the enhancement r a t i o s of combined n u c l e o p r o t e i n components. Isopycnic banding i n the Model-E a n a l y t i c a l u l t r a c e n t r i f u g e P u r i f i e d preparations of the E- and R - s t r a i n s were made to A0^ ,» = zoU _3 0.75 i n l.mL of 'Analar' CsCl s o l u t i o n w i t h a de n s i t y of 1.481 g«cm i n 0.02M sodium phosphate pH 7.0. Determinations of i s o p y c n i c buoyant d e n s i t i e s of the,nucleoprotein components were made from the S c h l i e r e n o p t i c a l d i f f r a c t i o n patterns a f t e r c e n t r i f u g a t i o n f o r at l e a s t 18 h at 44,770 rpm. 22 Preparation of RNA f o r polyacrylamide g e l e l e c t r o p h o r e s i s The d i s s o c i a t i o n b u f f e r employed f o r the pr e p a r a t i o n of RNA f o r e l e c t r o p h o r e s i s adapted from that of Dodds et a l . (1977), c o n s i s t e d of 0.08M T r i s - H C l pH 9.0, 0.004M Na^EDTA, 4.0 mg/mL ben t o n i t e , 4M urea, 4% SDS, 16% sucrose and 0.2% mercaptoethanol. I t was added to the nuc l e o p r o t e i n component suspensions described above, to achieve a f i n a l RNA concentration of about 1 mg/mL, assuming, c o n s i s t e n t w i t h other nepoviruses, an RNA content of about 40%. The nucleoproteins i n d i s s o c i a t i o n b u f f e r were heated i n a water bath at 50 C f o r t h i r t y minutes. Immediately before a p p l i c a t i o n of the samples to the g e l s , one drop of 1% bromo-phenol blue t r a c k e r dye was added. Samples of 40 and 80 yL were a p p l i e d to 2.5% polyacrylamide gels using a G i l s o n 'Pipetman'. The gels were prepared i n a procedure adapted from that of Dodds et a l . (1977). A 'plug' g e l of 0.5 mL of 10% polyacrylamide was f i r s t poured i n t o Perspex tubes whose bottom ends had been sealed w i t h P a r a f i l m . The 10% polyacrylamide, s u f f i c i e n t f o r a run of 12 tubes, c o n s i s t e d of 2.07 mL SE e l e c t r o p h o r e s i s b u f f e r (Dodds e t al., 1977), 0.65 mL of 1% (v/v) N,N,N',N'-tetramethylethylene diamine (TEMED), 4.0 mL of 15% acrylamide/0.75% b i s - a c r y l a m i d e and 0.1 mL of 10% ammonium p e r s u l f a t e s o l u t i o n . The 3E b u f f e r had been prepared by a 3:7 d i l u t i o n with d i s t i l l e d water from a 10E stock c o n t a i n i n g 0.36M T r i s , 0.18M sodium acetate t r i h y d r a t e , 0.01M Na^EDTA and adjusted to pH 7.2 w i t h g l a c i a l a c e t i c a c i d . When the plug g e l had s e t , 3 mL of 2.5% polyacrylamide s o l u t i o n c o n s i s t i n g of 12.14 mL d i s t i l l e d H^O, 9.95 mL of 3E b u f f e r , 2.4 mL of 1% (v/v) TEMED, 5.01 mL of 15% acrylamide/0.7/5% bisacrylamide and 0.2 mL of 10% ammonium p e r s u l f a t e was poured i n t o each tube. Before the s o l u t i o n had gelled", the top was c a r e f u l l y layered w i t h IE b u f f e r 23-to form a smooth g e l top surface. Before use, the gels were pre-electrophoresed at 75V and about 8mA per tube (ISCO Model 490 power supply) f o r 30 minutes. E l e c t r o p h o r e s i s , using IE tank b u f f e r , was c a r r i e d out f o r 150 min at 75V and about 8 mA per tube. At the end of the e l e c t r o p h o r e s i s , gels were removed from the tubes, s t a i n e d overnight i n 0.01% t o l u i d i n e blue and destained w i t h s e v e r a l changes of d i s t i l l e d water. P r e p a r a t i o n of RNA f o r inoculum The d i s s o c i a t i o n b u f f e r employed f o r the p r e p a r a t i o n of RNA f o r inoculum, a m o d i f i c a t i o n of the b u f f e r used by Dodds e_t a l . (1977) , c o n s i s t e d of 0.1M T r i s - H C l pH 9.0, 1% SDS, 0.025M Na^DTA and 0.15M NaCl. I t was added d i r e c t l y to the b l u i s h - w h i t e n u c l e o p r o t e i n PEG p e l l e t s to give a resuspension volume of 0.5 mL. The resuspensions were heated at 50 C f o r t h i r t y minutes i n a water bath before being layered onto sucrose gradients prepared as p r e v i o u s l y described. The RNA preparations were c e n t r i f u g e d at 38,000 rpm and 5 C f o r 240 min i n the SW41 r o t o r and scanned at 254 nm w i t h an ISCO UA-4 d e n s i t y - g r a d i e n t monitor i n order to c o l l e c t the RNA peak f r a c t i o n s . The d e n s i t y - g r a d i e n t absorbance p r o f i l e s of RNA prepared from separated n u c l e o p r o t e i n com-ponents, the remixed components and the o r i g i n a l l y p a r t i a l l y - p u r i f i e d v i r u s p r e p a r a t i o n were compared i n t h i s way. In order to determine that the moiety corresponding to the absorbance peak was RNA r a t h e r than n u c l e o p r o t e i n , the A /A r a t i o s were zoU zoU measured. In one experiment, the absorbance p r o f i l e from 320-220 nm of RNA prepared as j u s t described was determined i n a Cary Model 15 scanning spectrophotometer and compared w i t h published p r o f i l e s of RNA. The RNA 24 i n sucrose from the de n s i t y - g r a d i e n t c e n t r i f u g a t i o n was u s u a l l y stored at -2.0.C u n t i l needed. For i n o c u l a t i o n , the frozen RNA component preparations were thawed and the necessary amounts of each sample d i l u t e d to A = 0.1 with PBS zoU 2 co n t a i n i n g 5 mg/mL of bento n i t e . An e x t i n c t i o n c o e f f i c i e n t of 25 cm /mg at 260 nm was used to determine RNA concentration (Dodds et a l . , 1977). To t e s t the e f f i c i e n c y of RNA component separations and the s p e c i f i c i n f e c t i v i t y of the RNA pr e p a r a t i o n s , C_. amaranticolor, C_. quinoa, _N. c l e v e l a n d i i , and Samsun and Xanthi tobacco p l a n t s were i n o c u l a t e d (using s t e r i l i z e d cotton swabs to l i m i t r ibonuclease a c t i v i t y ) w i t h e i t h e r c o l d M-RNA, B-RNA, an equimolar combination of M-RNA and B-RNA, or RNA prepared d i r e c t l y from p a r t i a l l y - p u r i f i e d v i r u s p r e p a r a t i o n s . I n some experiments, the i n f e c t i v i t y of combined M- and B-RNA was compared to that of each separate component RNA using opposite h a l f - l e a v e s of C. amaranticolor, C_. quinoa, and Xa n t h i and Samsun tobacco. Generation of pseudorecombinants Pseudorecombinants were generated from M-and B-RNA components of the E- and R - s t r a i n s . M-RNA of the E - s t r a i n and B-RNA.of the R - s t r a i n , both at An/,n = 0.1, were mixed (-) as were M-RNA of the R - s t r a i n and zoU R ' R' B-RNA of the E - s t r a i n (-) and in o c u l a t e d to C. amaranticolor, N. c l e v e l a n d i i , and Samsun and Xanthi tobacco. I n o c u l a t i o n s of the i n d i v i d u a l component RNAs and homologous RNA combinations to i n d i v i d u a l p l a n t s and opposite h a l f - l e a v e s served as c o n t r o l s . New pseudore-combinant l o c a l l e s i o n types appearing on N> c l e v e l a n d i i were passaged three times through l o c a l l e s i o n s to generate pure pseudorecombinant l i n e s w i t h uniform symptomatologies i n the plants of the above host range. The pseudorecombinant i s o l a t e s were then maintained, l i k e the p a r e n t a l i s o l a t e s , i n N_. c l e v e l a n d i i by s e r i a l l y t r a n s f e r r i n g young s y s t e m i c a l l y i n f e c t e d leaves to young p l a n t s . Comparison of parental:,andfcpseudorecombinantisolates Component d i s t r i b u t i o n E R The E-, R-, T g — , and - i s o l a t e s were each propagated i n C. quinoa, i IS. £j p a r t i a l l y p u r i f i e d by the methods described e a r l i e r and subjected to l i n e a r - l o g sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n to ob t a i n p a r t i c l e component r a t i o s . This process a l s o served to produce p u r i f i e d v i r u s preparations f o r subsequent comparisons of symptomatology and s e r o l o g i c a l r e l a t i o n s h i p s . Symptomatology P u r i f i e d v i r u s of the four i s o l a t e s was d i l u t e d to An.n = 0.1 and 260 i n o c u l a t e d to N. c l e v e l a n d i i , Samsun and Xanthi tobacco, G.. globosa, C_. quinoa and C_. amaranticolor to compare l o c a l and systemic symptom types. Opposite h a l f - l e a f i n o c u l a t i o n s of C_. amaranticolor, C_. quinoa, Samsun and Xanthi tobacco were done to confirm r e l a t i o n s of l o c a l - l e s i o n symptom types. Serology A n t i s e r a were prepared against the parent E- and R - s t r a i n s from two young, white New Zealand r a b b i t s each immunized w i t h three i n t r a -muscular i n j e c t i o n s of e i t h e r p u r i f i e d E- or R - s t r a i n (lmg/mL) e m u l s i f i e d 1:1 w i t h Freund's complete adjuvant. The f i r s t and second i n j e c t i o n s were 2 weeks apart, with—booster i n j e c t i o n s given at 10 weeks. A f t e r 26 the second i n j e c t i o n , bleedings were done at weekly i n t e r v a l s f o r the next 11 weeks and t e s t e d by Ouchterlony d o u b l e - d i f f u s i o n serology to determine the t i t r e of the antiserum against i t s homologous antigen at a co n c e n t r a t i o n of A.,„ =1.0. 260 S e r o l o g i c a l comparisons of the four i s o l a t e s were done w i t h Ouchterlony d o u b l e - d i f f u s i o n i n agar g e l on glass s l i d e s (Mansi, 1958). Each p a r e n t - s t r a i n antiserum, d i l u t e d to a concentration 25 times that of i t s t i t r e d i l u t i o n , was placed i n a c e n t r a l w e l l surrounded by eight p e r i p h e r a l w e l l s which were f i l l e d i n p a i r s w i t h p u r i f i e d v i r u s of the four i s o l a t e s each at A„,« = 1.0. The s l i d e s were then incubated at 260 room temperature f o r 48 h i n a moist chamber to ob t a i n p r e c i p i t i n r e a c t i o n s . Regeneration of parents from pseudorecombinants E R The - and - pseudorecombinant i s o l a t e s were propagated i n N. c l e v e l a n d i i , p u r i f i e d , and the component RNAs of the two i s o l a t e s prepared as described f o r the two parent s t r a i n s . The M-RNA of the -R and the B-RNA of the - pseudorecombinant i s o l a t e s were mixed to generate a p r e d i c t e d E - s t r a i n p a r e n t a l combination, as were the r e c i p r o c a l RNAs to generate a p r e d i c t e d R - s t r a i n combination. These combinations and separate component RNAs were i n o c u l a t e d to opposite h a l f - l e a v e s of C_. quinoa, C_. amaranticolor and Samsun and Xanthi tobacco; N. c l e v e l a n d i i ,was a l s o i n o c u l a t e d w i t h these combinations as w e l l as the pseudore-combinants, to give d i r e c t symptom evidence of regeneration of p a r e n t a l from pseudorecombinant i s o l a t e s . The a p i c a l t i p s of C_. quinoa p l a n t s s y s t e m i c a l l y i n f e c t e d w i t h 'predicted-parent' RNA combinations were used as inoculum f o r propagation in C_. quinoa. The purified viruses obtained from this propagation were examined for component distribution, symptomatology and serology. These observations were compared with those made previously of known parental isolates to. confirm that parents had been generated from pseudore-combinants and that the pseudorecombinants had the genetic make-up ascribed to them. 2.8 RESULTS Comparative symptomatologies of CLRV s t r a i n s No obvious l o c a l or systemic symptoms were induced by the D-,C-',E-and R - s t r a i n s of CLRV i n N. g l u t i n o s a , N. glauca, Z i n n i a elegans and Datura stramonium. N. s y l v e s t r i s , N. r u s t i c a , Capsicum annum, Vigna  unguiculata cv. Blackeye, and Phaseolus v u l g a r i s cvs. B o u n t i f u l and P i n t o showed l o c a l and systemic symptoms when i n o c u l a t e d w i t h the four s t r a i n s but d i f f e r e n c e s i n symptom types among the four s t r a i n s , i f any, were n e i t h e r c l e a r nor e a s i l y r e p r o d u c i b l e . L o c a l and systemic symptoms of the four s t r a i n s on N. c l e v e l a n d i i , KL tabacum cvs. Samsun and X a n t h i , C_. quinoa and C_. amaranticolor (Table 1) were s u f f i c i e n t l y d i s t i n c t and repr o d u c i b l e to i n d i c a t e t h e i r usefulness as genetic markers. The systemic symptoms i n Samsun and Xanthi tobacco appeared as various forms of r a d i a t i n g c o n c e n t r i c curves of c h l o r o s i s and were described as 'map-contour' symptoms (Table 1). The long term (beyond 60 days a f t e r i n o c u l a t i o n ) systemic symptom types of the E- and R - s t r a i n s i n C_. amaranticolor were so markedly d i f f e r e n t ( F i g . 1) t h a t , p a r t l y because of t h i s v a l u a b l e marker, they were the s t r a i n s s e l e c t e d f o r pseudorecombi-n a t i o n genetic a n a l y s i s . P u r i f i c a t i o n methods S i g n i f i c a n t l o s s e s of v i r u s m a t e r i a l are to be expected i n the various procedures required f o r n u c l e o p r o t e i n - r e s o l u t i o n and RNA-e x t r a c t i o n . P u r i f i c a t i o n procedures should t h e r e f o r e be chosen on the ba s i s of t h e i r amenability to r a p i d (1 day or l e s s ) l a r g e s c a l e p u r i f i -c a t i o n of the different^CLRV s t r a i n s . Table 1 : Comparative symptomatologies of the strains of CLRV in l o c a l and systemic infections Strain Infection Reactions on d i f f e r e n t i a l hosts N. c l e v e l a n d i i N. tabacum cv. Samsun N^ . tabacum cv. Xanthi C. amaranticolor C. quinoa Dogwood l o c a l large necrotic disc lesions systemic extensive necrosis, death necrotic rings small, tight map-contour necrotic rings ' r i p p l i n g ' necrosis necrotic point lesions mottle, straight spindly growth necrotic point lesions a p i c a l w i l t , slow death Cherry l o c a l small necrotic ring lesions systemic f l e c k necrosis followed by recovery small necrotic lesions 'blurry' map-contour small necrotic lesions ' r i p p l i n g ' mild chlorosis necrotic/chlorotic point lesions mottle, fascicu-late growth n e c r o t i c / c h l o r o t i c point lesions severe w i l t , slow death Elderberry l o c a l small necrotic/ c h l o r o t i c rings systemic point necrosis followed by recovery nec r o t i c / c h l o r o t i c rings f i n e - l i n e map-contour necrotic/chlorotic c h l o r o t i c , pin-rings f i n e - l i n e map-contour point lesions apical t u f t i n g , dwarfing c h l o r o t i c , pin-point lesions c h l o r o s i s , survives and bears seed Rhubarb l o c a l large necrotic disc lesions large necrotic lesions large necrotic lesions necrotic disc lesions necrotic disc lesions systemic severe fleck necrosis followed by recovery coarse-line map-contour coarse-line map-contour mottle, fascicu-late growth severe w i l t , rapid death ho 30 F i g . 1. Systemic symptoms of CLRV i n f e c t i o n i n C_. amaranticolor. A. i n o c u l a t e d w i t h e l d e r b e r r y (E) s t r a i n : dwarfing and a p i c a l t u f t i n g a f t e r 60 days; B. i n o c u l a t e d w i t h rhubarb (R) s t r a i n : systemic mottle and p a r t l y f a s c i c u l a t e growth a f t e r 60 days 31 The p u r i f i c a t i o n procedure described as 'Method 2' by Jones and Mayo (1972) was the f i r s t attempted. P u r i f i e d , i n f e c t i o u s preparations of the C- and R - s t r a i n s were obtained from s y s t e m i c a l l y i n f e c t e d C_. quinoa l e a f and stem t i s s u e . The need f o r overnight f r e e z i n g of t i s s u e sap (and f o r subsequent thawing) and the use of large q u a n t i t i e s of f a i r l y expensive ammonium s u l f a t e , however, d i d not make i t seem a p a r t i c u l a r l y convenient method f o r the l a r g e - s c a l e r o u t i n e p u r i f i c a t i o n that would be required l a t e r i n the work. Walkey ej: a_l. (1973) had a l s o described an e f f e c t i v e p u r i f i c a t i o n method s u i t a b l e f o r a number of CLRV s t r a i n s , but the requirement f o r large volumes of chloroform (1 volume per weight of t i s s u e ) made i t i n -convenient f o r l a r g e - s c a l e p u r i f i c a t i o n s . More s e r i o u s l y , the middle component n u c l e o p r o t e i n of the cherry s t r a i n appeared (as r e f l e c t e d i n sucrose-gradient scanning p r o f i l e s ) to be p a r t i c u l a r l y adversely a f f e c t e d ; i t s c o n c e n t r a t i o n , n e a r l y equimolar to the bottom component i n other methods, was reduced to a mere shoulder at the base of the bottom component peak. The method s e l e c t e d f o r p a r t i a l p u r i f i c a t i o n i n v o l v e d homogenization w i t h h i g h - m o l a r i t y phosphate b u f f e r , c l a r i f i c a t i o n aided by s m a l l proportions by volume of chloroform/butanol, and concentration by PEG p r e c i p i t a t i o n and d i f f e r e n t i a l c e n t r i f u g a t i o n . I t was r a p i d ( l e s s than one day), e a s i l y a p p l i e d to s m a l l and l a r g e amounts of t i s s u e and gave reasonable y i e l d s (20-40 mg/kg) of i n f e c t i o u s p a r t i c l e s . V i r u s p u r i f i e d by t h i s method a l s o stood up to the r i g o r s of subsequent CsCl near-e q u i l i b r i u m c e n t r i f u g a t i o n s . The A„^_/A-ori r a t i o s and i n f e c t i v i t y assays on 100-fold d i l u t i o n s ZoU Z o U of p a r t i a l l y p u r i f i e d preparations were u s e f u l i n d i c a t o r s of p u r i t y and q u a l i t y . The A 26c / A 280 r a t i o s u s u a l l y ranged from 1.7 - 1.9; preparations w i t h lower r a t i o s s t i l l contained l a r g e p r o p o r t i o n s of host p r o t e i n w h i l e higher r a t i o s i n d i c a t e d host ribosome contamination as confirmed by subsequent sucrose gradient scanning p r o f i l e s and e l e c t r o n microscopy. Preparations made from s y s t e m i c a l l y i n f e c t e d t i s s u e s of C_. quinoa r a t h e r than N. c l e v e l a n d i i t i s s u e tended to have higher A„, n/A n r a t i o s (1.9 - 2.1) and higher l e v e l s of host ribosome zoU zo(J contamination. Sucrose gradient c e n t r i f u g a t i o n and scanning p r o f i l e s of p a r t i c l e s Sucrose d e n s i t y - g r a d i e n t scanning p r o f i l e s of p a r t i a l l y p u r i f i e d p reparations of the E- and R - s t r a i n s propagated from N. c l e v e l a n d i i ( F i g . 2) and C_. quinoa ( F i g . 3) revealed that w h i l e r e l a t i v e sediment-a t i o n values of the p a r t i c l e components of the two s t r a i n s i n the two hosts were constant, r e l a t i v e component d i s t r i b u t i o n was a property of the v i r u s s t r a i n as w e l l as a f u n c t i o n of the propagation host. The E-s t r a i n c o n s i s t e n t l y showed a lower p r o p o r t i o n of middle component than the R - s t r a i n but the d i f f e r e n c e was f a r more s t r i k i n g i n preparations from fJ. quinoa than from IJ. c l e v e l a n d i i . In a d d i t i o n , the R - s t r a i n c o n s i s t e n t l y produced d e t e c t a b l e amounts of RNA-free top component p a r t i c l e s (confirmed by e l e c t r o n microscopy) w h i l e such a top component was never detected i n E - s t r a i n preparations ( F i g s . 2 and 3). The sucrose d e n s i t y - g r a d i e n t s were a l s o simultaneously employed to prep-a r a t i v e l y p u r i f y the n u c l e o p r o t e i n and a l s o achieve a f i r s t - o r d e r separation of the components. Preparative near-equilibrium CsCl density-gradient c e n t r i f u g a t i o n . Centrifugation of p u r i f i e d nucleoprotein components through -3 CsCl (p = 1.45 g-cm ) for about 18 h to near-equilibrium gave much better r e s o l u t i o n than rate-zonal sucrose-gradient c e n t r i f u g a t i o n . (Fig. 4). In both the E- and R-strains, the near-equilibrium buoy-ant densities corresponding to the component absorbance peaks, calculated by i n t e r p o l a t i o n of dens i t i e s obtained for f i v e or s i x 0.25 mL f r a c t i o n s from the lower (CsCl-containing) 9 mL of the gradient, -3 were found to be p = 1.44 and 1.48 g-cm for the middle and bottom components, r e s p e c t i v e l y . 'Pure' middle and bottom component preparations obtained, respect-i v e l y , by pooling leading leading edge to apex, and apex to t r a i l i n g edge f r a c t i o n s of the two components were assessed for fu n c t i o n a l nucleoprotein-component r e s o l u t i o n by i n f e c t i v i t y measurements. The com-ponents i n CsCl, d i l u t e d 50-6©>200-fold with 0.02M sodium phosphate to render them non-plasmolytic, were inoculated separately and i n one-to-one combination to host plants. In one experiment, preparations of 'pure' ( l e . f u l l y resolved and separated) nucleoprotein components of the R.-strain were d i l u t e d 50-fold with buffer and Depth-F i g . 2. Absorbance scan patterns of sucrose density-gradients showing differences in p a r t i c l e component d i s t r i b u t i o n of the elderberry and rhubarb s t r a i n s of CLRV p u r i f i e d from JN. c l e v e l a n d i i . A. middle (M) and bottom (B) component nucleoproteins of the elderberry s t r a i n ; B. top (T) component RNA-free empty p a r t i c l e and middle (M) and bottom (B) component nucleoproteins of the rhubarb s t r a i n Absorbance scan patterns of sucrose density-gradients showing particle component distributions of the elderberry and rhubarb strains of CLRV purified from C_. quinoa. A. middle (M) and bottom (B) component nucleoproteins of the elderberry strain B. top (T) component RNA-free particles and middle (M) and bottom (B) component nucleoproteins of the rhubarb (R) strain 36 Depth -F i g . 4. Absorbance scan patterns showing extent of separation of middle and bottom n u c l e o p r o t e i n components (rhubarb s t r a i n ) . In each scan, the peak at the greatest depth corresponds to the bottom component. A. L i n e a r - l o g sucrose gradient c e n t r i f u g a t i o n ; B. 'Middle' component of 'A' a f t e r near-e q u i l i b r i u m CsCl d e n s i t y - g r a d i e n t c e n t r i -f u g a t i o n ; C. 'Bottom' component of 'A' a f t e r near-e q u i l i b r i u m CsCl d e n s i t y gradient c e n t r i -f u g a t i o n 37 i n o c u l a t e d separately and i n combination to C_. amaranticolor p l a n t s at the f o u r - l e a f stage; near-perfect f u n c t i o n a l r e s o l u t i o n was observed ( F i g . 5). In a l l experiments of t h i s type, the separated components appeared, on the b a s i s of induced symptoms, much l e s s i n f e c t i o u s than t h e i r a r t i f i c i a l one-to-one combination. In a d d i t i o n , l o c a l and systemic t i s s u e of hosts i n o c u l a t e d e a r l i e r w i t h pure middle component was almost never i n f e c t i o u s , w h i l e pure bottom component was often only i n f e c t i o u s a t very low l e v e l s . P l a n t s i n o c u l a t e d w i t h one-to-one combinations, however, y i e l d e d h i g h l y i n f e c t i o u s t i s s u e , which, when used as inoculum induced 10-50 times higher numbers of l o c a l l e s i o n s on C_. amaranticolor compared to s i m i l a r t i s s u e from hosts which had been i n o c u l a t e d w i t h pure bottom component. A n a l y s i s of n u c l e o p r o t e i n components by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n The determination of e q u i l i b r i u m buoyant d e n s i t i e s of the two n u c l e o p r o t e i n components by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n at 25 C was only p o s s i b l e w i t h the R - s t r a i n ; the E - s t r a i n appeared to form a p r e c i p i t a t e - l i k e aggregate w i t h i n a short time of beginning the run. The buoyant d e n s i t i e s of the middle and bottom components, c a l c u l a t e d from the S c h l i e r e n d i f f r a c t i o n p a t t e r n ( F i g . 6) were r e s p e c t i v e l y 1.471 -3 and 1.508 g-cm . As the run progressed, the middle component appeared to decrease and i t s S c h l i e r e n d i f f r a c t i o n p a t t e r n became wider than that of the bottom component. I s o l a t i o n and a n a l y s i s of n u c l e o p r o t e i n components For the f u r t h e r a n a l y s i s of the n u c l e o p r o t e i n components, the e x t r a c t i o n and p u r i f i c a t i o n of RNA and f o r medium-term storage of the 38 F i g . 5. C_. amaranticolor p l a n t s 45 days a f t e r i n o c u l a t i o n (at the f o u r - l e a f stage) w i t h middle (M), bottom (B) and combined (M+B) n u c l e o p r o t e i n components of the rhubarb s t r a i n of CLRV. The components had been separated by sucrose gradient c e n t r i f u g a t i o n f o l l o w e d by n e a r - e q u i l i b r i u m banding i n CsCl (p=1.45 g-cm~3). 39 Fig. 6. Schlieren pattern from a n a l y t i c a l ultracentrifugation of CLRV-rhubarb strai n i n CsCl density-gradient at equilibrium (44,770 rpm; 24h). Density increases from l e f t to ri g h t . Middle component nucleoprotein appears as smaller density-band near the centre of the gradient; bottom component nucleoprotein appears as the larger density band to the right of the middle component. 40 components, i t was d e s i r a b l e to have more concentrated preparations of separated n u c l e o p r o t e i n components that were a l s o C s C l - f r e e . The a d d i t i o n of PEG at 10% (w/v) to CsCl component p r e p a r a t i o n s , followed by low-speed c e n t r i f u g a t i o n achieved both concentration of the p a r t i c l e s and removal of CsCl i n a s i n g l e step w i t h minimal l o s s . Comparative spectrophotometric a n a l y s i s The middle and bottom component n u c l e o p r o t e i n s , r e s p e c t i v e l y , of both the E- and R - s t r a i n s showed A„^ n/A o o r i r a t i o s of 1.64-1.66 and zoU zou 1.81-1.83. In c o n t r a s t , the A /A r a t i o s of p u r i f i e d 'whole-virus' zoU zoU preparations (from N. c l e v e l a n d i i ) of the two s t r a i n s d i f f e r e d markedly: 1.76-1.77 f o r the E - s t r a i n and 1.71-1.72 f o r the R - s t r a i n . The A 0,.,A 0 0„ zoU/ zoU r a t i o s of the separated n u c l e o p r o t e i n components were c o n s i s t e n t w i t h t h e i r d i f f e r e n c e s i n absorbance p r o f i l e , uncorrected f o r l i g h t s c a t t e r i n g , between 225 and 295 nm (Fig-. 7A) . For both middle and bottom components the absorbance maximum was at 259 nm, the minimum at 239 nm. The A / max A . r a t i o s were 1.28 and 1.41 f o r the middle and bottom components, mm r r e s p e c t i v e l y . I n f e c t i v i t y of n u c l e o p r o t e i n components The r e l a t i v e i n f e c t i v i t i e s , at defined c o n c e n t r a t i o n s , of the middle and bottom component nucleoproteins of the R - s t r a i n and t h e i r e q u i -absorbant combination were measured mainly i n terms of number of l o c a l l e s i o n s induced and s e c o n d a r i l y by t h e i r a b i l i t y to induce systemic i n f e c t i o n s . Opposite h a l f - l e a v e s of C_. amaranticolor and C_. quinoa at the f o u r - l e a f stage and Xanthi tobacco at the two-leaf stage i n o c u l a t e d on one h a l f w i t h one component and on the other h a l f w i t h the eq u i -Absorbance scan patterns of CLRV nucleoproteins extracted RNA, uncorrected for light scattering, from 220-295 nm. A. separated middle component ( ) and bottom component ( ) nucleoproteins; B. extracted RNA 42 absorbant component combination c l e a r l y showed enhancement of i n f e c t i v i t y w i t h the presence of both components. In Xanthi tobacco, at A = 0.10 ZoU and 0.03, the equiabsorbant combinations induced r e s p e c t i v e l y , an average of 6.0 and 3.5 l e s i o n s per h a l f - l e a f w h i l e the pure components f a i l e d to induce any l e s i o n s on t h e i r i n o c u l a t e d h a l f - l e a v e s . C_. amaranticolor and C_. quinoa, more s e n s i t i v e h o s t s , were each t e s t e d i n d u p l i c a t e p l a n t s by the; opposite h a l f - l e a f method (Tables 2 and 3). In s e n s i t i v e h o s t s , such as C_. amaranticolor, a l o w - l e v e l CLRV i n f e c t i o n may f a i l to produce l o c a l l e s i o n s , but i t may e v e n t u a l l y produce systemic symptoms. To determine i f component r e s o l u t i o n had been obtained to the extent that n e i t h e r component on i t s own could induce even such a l o w - l e v e l i n f e c t i o n , separate C_. amaranticolor p l a n t s at the f o u r - l e a f stage were i n o c u l a t e d on four h a l f - l e a v e s w i t h middle, bottom and equiabsorbant combinations at three d i f f e r e n t component con-c e n t r a t i o n s . At A = 0.10, p l a n t s i n o c u l a t e d w i t h middle component, bottom component and t h e i r combination showed, r e s p e c t i v e l y , t o t a l s of 16, 67 and 316 l o c a l l e s i o n s , i n d i c a t i n g a moderate enhancement-of-infec-. t i v i t y r a t i o (7.6) s i m i l a r to that obtained by opposite h a l f - l e a f measurement (11.0 from Table 2). At A.rn = 0.03, the pl a n t i n o c u l a t e d zo(J w i t h middle component showed no l o c a l l e s i o n s and f a i l e d to l a t e r show any systemic symptoms; the bottom component induced 2 and the component combination 63 l e s i o n s , i n d i c a t i n g , as w i t h opposite h a l f - l e a v e s , that much higher r a t i o s are obtained at lower component concentrations. F i n a l l y , at A„.n = 0.01, n e i t h e r p l a n t i n o c u l a t e d w i t h separated com-zoU ponents showed any l o c a l l e s i o n s or l a t e r systemic i n f e c t i o n , w h i l e the component combination induced 11 l o c a l l e s i o n s , suggesting f u n c t i o n a l l y ' p e rfect' s e paration had"been achieved. Table 2: Enhancement of i n f e c t i v i t y by complementation of separated middle (M) and bottom (B) n u c l e o p r o t e i n components, as measured by i n d u c t i o n of l o c a l l e s i o n s ( I s . ) , on opposite h a l f - l e a v e s ( v ) of C. amaranticolor 43 R e p l i c a t e Number of l e s i o n s on i n o c u l a t e d h a l f - l e a v e s A260 =0.10 A260 0.03 A260" OJ 01 M v M+B B v M+B M v M+B B v M+B M v M+B B v M4 P l a n t 1 5 97 26 142 0 17 1 13 0 4 0 2 5 146 15 216 0 12 0 8 0 3 0 4 Pl a n t 2 19 191 28 207 0 19 1 15 0 2 0 5 10 103 3 115 0 11 0 12 0 3 1 6 Tot a l s 39 537 72 680 0 59 2 48 0 12 1 17 M+B I s . 537 + 680 11.0 59 + 48 63.5 12 + 17 29 M Is. +B I s . 39 + 72 0 + 2 0 + 1 Table 3: Enhancement of i n f e c t i v i t y by complementation of separated middle (M) and bottom (B) nu c l e o p r o t e i n components, as measured by i n d u c t i o n of l o c a l l e s i o n s ( I s . ) , on opposite h a l f - l e a v e s ( v ) of C_. quinoa R e p l i c a t e Number of l e s i o n s on i n o c u l a t e d h a l f - l e a v e s A260=°- 0 3 A 2 6 0 = 0 ' 0 1 M v M+B B v M+B M v M+B B + M+B Plan t 1 0 7 1 10 0 3 0 6 0 17 0 17 0 2 0 3 Plan t 2 0 2 0 3 0 4 1 3 Tot a l s 0 24 27 0 11 1 15 M+B I s . 24-+ 27 = M Is.+B Is. 0 + 1 44 RNA of separated components i n polyacrylamide g e l e l e c t r o p h o r e s i s Polyacrylamide g e l e l e c t r o p h o r e s i s of RNAs e x t r a c t e d from con-centrated (A„, =10-15) preparations of separated n u c l e o p r o t e i n components ZoU of the R - s t r a i n provided p h y s i c a l evidence confirming that v i r t u a l l y complete separation of the nucleoproteins had been achieved ( F i g . 8). The RNA i s o l a t e d from middle component co-migrated w i t h the sm a l l e r , f a s t e r m i g r a t i n g v i r a l RNA of a p a r t i a l l y p u r i f i e d p r e p a r a t i o n ( F i g . 8A, B), w h i l e RNA from bottom component co-migrated w i t h the l a r g e r , slower m i g r a t i n g RNA of a p u r i f i e d 'whole-virus' R - s t r a i n p r e p a r a t i o n ( F i g . 8C,D). RNA of separated components as inoculum The moiety obtained by sucrose gradient c e n t r i f u g a t i o n f o r use as inoculum was confirmed as being RNA r a t h e r than n u c l e o p r o t e i n by i t s A.^./A r a t i o s of 2.05-2.25 and i t s absorbance p r o f i l e , uncorrected zoU zoU f o r l i g h t s c a t t e r i n g , from 220-295 nm ( F i g . 7B). The absorbance maximum was at 259 nm and the minimum at 230 nm w i t h an A /A . =2.20. max min As an RNA c o n t r o l f o r a n a l y s i s and i n o c u l a t i o n , RNA was e x t r a c t e d from p u r i f i e d 'whole-virus' preparations of the E- and R - s t r a i n s (propagated i n N. c l e v e l a n d i i ) by the same methods used to e x t r a c t RNA from concentrated preparations of separated nucleoproteins ( F i g . 9). Although r e s o l u t i o n of CLRV RNA components i n sucrose d e n s i t y - g r a d i e n t s i s poor compared to the r e s o l u t i o n of p a r t i c l e s , i t i s p o s s i b l e to see that the RNA component d i s t r i b u t i o n r e f l e c t s the n u c l e o p r o t e i n component d i s t r i b u t i o n : the R - s t r a i n n u c l e o p r o t e i n ( F i g . 3B) and RNA ( F i g . 9B) components are about equimolar w h i l e f o r the E - s t r a i n both n u c l e o p r o t e i n 4 5 F i g . 8. Polyacrylamide g e l e l e c t r o p h o r e s i s of RNA e x t r a c t e d from separated n u c l e o p r o t e i n components and 'whole-v i r u s ' preparations of CLRV. A. RNA e x t r a c t e d from 'pure' middle component (M) n u c l e o p r o t e i n ; B. RNA ex t r a c t e d from a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of CLRV; C. RNA ex t r a c t e d from f u l l y p u r i f i e d CLRV; D. RNA ex t r a c t e d from 'pure' bottom component (B) nu c l e o p r o t e i n F i g . 9. Absorbance scan patterns of RNA obtained by sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . The RNA absorbance peaks are the doublets about o n e - t h i r d of the way down the gradient. A. RNA e x t r a c t e d from the e l d e r b e r r y (E) s t r a i n of CLRV; B. RNA ex t r a c t e d from the rhubarb (R) s t r a i n of CLRV 47 ( F i g . 3A) and RNA ( F i g . 9A) middle component are present i n one t h i r d to one-half the amount of r e s p e c t i v e bottom component. RNA made from concentrated (PEG-pelleted) separated middle (M) and bottom (B) component nucleoproteins gave mono-disperse peaks, t h e i r apices d i f f e r i n g by 1.8-2.0 mm (SW 41 c e n t r i f u g a t i o n tubes) i n sedimentation depth ( F i g . 10). When d i l u t e d to A..,. = 0.10 w i t h PBS-ZoU bentonite b u f f e r , such RNA component preparations were r e a d i l y used as inoculum. M-BNA or B-RNA prepared from t h e i r separated n u c l e o p r o t e i n components had very low l e v e l s of i n f e c t i v i t y , but had high l e v e l s i n equiabsorbant combination. When measured by l o c a l - l e s i o n counts on in o c u l a t e d h a l f - l e a v e s of C_. amaranticolor at the f o u r - l e a f stage, enhancement-of-infectivity r a t i o s ranged from 30-60. In the l e s s s e n s i -t i v e X anthi and Samsun tobacco hosts, each RNA component, on i t s own, u s u a l l y f a i l e d to produce any symptoms, w h i l e equiabsorbant mixtures produced l o c a l l e s i o n s and systemic symptoms. Generation of pseudorecombinants The pseudorecombinant i n o c u l a , made by mixing the equiabsorbant (A =0.10) RNA component preparations of one s t r a i n w i t h complementary ZoU RNA component preparations of the other s t r a i n , might induce r e s i d u a l l o w - l e v e l i n f e c t i o n s of one of the component s t r a i n s r a t h e r than true pseudorecombinant i n f e c t i o n s . To d i s t i n g u i s h between these outcomes, C_. amaranticolor at the f o u r - l e a f stage and Xanthi tobacco p l a n t s were in o c u l a t e d w i t h s i n g l e RNA components, homologous RNA component combi-nations as w e l l as the heterologous, pseudorecombinant RNA combinations (Table 4 ). 48 Dapth—ft> Fig. 10. Absorbance scan patterns of RNA (of the rhubarb s t r a i n of CLRV) obtained by sucrose density-gradient c e n t r i -fugation, showing the monodisperse nature and s l i g h t differences in sedimentation velocity of RNA extracted from separated middle (M-RNA) and bottom (B-RNA) component nucleoprotein. The RNA absorbance peaks are about one-third of the way down the gradient. A. RNA extracted from separated middle component nucleoprotein (M-RNA); B. RNA extracted from a re-mixture of separated middle and bottom component nucleoproteins; C. RNA extracted from separated bottom component nucleoprotein (B-RNA); D. RNA extracted from a 'whole-virus' preparation 49 Table 4: R e l a t i v e i n f e c t i v i t i e s at A =0.10 of middle (M) and bottom zoU (B) RNA components of CLRV e l d e r b e r r y (E) and rhubarb (R) s t r a i n s i n o c u l a t e d as separate components and i n homologous and heterologous equiabsorbant combinations RNA inoculum Sum of l e s i o n s on i n o c u l a t e d h a l f - l e a v e s M-RNA B-RNA C_. amaranticolor IJ. tabacum cv. Xanthi (4 h a l f - l e a v e s ) (2 h a l f - l e a v e s ) E - 1 0 - E 2 0 E E 69 4 R - 1 0 - R 3 0 R R 98 6 E R 69 17 R E 51 4 The data i n d i c a t i n g enhancement of i n f e c t i v i t y by complementation with heterologous RNA components (Table 4) corresponded to observations of unique types of l o c a l - l e s i o n induced by heterologous RNA combinations i n some of the d i f f e r e n t i a l hosts ( c f . Table 1). These new types were e s p e c i a l l y d i s t i n c t i n IJ. c l e v e l a n d i i . and predominated over p a r e n t a l l e s i o n types on leaves i n o c u l a t e d w i t h heterologous RNA combinations. Pure stock i n f e c t i o n s of the two pseudorecombinant i s o l a t e s were obtained by three l o c a l l e s i o n passages i n IJ. c l e v e l a n d i i ; the i n f e c t i o n s were deemed to be pure - a f t e r the t h i r d passage by v i r t u e of t h e i r mono-t y p i c l o c a l - l e s i o n symptoms a f t e r the second passage. The pseudore-combinant that had obtained i t s M-RNA from the E - s t r a i n and B-RNA from 'E' the R - s t r a i n was designated - , the i s o l a t e from the r e c i p r o c a l combi-'R' natxon, - . 50' Comparison of the parental arid pseudorecombinant i s o l a t e s Symptomatology The l o c a l lesions induced on N. c l e v e l a n d i i 4-6 days a f t e r inoculation were the c l e a r e s t , most r e l i a b l e s i n g l e markers to d i s -E R tin g u i s h the E-, R-, - - and - - i s o l a t e s . I t i s clear from the K. E comparison of the l o c a l - l e s i o n types induced i n t h i s host by each of E R the four i s o l a t e s (Fig. 11), that although - and - are d i s t i n c t from R E t h e i r parent E- and R-isolates, basic s i m i l a r i t i e s i n l e s i o n morphology exist between parents and pseudorecombinants; the lesions induced by E * R - are s i m i l a r to those induced by R, as those induced by - are to E. R E With t h i s i n d i c a t i o n that l o c a l l e s i o n morphology, at least i n c l e v e l a n d i i i s determined by the larger RNA, which i s derived from the bottom component nucleoprotein (B-RNA), other d i f f e r e n t i a l hosts (cf. Table 1) were examined i n t h i s regard. To ensure that r e s u l t s would be comparable, p u r i f i e d preparations (from N_. c l e v e l a n d i i propagations) at a concentration of A,,,- = 0.10 of each of these four i s o l a t e s were used. 260 Opposite half-leaves of Xanthi tobacco and C_. amaranticolor (Figs. 12 and 13, respectively),/were inoculated with the two parental i s o l a t e s , and parental versus pseudorecombinant i s o l a t e s i n order to examine the inheritance of l o c a l l e s i o n determination i n these hosts. From F i g . 12C i t appears - -induced lesions have the morphology of E those induced by the R-isolate, but - -induced lesions (Fig. 12B) are K so d i s t i n c t from those induced by ei t h e r parent (Figs. 12A,B), that no cl e a r assignment of inheritance can be made. Good comparisons of l o c a l -l e s i o n types on opposite half-leaves of Samsun are more d i f f i c u l t to I l l nm I I I I I I I I I I I I I [ I I I ME TRIC | 2 3 Fig. 11. Comparison of l o c a l - l e s i o n symptom types induced by parental and pseudorecombinant isolates 4 days after inoculation on N. c l e v e l a n d i i . B. R-strain (parent) A. E-strain (parent) C. - pseudorecombinant E D. - pseudorecombinant R Ml I RIC I Fig. 12. Comparisons on opposite half-leaves of l o c a l - l e s i o n symptom types induced by parental and pseudorecombinant isolates i n Xanthi tobacco. A. l e f t half: R-strain (parent); right h a l f : E-strain (parent); B. l e f t half: - pseudorecombinant; right half: E-strain (parent); C. l e f t half: - pseudorecombinant; right half: R-strain (parent) Fig. 13. Comparisons on opposite half-leaves of l o c a l - l e s i o n symptom types induced by parental and psuedorecombinant isolates i n C_. amaranticolor. A. l e f t half: R-strain (parent); right h a l f : E-strain parent; B. l e f t half: - -pseudorecombinant; right half: E-strain parent; C. l e f t h a l f : - -pseudorecombinant; right half: R-strain parent 54 o b t a i n , but a very s i m i l a r p a t t e r n of l o c a l l e s i o n symptomatology to that i n Xanthi tobacco i s observed. The pseudorecombinant i s o l a t e s appear to i n h e r i t t h e i r l o c a l - l e s i o n symptomatology i n C_. amaranticolor from the parent c o n t r i b u t i n g the smaller RNA, which i s dervied from the. middle component n u c l e o p r o t e i n (M-RNA) ( F i g . 13); a s i m i l a r p a t t e r n was a l s o observed i n C_. quinoa. The genetics of systemic-symptom determination i n the d i f f e r e n t i a l hosts were a l s o examined by comparing symptoms induced (at equal concen-t r a t i o n s : A 0. 0 = 0.10) by p u r i f i e d parent and pseudorecombinant i s o l a t e s . 2.0V Among the d i f f e r e n t i a l hosts of the genus N i c o t i a n a , Samsun tobacco gave the c l e a r e s t , r e a d i l y d i s t i n g u i s h a b l e systemic symptoms. The pseudorecombinants appear to i n h e r i t t h e i r type of systemic 'map-contour' symptoms from the parent c o n t r i b u t i n g the M-RNA ( F i g . 14). The - -K is.ol.at.e w h i l e a potent l o c a l - l e s i o n inducer on N i c o t i a n a spp. ( c f . Table 4 ) , i s a r e l a t i v e l y poor inducer of systemic symptoms and the in h e r i t a n c e does not appear as c l e a r as i n the R - - p a i r ( F i g s . 14B,D). E A s i m i l a r p a t t e r n of i n h e r i t a n c e i s observed i n Jy]_. c l e v e l a n d i i and Xanthi tobacco. In Chenopodium amaranticolor, where p a r t i c u l a r l y d i s t i n c t systemic symptoms are induced by the p a r e n t a l i s o l a t e s , a c l e a r - c u t determination E of symptom i n h e r i t a n c e i s evident ( F i g . 15). The - - i s o l a t e i n h e r i t s R the c h a r a c t e r i s t i c a p i c a l t u f t i n g and dwarfing from the parent E - i s o l a t e ( F i g s . 15A,C), though i t can be d i s t i n g u i s h e d from the parent by i t s c o n s i s t e n t r e t e n t i o n of dead a p i c a l leaves ( F i g . 15C); the - - i s o l a t e . E i n h e r i t s the p a l i n g l e a f - m o t t l e and s p i n d l y , p a r t l y f a s c i c u l a t e growth from the parent R - i s o l a t e . This p a t t e r n of systemic-symptom determination by M-RNA i s als o 55 F i g . 14. Comparison of systemic symptoms induced i n Samsun tobacco by the p a r e n t a l and pseudorecombinant i s o l a t e s . A. ' F i n e - l i n e ' map-contour symptom induced by E - s t r a i n (parent); B. 'Coarse-line' map-contour symptom induced by R - s t r a i n (parent); C. ' F i n e - l i n e ' map-contour symptom induced by § -pseudorecombinant; R D. 'Coarse-line' map-contour symptom induced by § -pseudorecombinant 56 57 (Fig. 15. Comparison of systemic symptoms induced i n C_. amaranticolor 60 days a f t e r i n o c u l a t i o n w i t h : A. buffered sap from healthy C_. amaranticolor; B. E - s t r a i n (parent); C. R - s t r a i n (parent); E D. - -pseudorecombinant; R E. -pseudorecombinant 59 observed i n C_. quinoa. P l a n t s i n o c u l a t e d w i t h the - pseudorecombinant or the E parent s u r v i v e a systemic m o t t l i n g i n f e c t i o n to bear seed. In c o n t r a s t , p l a n t s i n o c u l a t e d w i t h e i t h e r the - - or R - i s o l a t e r a p i d l y E succumb to an a p i c a l w i l t . Since Jones (1977) has i n d i c a t e d systemic symptoms i n G. globosa are i n h e r i t e d through the B-RNA of the R - s t r a i n , the genetics of symptom determination i n t h i s host were a l s o examined, although r a t h e r high (A^gQ = 0.50) doses of p u r i f i e d v i r u s are u s u a l l y needed to r e l i a b l y induce l e v e l s of symptoms s u i t a b l e f o r comparison, At A^ Q^ = 0.50, E - s t r a i n showed n e i t h e r l o c a l nor systemic symptoms, w h i l e R - s t r a i n showed l o c a l ' f i n e - l i n e ' c h l o r o t i c r i n g s and systemic f l e c k i n g , v e i n •g n e c r o s i s and w i l t . Of the pseudorecombinants, only the - - i s o l a t e R induced systemic symptoms and they appeared s i m i l a r to those induced by the parent R - s t r a i n . This pseudorecombinant a l s o produced l o c a l l e s i o n s but, c o n s i s t i n g of coarse anthocyanescent r i n g s , were c l e a r l y d i f f e r e n t from the R - s t r a i n l e s i o n s . The - - i s o l a t e o c c a s i o n a l l y induced s m a l l numbers of f a i n t f i n e - l i n e l o c a l l e s i o n s that bore resemblance to parent R - s t r a i n l o c a l l e s i o n s , but never induced systemic i n f e c t i o n s . These observations tend to confirm those of Jones (1977) that B-RNA determines the a b i l i t y to induce systemic symptoms i n Cl. globosa. P a r t i c l e component d i s t r i b u t i o n Absorbance (A^^^) scans of p a r t i a l l y - p u r i f i e d (from C_. quinoa) p a r e n t a l and pseudorecombinant i s o l a t e s c e n t r i f u g e d through sucrose gradients show that p a r t i c l e component d i s t r i b u t i o n i s i n h e r i t e d through M-RNA ( F i g . 16). The - - i s o l a t e has the p r o f i l e of i t s E R parent, the - - i s o l a t e that of i t s R parent. 60 K 1 tn s Dapth Fig. 16. Absorbance scan patterns of sucrose density gradients showing relationships of p a r t i c l e component dis t r i b u t i o n s among parental and pseudorecombinant isolates p a r t i a l l y p u r i f i e d from C_. quinoa. A. E-strain (parent) B. R-strain (parent) C. - -pseudorecombinant R D. R -pseudorecombinant 61 Serology The agar-gel Ouchterlony d o u b l e - d i f f u s i o n serology of the parent and pseudorecombinant i s o l a t e s i s shown i n F i g . 17. The p r e c i p i t i n - l i n e p atterns against each of the parent E- and R - s t r a i n a n t i s e r a i n d i c a t e , E by t h e i r confluence, s e r o l o g i c a l i d e n t i t y of the E- and - - i s o l a t e s and R of the R and - - i s o l a t e s . Thus serology, l i k e p a r t i c l e component E d i s t r i b u t i o n , appears determined by the smaller M-RNA. Regeneration of parents from pseudorecombinants .. I... Mixing of 'pure' M-RNA of the - - i s o l a t e w i t h 'pure' B-RNA of the R - - i s o l a t e was p r e d i c t e d to produce an i s o l a t e i d e n t i c a l to the parent E E - s t r a i n , as the r e c i p r o c a l combination was to produce one i d e n t i c a l to the parent R - s t r a i n . These p r e d i c t i o n s appeared to be f u l f i l l e d based on the a v a i l a b l e b i o l o g i c a l and p h y s i c a l evidence. Symptom evidence The d i f f e r e n t i a l hosts ( c f . Table 1) i n o c u l a t e d w i t h heterologous combinations of pseudorecombinant-isolate RNAs were p r e d i c t e d to induce symptoms c h a r a c t e r i s t i c of .:the p a r e n t a l s t r a i n s . The l o c a l - l e s i o n symptoms induced on C_. quinoa and C_. amaranticolor were indeed those of the p r e d i c t e d parents but since the l o c a l symptoms induced by E- • E R and - - i s o l a t e s are i d e n t i c a l , as those induced by the R- and - - i s o l a t e s are, a d d i t i o n a l confirming symptom evidence i s r e q u i r e d . Since the l o c a l - l e s i o n symptoms on N. c l e v e l a n d i i are c h a r a c t e r i s t i c f o r each i s o l a t e , 'regenerated' p a r e n t a l RNA combinations were examined f o r the in d u c t i o n of such symptoms of the p r e d i c t e d parent-type i n f e c t i o n ( F i g . Double-diffusion agar gel serology showing genetic determination of coat protein a n t i g e n i c i t y . A. Central well contains a 1:100 d i l u t i o n of antiserum ( t i t r e = 2560) made against p u r i f i e d E - s t r a i n . Peripheral wells contain preparations E R of p u r i f i e d E - strain(-) and R-strain (-) parents, E R and - - and - -pseudorecombinants at A , = 1.0; R E 2 oU B. Central well contains a 1:50 d i l u t i o n of antiserum ( t i t r e = 1280) made against p u r i f i e d R -strain. Peripheral wells are as i n F i g . 17A. 63 18). The l o c a l - l e s i o n symptoms induced by the regenerated parental RNA combinations are indeed i d e n t i c a l with those induced by inocula of the o r i g i n a l parent s t r a i n s (cf. F i g . 11). The l o c a l - l e s i o n symptoms induced i n Samsun and Xanthi tobacco, although less clear-cut and c h a r a c t e r i s t i c than those of N. c l e v e l a n d i i , conformed to th i s pattern. Systemic symptoms induced on the d i f f e r e n t i a l hosts were also used to test for the i d e n t i t y of 'regenerated' parental RNA combinations. On a l l the d i f f e r e n t i a l hosts examined, the types of induced systemic symptoms conformed to the predicted pattern. The systemic symptomatology on C_. amaranticolor was the most dec i s i v e evidence since the, otherwise s i m i l a r , systemic pathology induced by the parent E- and pseudorecombinant - - i s o l a t e s can be distinguished on the basis of retention of dead K. a p i c a l leaves by the pseudorecombinant (cf. F i g . 15). The regenerated 'parental' RNA combination i n cons i s t e n t l y f a i l i n g to r e t a i n dead a p i c a l leaves shows the systemic symptoms c h a r a c t e r i s t i c of the parent E - i s o l a t e . P u r i f i e d preparations of vi r u s at A = 0.50, propagated i n zoU C_. quinoa from the regenerated parental RNA combinations, and inoculated to CJ. globosa at the four-l e a f stage also produced the predicted parental-i s o l a t e l o c a l and systemic symptoms; the 'regenerated' E - i s o l a t e induced no symptoms while the symptoms of the 'regenerated' R-isolate were i d e n t i c a l with those of the o r i g i n a l parent R-strain inoculated under the same conditions. P a r t i c l e component d i s t r i b u t i o n evidence Absorbance scans at 254 nm a f t e r sucrose gradient c e n t r i f u g a t i o n of p u r i f i e d preparations from C_. quinoa propagations of the pseudore-combinants and the products of the regenerated parental RNA combinations 64 F i g . 18. Comparison of l o c a l - l e s i o n symptoms on _N. c l e v e l a n d i i demonstrating regeneration of p a r e n t a l from pseudorecombinant i s o l a t e s . A. Enlarged l e a f shows symptoms induced by 'regenerated' E - s t r a i n RNA combination. Inset shows symptoms induced by the ' o r i g i n a l ' parent E - s t r a i n (from F i g . 11); B. Enlarged l e a f shows symptoms induced by 'regenerated' R - s t r a i n RNA combination. Inset shows symptoms induced by the ' o r i g i n a l ' parent R - s t r a i n (from F i g . 11). 65 are shown i n F i g . 19. The product of the regenerated-parental RNA combinations showed the presence or absence of top component, and component d i s t r i b u t i o n patterns that would be the properties predicted on the basis of e a r l i e r comparisons of parent s t r a i n s ( Fig. 2) and parent and pseudorecombinant i s o l a t e s ( F ig. 16). Thus, the regenerated E - i s o l a t e , l i k e the natural E - s t r a i n , had no detectable top component and much more B- than M-nucleoprotein; the regenerated R-isolate, l i k e the natural R-strain, has a small top component and about equal amounts of the two nucleoprotein components. The 'regenerated' parental i s o l a t e s also show the same re l a t i o n s h i p s i n p a r t i c l e component d i s t r i b u t i o n s (inheritance through M-RNA) with pseudorecombinant i s o l a t e s as do natural parental i s o l a t e s (Figs. 16 and 19). S e r o l o g i c a l evidence Ouchterlony double-diffusion serology of p u r i f i e d v i r u s at = 1.0 of regenerated parental i s o l a t e s and the pseudorecombinant i s o l a t e s they were generated from, against antisera to the p u r i f i e d v i r u s of the o r i g i n a l parent s t r a i n s , produced a pattern of p r e c i p i t i n l i n e s i d e n t i c a l to that obtained when comparing p u r i f i e d v i r u s of the o r i g i n a l parent s t r a i n s and the pseudorecombinants that had been made from them (cf. F i g . 17). The p r e c i p i t i n l i n e of the regenerated E - i s o l a t e was confluent only with that of the - pseudorecombinant and spurred with that R of the - pseudorecombinant; correspondingly, the regenerated R-isolate E was s e r o l o g i c a l l y i d e n t i c a l to the - pseudorecombinant i t was generated E E from, but spurred with that of the - pseudorecombinant.. 66 Depth F i g . 19. Absorbance scan patterns of sucrose d e n s i t y gradients showing p r e d i c t e d r e l a t i o n s h i p s of p a r t i c l e component d i s t r i b u t i o n s among pseudorecombinant and 'regenerated' p a r e n t a l i s o l a t e s p u r i f i e d from C_. quinoa. A. C. R -pseudorecombinant - -pseudorecombinant E B. 'regenerated' E - i s o l a t e D. 'regenerated' R - i s o l a t e 67 DISCUSSION The observations made i n t h i s work confirm and extend e a r l i e r work on the s t r a i n s and f u n c t i o n a l l y - b i p a r t i t e genome of CLRV by Jones and Mayo (1972), Walkey et a l . (1973) and Jones (1977). The existence of r e l a t e d v i r u s i s o l a t e s w i t h sets of p r o p e r t i e s that d i f f e r c o n s i s t e n t l y i s an e s s e n t i a l requirement f o r any genetic a n a l y s i s by pseudorecombination. In r e p o r t i n g c o n s i s t e n t and s i g n i -f i c a n t d i f f e r e n c e s among the elm, dogwood, cherry, e l d e r b e r r y and rhubarb s t r a i n s of CLRV w i t h respect to serology and p a r t i c l e Immuno-e l e c t r o p h o r e s i s , the work of Walkey et a l . (1973) i n d i c a t e d some p o s s i b l e genetic markers. While the cherry and rhubarb s t r a i n s a l s o d i f f e r e d from the other three by t h e i r higher p r o p o r t i o n of coat p r o t e i n l y s i n e , a l l f i v e s t r a i n s appeared v i r t u a l l y i n d i s t i n g u i s h a b l e by p a r t i c l e sedimentation v e l o c i t i e s or p r o t e i n subunit and .nucleic a c i d component molecular weights. Thus, these l a t t e r p r o p e r t i e s cannot serve as genetic markers of CLRV s t r a i n s . Of the p o s s i b l e markers of d i f f e r e n c e s between r e l a t e d v i r a l i s o l a t e s , symptomatology i n d i f f e r e n t i a l hosts i s probably the most a c c e s s i b l e to examination. I t was a fortunate circumstance that some markedly d i f f e r e n t symptoms on the d i f f e r e n t i a l hosts were observed ( c f . Table 1) when comparing the s e r o l o g i c a l l y - d i s t i n c t but c l o s e l y -r e l a t e d (Walkey et a l . , 1973) e l d e r b e r r y and rhubarb s t r a i n s of CLRV; success i n a c h i e v i n g pseudorecombination appears, g e n e r a l l y , to increase with i n c r e a s i n g s e r o l o g i c a l relatedness of the i s o l a t e s concerned (Jaspars, 1974; Bruening, 1977). Thus the s t r a i n s of CLRV p r e d i c t e d to be most l i k e l y to form productive pseudorecombinants a l s o had the 68 best d i f f e r e n t i a l symptom markers. D i f f e r e n c e s between i s o l a t e s i n p a r t i c l e component d i s t r i b u t i o n have been used as markers i n pseudorecombination genetic analyses. For example, Hartmann e_t a l . (1976), i n analysing the genetics of a l f a l f a mosaic v i r u s (AMV), which has four d i s t i n c t RNA species and a f u n c t i o n a l l y t r i p a r t i t e genome, found the p a r t i c l e component r a t i o to be i n h e r i t e d w i t h the RNA that determines t r y p t i c peptide f i n g e r p r i n t s and thus the coat p r o t e i n . Jones and Mayo (1972) have shown that w i t h the e l d e r b e r r y s t r a i n of CLRV r e l a t i v e proportions of the p a r t i c l e components do not change with i n c r e a s i n g time of i n f e c t i o n . This has been confirmed f o r the rhubarb s t r a i n as w e l l , i n the course of the work reported here. This s t a b i l i t y of p a r t i c l e component d i s t r i b u t i o n w i t h time allows the use of t h i s property as a genetic marker w i t h these two s t r a i n s of CLRV, but may not be taken f o r granted i n s i m i l a r nepovirus systems; Schneider and Diener (1966) have shown that the d u r a t i o n of i n f e c t i o n before h a r v e s t i n g of i n f e c t e d Phaseolus v u l g a r i s cv. Black V a l e n t i n e a f f e c t s the r e l a t i v e proportions of p u r i f i e d p a r t i c l e components of two s t r a i n s of TRSV. ' The other e s s e n t i a l requirement f o r s u c c e s s f u l pseudorecombination genetic a n a l y s i s , the "separation of components, n u c l e o p r o t e i n or RNA, that are complementary i n i n f e c t i o n , i s r e l a t i v e l y d i f f i c u l t to f u l f i l l i n CLRV because of the p r o p o r t i o n a l l y small d i f f e r e n c e s i n molecular weight and sedimentation v e l o c i t y of the n u c l e o p r o t e i n and RNA components. Walkey et a l . (1973), using s i n g l e - c y c l e s e p a r a t i o n by sucrose d e n s i t y -gradient c e n t r i f u g a t i o n , d i d not o b t a i n separation of the two nucleo-p r o t e i n components and found each 'component' to be h i g h l y i n f e c t i o u s 69 on i t s own. Jones and Mayo (1972) employed m u l t i p l e - c y c l e sucrose gradient c e n t r i f u g a t i o n and obtained enhancement-of-infectivity r a t i o s from 4 to 8. Moreover, i n some experiments, they succeeded i n o b t a i n i n g middle component nu c l e o p r o t e i n f r e e of i n f e c t i v i t y . Based on the p e r s i s t e n t r e s i d u a l i n f e c t i v i t y of bottom component n u c l e o p r o t e i n , however, i t appeared the technique was unable to produce bottom component preparations e n t i r e l y f r e e of middle component. Nucleoprotein components can o f t e n be more h i g h l y r e s o l v e d by sepa r a t i o n techniques based on d i f f e r e n c e s i n p a r t i c l e d e n s i t y r a t h e r than sedimentation v e l o c i t y . Thus, bromoviruses, which sediment as s i n g l e components i n r a t e - z o n a l sucrose g r a d i e n t s , can be resolved i n t o three c l o s e l y - s p a c e d e q u i l i b r i u m - d e n s i t y bands i n CsCl (Lane and Kaesberg, 1971). In CsCl e q u i l i b r i u m a n a l y t i c a l u l t r a c e n t r i f u g a t i o n , the n u c l e o p r o t e i n components of the rhubarb s t r a i n of CLRV were c l e a r l y -3 resolved as bands at 1.471 (middle) and 1.508 (bottom) g»cm . The RNA prop o r t i o n s by weight of the middle and bottom component n u c l e o p r o t e i n s , obtained by ap p l y i n g these e q u i l i b r i u m d e n s i t y values to the e m p i r i c a l formula of Sehgal et^ a l . (1970) are 38.5 and 42.3%, r e s p e c t i v e l y . Using values f o r n u c l e o p r o t e i n p a r t i c l e and RNA molecular weights of the el d e r b e r r y and rhubarb s t r a i n s of CLRV, obtained by c a l c u l a t i o n from the r e s u l t s of Walkey eX a_l. (1973), and confirmed f o r the preparations of these two s t r a i n s a c t u a l l y used i n t h i s work (Ramsdell; unpublished r e s u l t s ) , the middle component n u c l e p r o t e i n c o n s i s t e d of 38.8%, the bottom component 42.0% RNA by weight. This i s i n good agreement w i t h the r e s u l t s c a l c u l a t e d from the buoyant d e n s i t i e s . The buoyant de n s i t y values obtained i n the p r e p a r a t i v e CsCl c e n t r i -fugations are lower than those observed i n a n a l y t i c a l u l t r a c e n t r i f u g a t i o n 70 because the n u c l e o p r o t e i n preparations were c e n t r i f u g e d through CsCl f o r about 18 h, s u f f i c i e n t l y long to achieve good r e s o l u t i o n . Longer c e n t r i f u g a t i o n times were not used, so as to avoid p a r t i c l e d i s i n t e g r a t i o n , e s p e c i a l l y of the middle component, and the l o s s of p a r t i c l e i n f e c t i v i t y . A true e q u i l i b r i u m density under the p r e p a r a t i v e c o n d i t i o n s described would take over 400 h to achieve ( B i r n i e , 1978). Thus the p r e p a r a t i v e separation i n CsCl brings the components only to a n e a r - e q u i l i b r i u m d e n s i t y . In both s t r a i n s examined i n t h i s work, the A„^_/A r a t i o s of the zoU zoU p u r i f i e d middle and bottom component nucleoproteins were 1.64-1.66 and 1.81-1.83, r e s p e c t i v e l y . These r a t i o s and the n u c l e o p r o t e i n component r a t i o s account together f o r the A„rr./A r a t i o s of the p u r i f i e d 'whole-Z o U z o U v i r u s ' preparations (from N. c l e v e l a n d i i ) of the two s t r a i n s . The rhubarb s t r a i n w i t h i t s about equimolar proportions of n u c l e o p r o t e i n components has an A„.n/A r a t i o of 1.71-1.72 ( i n agreement with Walkey Z o U zoU et a l . , 1973), about half-way between the values f o r the separated components, w h i l e the e l d e r b e r r y s t r a i n w i t h an approximately two-to-one preponderance of the bottom component ( c f . F i g . 3) has a p r o p o r t i o n a l l y higher r a t i o of 1.76-1.77. A l l the r e s o l u t i o n of components was achieved by CsCl near-e q u i l i b r i u m c e n t r i f u g a t i o n at the n u c l e o p r o t e i n l e v e l . The technique used f o r e x t r a c t i n g the RNA o r d i n a r i l y used f o r a n a l y t i c a l purposes gave, i n the A^ ,.^  scans of the sucrose g r a d i e n t s , i n f o r m a t i o n that could confirm that the e x t r a c t e d RNAs were monodisperse products of separate components, and i n d i c a t e by the sharpness of the absorbance peak on the l e a d i n g edge, the q u a l i t y or l a c k of degradation i n the RNA preparation 71 (cf F i g . 9). No a d d i t i o n a l r e s o l u t i o n per se, however, was accomplished at t h i s step.. The RNA preparations obtained by t h i s technique are easy to measure fo r concentration by absorbance at 260 nm, immediately a v a i l a b l e i n a form s u i t a b l e f o r i n o c u l a t i o n and are r e a d i l y stored by f r e e z i n g at -20 C without apparent l o s s of i n f e c t i v i t y . In a d d i t i o n , t h i s RNA-component pr e p a r a t i o n p r o t o c o l , u n l i k e the separation of RNA components by polyacrylamide g e l e l e c t r o p h o r e s i s (Murant e_t a l . , 1972) , gives separated nucleoproteins as w e l l as separated RNA-components i n the same run. A major o b j e c t i o n to the use of CsCl f o r p r e p a r a t i v e purposes, the high cost of s u i t a b l e p u r i f i e d grades, i s p a r t i a l l y met by the use of a cheaper, t e c h n i c a l grade s u f f i c i e n t l y p u r i f i e d by f i l t r a t i o n and c h e l a t i o n of contaminating d i v a l e n t c a t i o n s ( B i r n i e , 1978). The pseudorecombination genetics of CLRV described i n t h i s study are of a s i m i l a r p a t t e r n to those observed i n e a r l i e r work on other nepoviruses. As w i t h RRV (Harrison et_ a l . , 1972; H a r r i s o n et a l , 1974b) and TBRV (Randies et a l . , 1977; H a r r i s o n and Murant, 1977b) and pr e l i m i n a r y work on CLRV (Jones, 1977), the smaller M-RNA determines serology and thus the a n t i g e n i c i t y of the c o a t - p r o t e i n polypeptide. Indeed, the only f u n c t i o n a l l y multipartite-genome p l a n t v i r u s e s known not to have p r o p e r t i e s of the coat p r o t e i n determined by the s m a l l e s t f u n c t i o n a l RNA species are CRSV (Dodds et a l . , 1977) and PEMV ( H u l l and Lane, 1973). The presence or absence of detectable amounts of RNA-free top-component p a r t i c l e s was a l s o found i n t h i s study to be i n h e r i t e d through M-RNA. Although the genetics of t h i s property have not been examined w i t h other nepoviruses, a s i m i l a r p a t t e r n >o£ i n h e r i t a n c e through the smaller RNA was described i n CPMV-yellow s t r a i n by Bruening 72 (1969), and i n t e r p r e t e d as a f u n c t i o n of capsid p r o t e i n production and not as an a l l e l e of another gene on the smaller RNA. The genetic determination of p a r t i c l e component d i s t r i b u t i o n , found i n t h i s work to be as s o c i a t e d w i t h M-RNA, has not been examined i n other nepoviruses. Such examinations have been c a r r i e d out with PEMV ( H u l l and Lane, 1973), AMV (Hartmann et a l . , 1976), CPMV-yellow s t r a i n (Wood, 1972) and CPMV-severe s t r a i n (Thongneearkom and Goodman, 1978). In the f i r s t three cases, p a r t i c l e component d i s t r i b u t i o n appears determined by the same RNA that determines coat p r o t e i n , suggesting, that l i k e the formation of top component, t h i s property may be a f u n c t i o n of the capsid p r o t e i n . In the case of CPMV-severe s t r a i n , one of the pseudorecombinants had a p a r t i c l e component d i s t r i b u t i o n u n l i k e that of e i t h e r parent (Thongmeearkom and Goodman, 1978). Since the two parent s t r a i n s d i d not d i f f e r i n p a r t i c l e component d i s t r i b u t i o n , i t was suggested that each RNA component might i n some manner be determining the r a t e of synthesis or encapsidation of the other RNA. The determination of symptom types by d i f f e r e n t RNAs, depending on host, and i n the N i c o t i a n a spp. on type of i n f e c t i o n , l o c a l or systemic, lac k s d i r e c t counterparts i n e a r l i e r genetic analyses of other nepoviruses. I t does correspond i n p a t t e r n to the observation i n RRV (Harrison et^ a l . , 1974b), where the l a r g e r RNA determines the a b i l i t y to i n f e c t non-inoculated leaves of Phaseoliis v u l g a r i s L., w h i l e the smaller RNA (except when i n a s s o c i a t i o n w i t h the l a r g e r RNA of the Llo y d George s t r a i n ) determines systemic y e l l o w i n g symptoms i n Petunia  hybrida Vilm. The observation i n the present work, that the lar-iger RNA (B-RNA) of the rhubarb s t r a i n confers the a b i l i t y to s y s t e m i c a l l y i n f e c t G_. globosa agrees w i t h the p r e l i m i n a r y report of Jones (1977) . 73 Pseudorecombination genetic, a n a l y s i s can only be v a l i d i f the i s o l a t e s prepared by mixing heterologous, complementing nucleoproteins or RNAs of two d i f f e r e n t v i r a l i s o l a t e s are t r u l y pseudorecombinant and not merely f o r t u i t o u s l y modified by the treatment undergone to e x h i b i t a l t e r e d p h y s i c a l and b i o l o g i c a l p r o p e r t i e s . Regenerating i s o l a t e s , i d e n t i c a l i n a l l observable p r o p e r t i e s to the p a r e n t a l i s o l a t e t hat i s the p r e d i c t e d outcome of the 'pseudorecombination a p p l i e d to the pseudorecombinants', provides strong confirming evidence of the v a l i d i t y of the genetic a n a l y s i s . Besides the present work, such a con f i r m a t i o n of nepovirus pseudorecombinant genetic a n a l y s i s by backcrossing to regenerate the p r e d i c t e d p a r e n t a l types has been reported f o r RRV (Harrison et_ al., 1974b) but not f o r TBRV, the only other nepovirus h i t h e r t o reported i n the l i t e r a t u r e to have undergone pseudorecombination genetic a n a l y s i s (Randies e_t a l . , 1977) . T his i s because only one of the two p a r e n t a l pseudorecombinants could be generated i n the f i r s t place. There are s t i l l many aspects of nepovirus genetics that await i n v e s t i g a t i o n by pseudorecombinant genetic a n a l y s i s . For CLRV i n p a r t i c u l a r , the genetics of nematode- and s e e d - t r a n s m i s s i b i l i t y remain to be determined. Determination of the former i s u n l i k e l y to be a p a r t i c u l a r l y p r o f i t a b l e l i n e of i n q u i r y . In the f i r s t p l a c e , the s p e c i f i c t ransmission of CLRV by nematodes has yet to be unequivocally demonstrated. A l s o , the evidence that s p e c i f i c nematode trans m i s s i o n i s a f u n c t i o n of the capsid p r o t e i n (Harrison e t a l . , 1974a) and the observed determination of t h i s property i n RRV (Harrison et a l . , 1974a,b) and TBRV (Ha r r i s o n , 1977b) by the c o a t - p r o t e i n determining smaller RNA, suggests that no new i n s i g h t i n t o nepovirus genetics would be gained. The genetic a n a l y s i s of "seed transmission i s another matter, however, 74 since the analyses of t h i s property i n RRV and TBRV (Hanada and H a r r i s o n , 1977), w h i l e suggesting a predominant i n f l u e n c e of the l a r g e r RNA, are not c o n c l u s i v e . Moreover, u n l i k e TBRV (Hanada and H a r r i s o n , 1977), the p o s s i b i l i t y e x i s t s w i t h CLRV to confirm r e s u l t s w i t h at l e a s t two pseudorecombinant i s o l a t e s and corresponding backcrosses that r e -generate p r e d i c t e d p a r e n t a l i s o l a t e s . Recent d i s c o v e r i e s about two d e f i n i t i v e nepoviruses, TRSV and TomRSV, suggest e x c i t i n g areas of f u t u r e i n q u i r y . H a r r i s o n and Barker (1978) have shown that a small p r o t e a s e - s e n s i t i v e s t r u c t u r e , c o v a l e n t l y attached (probably at the 5'-end) to both RNA components, i s r e q u i r e d f o r i n f e c t i v i t y . I f , by o l i g o p e p t i d e a n a l y s i s , or some other form of a n a l y s i s capable of d i s t i n g u i s h i n g small s i m i l a r peptides, t h i s RNA-peptide can be shown to be s t r a i n - s p e c i f i c , pseudorecombination a n a l y s i s might r e v e a l which RNA codes f o r i t . Of course, enormous t e c h n i c a l d i f f i c u l t i e s would e x i s t , mainly i n o b t a i n i n g enough of t h i s RNA-peptide to work w i t h i n the f i r s t p l ace. Probably beyond the reach of pseudorecombination a n a l y s i s alone i s the question: What are the immediate gene products coded f o r by the p l a n t v i r a l genome? I t now appears l i k e l y , from the recent work of Chu and F r a n c k i (1979) that only one-quarter of the p r e v i o u s l y estimated coding c a p a c i t y i n the smaller RNA i s r e q u i r e d i n nepoviruses f o r the coat p r o t e i n polypeptide subunit. There i s thus, as w i t h a l l p l a n t v i r u s e s , a great deal of genetic information to which we as yet can a s c r i b e no s p e c i f i c product or f u n c t i o n . SUMMARY The e l d e r b e r r y (E) and rhubarb (R) s t r a i n s of cherry l e a f r o l l v i r u s , a nepovirus, were found to d i f f e r c o n s i s t e n t l y i n p a r t i c l e com-ponent d i s t r i b u t i o n , serology and symptomatology i n a number of i n d i c a t o r host species. The separation of the two genetic components, middle- (M) and bottom- (B) component RNA, of both E- and R - s t r a i n s were confirmed by evidence from sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n and p o l y a c r y l -amide g e l e l e c t r o p h o r e s i s of RNA as w e l l as by the much greater i n f e c t -i v i t y of combined-component compared to separated-component i n o c u l a . Stable pseudorecombinant i s o l a t e s generated by heterologous combination of complementing RNA components, when compared w i t h the o r i g i n a l p a r e n t a l s t r a i n s , i n d i c a t e d the genetics of the marker p r o p e r t i e s that were used to d i s t i n g u i s h between the two s t r a i n s . M-RNA determined s e r o l o g i c a l s p e c i f i c i t y , p a r t i c l e component d i s t r i b u t i o n , systemic symptoms i n N i c o t i a n a c l e v e l a n d i i and N. tabacum cvs. Samsun and X a n t h i , and l o c a l and systemic symptoms i n Chenopodium amaranticolor and C. quinoa. B-RNA determined the a b i l i t y to induce systemic symptoms i n Gomphrena globosa and the type of l o c a l l e s i o n i n N. c l e v e l a n d i i . 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