PUCCINIA GRAMINIS f . s p . TRITICI, RACE C17: PHYSIOLOGY OF UREDOSPORE GERMINATION AND GERMTUBE DIFFERENTIATION By S a r a h J . Hopkinson B.Sc, The U n i v e r s i t y o f V i c t o r i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © Sarah Jane Hopkinson, 1988 In presenting degree this at the thesis in University of partial fulfilment British Columbia, freely available for reference and study. copying of department this or thesis by for scholarly his or publication of this thesis her P\c\rVv DE-6 (2/88) hnw \Q requirements for an advanced I agree that the Library shall make it purposes may representatives. It be is granted by the head understood that of my copying or for financial gain shall not be allowed without my written *"Sr.\cy,r\c v>. The University of British Columbia Vancouver, Canada Date the I further agree that permission for extensive permission. Department of of ABSTRACT G e r m i n a t i n g u r e d o s p o r e s o f r a c e C17 of Puccinia graminis f . s p . t r i t i c i form c h a r a c t e r i s t i c i n f e c t i o n s t r u c t u r e s ( a p p r e s s o r i u m , i n f e c t i o n peg, r e s p o n s e t o a 1.5 germination a t 19° h h e a t shock a t 29° C. The i n f e c t i o n s t r u c t u r e s was a l c o h o l and, v e s i c l e , i n f e c t i o n hypha) i n C administered 2 h after p r o p o r t i o n of s p o r e l i n g s forming augmented by n u t r i e n t s , n - n o n y l an a p p r o p r i a t e l y t i m e d h e a t shock. The heat shock t e m p e r a t u r e r e q u i r e d t o i n d u c e maximum d i f f e r e n t i a t i o n had a v e r y p r e c i s e optimum w h i c h v a r i e d s l i g h t l y f o r each spore l o t . V a r i a t i o n s one degree above o r below t h i s optimum r e d u c e d t h e p e r c e n t d i f f e r e n t i a t i o n by g r e a t e r t h a n 40%. The p r e s e n c e o f an i n h i b i t o r o f p r o t e i n s y n t h e s i s , p u r o m y c i n , i n the germination medium: (1) p r e v e n t e d uredosporeling d i f f e r e n t i a t i o n b u t had no e f f e c t on g e r m i n a t i o n , (2) s i g n i f i c a n t l y reduced t h e p r o p o r t i o n o f germtubes f o r m i n g a p p r e s s o r i a , and germtube n u c l e i . (3) i n most c a s e s p r e v e n t e d t h e d i v i s i o n o f I t was concluded t h a t e s s e n t i a l d i f f e r e n t i a t i o n - s p e c i f i c p r o t e i n s are synthesized onset of germination, throughout the formation and t o t h e c o m p l e t i o n o f d i f f e r e n t i a t i o n . of from t h e appressoria These r e s u l t s were c o n s i s t e n t w i t h t h e observed e f f e c t s o f h e a t shock on t h e of p r o t e i n h y d r o l y s i s . During germination t h e r e was a h y d r o l y s i s o f p r o t e i n l e a d i n g t o an i n c r e a s e i n s i z e o f endogenous p o o l o f f r e e amino a c i d s and t o an increased rate net the leakage of amino acids to the germination medium. Heat shock e f f e c t i v e l y reduced the amount of endogenous free amino acids and the extent to which amino acids were l o s t to the medium. I t was concluded that i n heat shocked sporelings protein synthesis was increased r e l a t i v e to protein hydrolysis by comparison with the r e l a t i v e rates of these two processes i n germinating was (non-shocked) uredosporelings. Moreover, there no net protein synthesis during the formation of structures induced by heat shock. the germination medium was shocked sporelings. infection The loss of amino acids to selective, p a r t i c u l a r l y i n heat iv TABLE OF CONTENTS Page Number Abstract i i L i s t of Tables viii L i s t of Figures x Acknowledgements xiii 1. Introduction 1 2. L i t e r a t u r e Review 6 2.1 2.2 2.3 U r e d o s p o r e G e r m i n a t i o n and Morphogenesis 2.1.1 G e r m i n a t i o n I n h i b i t o r s and S t i m u l a n t s .... 6 2.1.2 Thigmodifferentiation 7 2.1.3 Thermodifferentiation 8 2.1.4 Chemodifferentiation 9 2.1.5 Mechanisms o f D i f f e r e n t i a t i o n 9 2.1.6 Role of I n f e c t i o n Structures 10 C y t o l o g i c a l Events 2.2.1 N u c l e a r Changes 11 2.2.2 P r o t e i n Metabolism 13 N u t r i t i o n a l Requirements o f R u s t F u n g i 2.3.1 P h y s i o l o g y o f t h e H o s t - P a r a s i t e Complex . 2.3.2 A x e n i c C u l t u r e and M e t a b o l i s m 17 N u t r i t i o n a l Requirements 20 Synthetic Capacity 21 Sulphur Metabolism 22 M e t a b o l i t e Leakage 22 Endogenous F r e e Amino A c i d s 24 E x p e r i m e n t a l Methods 26 3.1 P r o d u c t i o n and C o l l e c t i o n o f Spores 26 3.2 Spore G e r m i n a t i o n 27 3.3 Spore G e r m i n a t i o n w i t h t h e I n d u c t i o n t o D i f f e r e n t i a t e 27 3.4 C r i t e r i a f o r t h e Assessment o f S p o r e l i n g Development 27 3.5 S t a i n i n g and Counts 30 3.6 Temperature Range T r i a l s 30 3.7 E s s e n t i a l P r o t e i n S y n t h e s i s : Puromycin 33 3.8 N u c l e a r S t a i n i n g : DAPI 35 3.9 Amino A c i d A n a l y s i s 37 3.9.1 H i g h Performance L i q u i d Chromatography .. 37 3.9.2 Reagents 39 3.9.3 Instrument 40 3.9.4 Sample C o l l e c t i o n 40 3.9.5 Sample Clean-up 43 3.9.6 HPLC o f Amino A c i d s as P h e n y l t h i o c a r b a m o y l Derivatives B u f f e r System 45 Pre-Column D e r i v a t i z a t i o n 45 Sample P r e p a r a t i o n 46 Chromatography 46 VI Results 50 4.1 Uredospore G e r m i n a t i o n and D i f f e r e n t i a t i o n 50 4.2 Temperature Range T r i a l s 4.3 I n f l u e n c e o f N u t r i e n t s on S p o r e l i n g D i f f e r e n t i a t i o n 53 60 4.4 Essential Protein Synthesis 62 4.5 C y t o l o g y o f U r e d o s p o r e l i n g Development 4.5.1 Nuclear Staining 67 4.5.2 Nuclear Behaviour d u r i n g Germination and Differentiation 4.6 68 Amino A c i d A n a l y s i s 75 4.6.1 Experimental Plan 75 4.6.2 Complications 78 4.6.3 Exogenous F r e e Amino A c i d s : R e s t i n g Spore Wash 81 4.6.4 Endogenous F r e e Amino A c i d s : E x t r a c t s o f Unwashed R e s t i n g Uredospores 4.6.5 83 E f f e c t o f Heat Shock on t h e F r e e Amino A c i d s i n Germinated Uredospores A f t e r Germination and i n t h e Leachate 85 V l l Discussion 5.1 89 Temperature Requirements f o r the D i f f e r e n t i a t i o n of P. g r a m i n i s t r i t i c i U r e d o s p o r e l i n g s 5.2 The I n f l u e n c e o f N u t r i e n t s on 89 Uredospore Differentiation 90 5.3 The T i m i n g o f E s s e n t i a l P r o t e i n S y n t h e s i s 92 5.4 N u c l e a r B e h a v i o u r Accompanying G e r m i n a t i o n and 5.5 Differentiation 95 5.4.1 97 S t a i n i n g w i t h DAPI The E f f e c t o f Heat Shock on t h e Amounts and K i n d s o f F r e e Amino A c i d s i n G e r m i n a t i n g Uredospores and Leachates their 99 Summary 107 Literature Cited 109 V l l l LIST OF TABLES Page Number I. The c o m p o s i t i o n and p r e p a r a t i o n o f t h e Ca-K-P0 4 buffer and t h e v o l a t i l e g e r m i n a t i o n s t i m u l a n t , n - n o n y l a l c o h o l 28 II. The c o m p o s i t i o n and p r e p a r a t i o n of the d i f f e r e n t i a t i o n medium, MPG III. 29 L a c t o p h e n o l - T r y p a n b l u e ; a mounting and s t a i n i n g medium f o r semipermanent mounts o f f u n g i IV. Timing o f e s s e n t i a l p r o t e i n synthesis: 31 Experimental design 34 V. A d e s c r i p t i o n of the f i x a t i o n schedules t e s t e d 36 VI. Pump method; t h e b u f f e r g r a d i e n t system and f l o w rate VII. 48 A summary o f e x p e r i m e n t s i n v e s t i g a t i n g t h e i n f l u e n c e o f t h e components o f MPG, n-nonyl a l c o h o l , and h e a t shock on t h e p e r c e n t a g e o f g e r m l i n g s d e v e l o p i n g i n f e c t i o n structures 61 V I I I . The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin, on t h e development o f i n f e c t i o n s t r u c t u r e from g e r m i n a t i n g uredospores 63 ix IX. The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin, on t h e development o f i n f e c t i o n s t r u c t u r e from g e r m i n a t i n g uredospores. E x p r e s s e d as a mean p e r c e n t o f t h e preceding structure X. 66 PITC-amino a c i d peaks i d e n t i f i e d by number and r e t e n t i o n time XI. 77 The f r e e amino a c i d c o m p o s i t i o n o f t h e b u f f e r wash from ungerminated u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i XII. ... 82 Endogenous f r e e amino a c i d l e v e l i n unwashed r e s t i n g u r e d o s p o r e s , and i n non-shocked u r e d o s p o r e l i n g s a f t e r 8 h and 20 h 84 X I I I . The f r e e amino a c i d c o n t e n t o f unwashed r e s t i n g u r e d o s p o r e s , and i n h e a t shocked u r e d o s p o r e l i n g s a f t e r 8 h and 20 h XIV. 86 The f r e e amino a c i d c o m p o s i t i o n o f l e a c h a t e from h e a t shocked and non-shocked u r e d o s p o r e l i n g s a f t e r an 8 h and 20 h i n c u b a t i o n p e r i o d XV. 87 The d i s t r i b u t i o n o f t h e f r e e amino a c i d s a s s o c i a t e d w i t h r e s t i n g s p o r e s , non-shocked and h e a t uredosporelings shocked 100 X LIST OF FIGURES Page Number 1. G e r m i n a t i o n and d i f f e r e n t i a t i o n o f t h e wheat r u s t pathogen, P. g r a m i n i s t r i t i c i , on t h e c e r e a l h o s t . . 2. The c h e m i c a l s t r u c t u r e o f DAPI ( 4 ' , 6 - d i a m i d i n o - 2 phenyl indole) 3. 3 38 HPLC amino a c i d a n a l y s i s system c o n f i g u r a t i o n f o r RPa n a l y s i s o f amino a c i d s w i t h post-column u l t r a v i o l e t detection 4. Sample p r e p a r a t i o n scheme f o r c r u d e amino a c i d u s i n g Sep-Pak C 5. 41 1 8 cartridges 44 R e a c t i o n scheme o f amino compound by pre-column d e r i v a t i z a t i o n w i t h PITC 6. samples . 47 The p e r c e n t g e r m i n a t i o n o f u r e d o s p o r e s on Ca-K b u f f e r i n b o t h t h e p r e s e n c e and t h e absence o f n - n o n y l alcohol 51 7. T i m i n g o f u r e d o s p o r e l i n g morphogenesis 52 8. Temperature r e q u i r e m e n t s f o r a p p r e s s o r i u m f o r m a t i o n from u r e d o s p o r e germtubes 9. 54 Temperature r e q u i r e m e n t s f o r t h e complete d i f f e r e n t i a t i o n of uredospore germlings 10. The r e l a t i o n s h i p between h e a t shock t e m p e r a t u r e and t h e t r a n s f o r m e d p e r c e n t o f a p p r e s s o r i a formed 11. 56 58 The r e l a t i o n s h i p between h e a t shock t e m p e r a t u r e and t h e transformed percent of t o t a l differentiation sporeling 59 XI 12. The p r e s e n c e o f a p r o t e i n i n h i b i t o r (puromycin) i n the g e r m i n a t i o n medium s i g n i f i c a n t l y reduced t h e number o f germtubes f o r m i n g a p p r e s s o r i a 13. 64 F o l l o w i n g 2 h g e r m i n a t i o n two i n t e r p h a s e n u c l e i . m i g r a t e d from t h e u r e d o s p o r e i n t o t h e e l o n g a t i n g germtube... 14. The germtube o f non-shocked u r e d o s p o r e l i n g s remained b i n u c l e a t e f o r up t o 20 h 15. 69 69 I n some c a s e s t h e n u c l e i w i t h i n 2 0 - h o u r - o l d u r e d o s p o r e l i n g s were e l o n g a t e d i n form and had a "ragged" appearance 16. 69 Four h o u r s f o l l o w i n g g e r m i n a t i o n t h e two n u c l e i m i g r a t e d i n t o t h e newly formed a p p r e s s o r i u m i n i t i a l . Their e l o n g a t e d form suggested t h a t t h e s e n u c l e i were i n c o n j u g a t e t e l o p h a s e and p r e p a r i n g t o d i v i d e 17. The f i r s t round o f m i t o s i s u s u a l l y o c c u r r e d w i t h i n t h e mature a p p r e s s o r i u m 5 t o 8 h a f t e r g e r m i n a t i o n 18. 72 Ten h o u r s f o l l o w i n g g e r m i n a t i o n t h e s i x - n u c l e a t e v e s i c l e was d e l i m i t e d from t h e a p p r e s s o r i u m by a septum.... 21. 70 O c c a s i o n a l l y two n u c l e i d i v i d e d w i t h i n t h e appressorium 20. 70 The mature a p p r e s s o r i u m c o n t a i n e d f o u r d a u g h t e r nuclei 19. 70 72 I n most c a s e s a n u c l e a r p a i r d i v i d e d i n t h e mature v e s i c l e to y i e l d a t o t a l of eight nuclei 72 X l l 22. The o r i e n t a t i o n o f n u c l e a r m i g r a t i o n a l p a t t e r n s was found t o be r e l a t e d t o t h e p o l a r i t y o f t h e vesicle 23. 73 The i n f e c t i o n hypha c o n t a i n e d e i g h t , s i x , o r more commonly, f o u r n u c l e i 24. 73 I n some c a s e s a n u c l e a r p a i r f a i l e d t o m i g r a t e with o t h e r n u c l e i and remained i n t h e v e s i c l e 25. Twenty t h r e e h o u r s f o l l o w i n g g e r m i n a t i o n t h e i n f e c t i o n hypha most o f t e n c o n t a i n e d f o u r expanded n u c l e i . . . . 26. 73 S e p a r a t i o n o f amino a c i d s t a n d a r d by r e v e r s e - p h a s e 73 high- p r e s s u r e l i q u i d chromatography 27. 76 R e p r e s e n t a t i v e RP-HPLC s e p a r a t i o n s o f amino a c i d s i s o l a t e d from non-shocked 2 0 - h o u r - o l d and t h e l e a c h a t e o f 2 0 - h o u r - o l d uredosporelings h e a t shocked uredosporelings 28. 79 Diagrammatic r e p r e s e n t a t i o n o f t h e n u c l e a r b e h a v i o u r o f d i f f e r e n t i a t i n g u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i , C17 race 96 xiii Acknowledgements I would l i k e t o t h a n k t h e p e o p l e t o whom I owe my p e r s o n a l s a n i t y and t h e f i n a l c o m p l e t i o n o f t h i s t h e s i s . A special t h a n k y o u t o Dr. L i n d a Boasson f o r h e r companionship i n t h e l a b and h e r a l w a y s h e l p f u l suggestions. Thankyou t o Dr. M i c h a e l Shaw f o r t h e many days s p e n t r e v i e w i n g t h e r e s u l t s and h i s i n v a l u a b l e a s s i s t a n c e t h r o u g h a l l aspects o f t h i s t h e s i s . T h i s work was s u p p o r t e d by a g r a n t from t h e N a t u r a l S c i e n c e and E n g i n e e r i n g Research Committee t o Dr. M i c h e a l Shaw. 1 1. INTRODUCTION R u s t f u n g i a r e b i o t r o p h i c pathogens on p l a n t s , g r e a t l o s s e s t o many c u l t i v a t e d c r o p s . The wheat pathogen, P u c c i n i a g r a m i n i s P e r s . f.sp. t r i t i c i b l a c k stem r u s t o f and E. Henn. r a n k s among t h e more s e r i o u s o f c e r e a l The stem r u s t i s m a c r o c y c l i c causing and h e t e r o e c i o u s ; Eriks diseases. t h a t i s , the l i f e c y c l e e x h i b i t s f i v e d i s t i n c t s p o r e s t a g e s on two The u r e d i a l s t a g e , w h i c h i s t h e most d e s t r u c t i v e hosts. economically, p e r p e t u a t e s t h e fungus t h r o u g h o u t t h e g r o w i n g season by r e i n f e c t i n g the c e r e a l host. R u s t f u n g i t h a t appear m o r p h o l o g i c a l l y identical but i n f e c t d i f f e r e n t h o s t genera a r e c l a s s e d as formae s p e c i a l e s (f.sp.). W i t h i n each forma s p e c i a l i s t h e r e a r e many p h y s i o l o g i c a l r a c e s w h i c h a r e p a t h o g e n i c on o n l y c e r t a i n v a r i e t i e s w i t h i n the host species. r a c e C17 study. ( f o r m e r l y r a c e 56), was The s u b r a c e 56A P. g r a m i n i s f . s p . employed i n t h e tritici. current r a c e i s g e n e t i c a l l y s t a b l e w i t h o n l y one rare known. and I t i s highly aggressive on wheat c a p a b l e o f c a u s i n g huge economic l o s s e s i n N o r t h A m e r i c a . The s p e c i f i c r e a c t i o n of a host c u l t i v a r t o a s e r i e s of p h y s i o l o g i c a l r a c e s i s d e t e r m i n e d by complementary genes f o r r e s i s t a n c e i n t h e h o s t and v i r u l e n c e genes i n t h e pathogen (i.e. by gene-for-gene r e a c t i o n s ) ( F l o r 1971). I n most c a s e s i n c o m p a t i b i l i t y ( r e s i s t a n t r e a c t i o n ) r e s u l t s from t h e i n t e r a c t i o n o f a dominant gene f o r r e s i s t a n c e (R) i n t h e h o s t 2 and a complimentary dominant gene f o r avirulence pathogen. A l l other reactions (P) i n the (R-pp, rrP-, and rrpp) r e s u l t i n host-pathogen compatibility (susceptible reaction). Infection of a host plant by the rust fungi i s characterized by a series of morphological events (Figure 1). The uredospore germinates r e a d i l y on water. The germtube of wheat stem rust grows across the c u t i c u l a r ridges of the l e a f blade; upon contact with a stomatal pore germtube elongation ceases and d i f f e r e n t i a t i o n begins. D i f f e r e n t i a t i o n involves the sequential development of i n f e c t i o n structures: appressorium (app), the i n f e c t i o n peg the ( i p ) , the substomatal v e s i c l e (ssv), and the i n f e c t i o n hypha ( i h ) . Once established, the mycelium grows primarily i n an i n t r a c e l l u l a r fashion, obtaining nutrients from host c e l l s through s p e c i a l i z e d structures c a l l e d haustoria. Nutrients are also absorbed by i n t e r c e l l u l a r mycelium. The susceptible host i s able to both induce or a s s i s t fungal morphogenesis and provide the balance of nutrients e s s e n t i a l f o r further growth. The a b i l i t y to culture the wheat rust pathogen axenically was f i r s t demonstrated using an Australian race of wheat stem rust (Williams et a l . 1966). Research u t i l i z i n g axenic culture can provide regarding the biology the rust fungi. information (metabolism, n u t r i t i o n , and genetics) of Unfortunately, serious obstacles such as the very slow growth rates of these fungi, and t h e i r unpredictable n u t r i t i o n a l requirements have l i m i t e d the widespread use of 3 Fig. 1. Germination and d i f f e r e n t i a t i o n of the rust pathogen, P. graminis t r i t i c i , on the cereal host: The wheat stem rust uredospore (Ur) germinates readily on the leaf surface. Contact with a stomatal pore causes the germtube (gt) to cease l i n e a r growth and begin d i f f e r e n t i a t i o n : an appressorium (app) forms over the stomate, an i n f e c t i o n peg (ip) grows between the guard c e l l s and expands to form the substomatal v e s i c l e (ssv), the v e s i c l e gives r i s e to the infection hypha (ih) • 4 a x e n i c c u l t u r e methods (Maclean 1982). Furthermore, a p p a r e n t , a t l e a s t w i t h P. g r a m i n i s t r i t i c i , a b e r r a n t forms a r e encouraged i t seems that genetically t o develop, t h a t s p o r u l a t i o n i s a t b e s t e r r a t i c and t h a t fewer t h a n h a l f t h e r a c e s t h a t have been t e s t e d have s u r v i v e d under t h e c u l t u r e c o n d i t i o n s employed ( W i l l i a m s 1984). A c c o r d i n g t o W i l l i a m s (1971) d i f f e r e n t i a t i o n i s a n e c e s s a r y p r e l u d e towards o b t a i n i n g g e n e t i c a l l y (haploid, d i k a r y o t i c ) pathogenic c o l o n i e s . normal The p r o p o r t i o n o f g e r m l i n g s t h a t form i n f e c t i o n s t r u c t u r e s i s i n f l u e n c e d by n u t r i e n t s , h e a t shock, t h e t i m e o f i n o c u l u m c o l l e c t i o n , and the g e n e t i c c o n s t i t u t i o n of the spore. differentiation by Sporeling i s most o f t e n i n d u c e d by a t i m e d h e a t shock (30°C, 1.5 h) a d m i n i s t e r e d 2 h f o l l o w i n g g e r m i n a t i o n a t 19°C. I n o r d e r t o e s t a b l i s h a g i v e n r u s t fungus i n c u l t u r e , s p e c i f i c n u t r i t i o n a l r e q u i r e m e n t s must be met. The medium must c o n t a i n i n o r g a n i c s a l t s , a s o u r c e o f c a r b o h y d r a t e , reduced n i t r o g e n , and reduced s u l p h u r (e.g. c y s t e i n e ) . Howes r e p o r t e d t h a t a wide range o f amino compounds were e x c r e t e d i n t o c u l t u r e f i l t r a t e s o f P. g r a m i n i s t r i t i c i i n c u b a t i o n (see Howes and S c o t t 1973). a f t e r 9 days The f u l l e x t e n t o f amino a c i d l e a k a g e d u r i n g t h e f i r s t 20 h o f g e r m i n a t i o n and d i f f e r e n t i a t i o n was n o t a s s e s s e d . T h i s s t u d y was d e s i g n e d t o i n v e s t i g a t e t h e l e a k a g e o f f r e e amino a c i d s from uredospore g e r m l i n g s c o n c o m i t a n t w i t h t h e p r o c e s s e s o f g e r m i n a t i o n and differentiation ( i n d u c e d by h e a t s h o c k ) . The changes i n t h e 5 endogenous free amino acid pool were also assessed. The objectives of the present study thus were: 1. To reexamine the temperature requirement f o r the process of d i f f e r e n t i a t i o n of the wheat stem rust fungus. 2. To examine the influence of nutrients on the proportion of germlings that form i n f e c t i o n structures. 3. To determine the timing of essential protein synthesis during d i f f e r e n t i a t i o n . 4. To investigate nuclear behaviour accompanying germination and d i f f e r e n t i a t i o n of P. graminis t r i t i c i , race C17: nuclear d i v i s i o n , nuclear migration patterns, and nuclear d i s t r i b u t i o n . 5. To determine the amount and kinds of free amino acids present during the germination of wheat stem rust uredospores (race C17) and the d i f f e r e n t i a t i o n of germ tubes into i n f e c t i o n structures. 5.1 To characterize the free amino acids within nongerminated uredospores and those adhered to the outer spore wall. 5.2 To assess the changes i n the endogenous free amino acid pools of uredospore germlings during germination and d i f f e r e n t i a t i o n . 5.3 To determine the amounts and kinds of amino acids leached into the medium during the process of germination and d i f f e r e n t i a t i o n . 6 2. LITERATURE REVIEW 2.1 2.1.1 U r e d o s p o r e G e r m i n a t i o n and Morphogenesis G e r m i n a t i o n I n h i b i t o r s and S t i m u l a n t s U r e d o s p o r e g e r m i n a t i o n and germtube d i f f e r e n t i a t i o n i s s u b j e c t t o r e g u l a t i n g mechanisms i n v o l v i n g endogenous i n h i b i t o r s and s t i m u l a t o r s . Germination i n h i b i t o r s are dormancy a g e n t s w h i c h resemble hormones i n t h e i r m o b i l i t y from s p o r e t o s p o r e , t h e i r r e g u l a t o r y a c t i o n and t h e i r h i g h biological activity ( A l l e n 1976). Sporostatic levels of c i s - cinnamates a r e found i n u r e d o s p o r e s . The e f f e c t o f t h e i n h i b i t o r s i s r e a d i l y r e v e r s i b l e , f o r example, during h y d r a t i o n , and p r i o r t o g e r m i n a t i o n , t h e a c t i v e c i s - c i n n a m a t e s a r e r e l e a s e d and a r e e i t h e r d i l u t e d t o below a c t i v e l e v e l s o r c o n v e r t e d t o i n a c t i v e t r a n s - i s o m e r s by u l t r a v i o l e t l i g h t (420 nm) ( A l l e n 1972). The g e r m i n a t i o n i n h i b i t o r o f wheat stem r u s t i s m e t h y l c i s - f e r u l a t e : a m e t h y l e s t e r o f 3-methoxy-4hydroxycinnamate (Macko e t a l . 1971). I t has an E D 5 0 o f 0.2 ng/ml (Macko e t a l . 1972) and i s a c t i v e o n l y d u r i n g g e r m i n a t i o n ( m y c e l i a l growth i s n o t a f f e c t e d [ A l l e n 1 9 7 6 ] ) . The i n h i b i t o r p r e v e n t s t h e d i s s o l u t i o n o f t h e germ p o r e p l u g b u t i t s m o l e c u l a r s i t e o f a c t i o n i s unknown (Hess 1975). Endogenous g e r m i n a t i o n s t i m u l a n t s such as n - n o n y l a l c o h o l (nonanal) ( F r e n c h and Weintraub 1957), 6-methyl-5-hepten-2-one ( R i n e s e t a l . 1974), coumarins, and p h e n o l s (Van Sumere e t a l . 7 1957) are not species s p e c i f i c . Macko and c o - w o r k e r s (1976) suggest t h a t t h e s t i m u l a n t s f a c i l i t a t e germination by p r o m o t i n g t h e r e l e a s e o f t h e i n h i b i t o r s from t h e s p o r e . 2.1.2 Thigmodifferentiation Uredosporeling physical stimuli. d i f f e r e n t i a t i o n may be i n d u c e d i n v i t r o by I n a s e r i e s o f e x p e r i m e n t s e m p l o y i n g bean r u s t g e r m l i n g s and a r t i f i c i a l membranes D i c k i n s o n (1949, 1971) demonstrated t h a t t h e formation o f i n f e c t i o n s t r u c t u r e s r e l i e s on a r e c o g n i t i o n e v e n t t h a t i s c o r r e l a t e d w i t h thigmotropic s t i m u l i . specific D i f f e r e n t i a t i o n thus stimulated ( t h i g m o d i f f e r e n t i a t i o n ) requires f i r m attatchment o f t h e germtube t o t h e i n d u c t i v e s u r f a c e (Wynn and S t a p l e s 1981). Recent d a t a s u g g e s t t h a t , i n bean r u s t , p r o t e i n s on t h e h y p h a l s u r f a c e a r e i n v o l v e d i n s u b s t r a t e a d h e s i o n and t h e transmission o f a s i g n a l t o the cytoskeleton t o begin nuclear d i v i s i o n ( E p s t e i n e t a l . 1985). According t o Hoch e t a l . (1987) t h e t o p o g r a p h i c a l s i g n a l r e q u i r e d t o t r i g g e r maximum c e l l d i f f e r e n t i a t i o n i n Uromyces appendiculatus surface. i s a 0.5 m h i g h r i d g e on t h e s u b s t r a t e The e l e v a t i o n o f t h e r i d g e i s c r i t i c a l , g r e a t e r t h a n 1.0 m o r l e s s t h a n 0.25 ridges m are not e f f e c t i v e s i g n a l s and a r e u n a b l e t o promote maximum d i f f e r e n t i a t i o n . It was a l s o r e p o r t e d t h a t t h e growth o f t h e germtubes i s o r i e n t e d by r i d g e s p a c i n g s o f 0.5 t o 6.7 m. A l t h o u g h c o l l o d i o n membranes c o n t a i n i n g p a r a f f i n o i l 8 induced bean rust germlings to d i f f e r e n t i a t e , they had to no e f f e c t on P. q. t r i t i c i (Maheshwari et a l . little 1967b). Staples and h i s group (1983) found that contact s t i m u l i are responsible f o r the induction and positioning of the wheat stem rust appressorium. However, further development of the stem rust germling requires other environmental factors most l i k e l y of host o r i g i n . 2.1.3 Thermodif f e r e n t i a t i o n Another type of induction was reported f o r P. q. t r i t i c i . The exposure of germinated uredospores to elevated temperatures of 30 to 31°C f o r 90 min followed by the return to lower temperatures promoted up to 90% d i f f e r e n t i a t i o n (Maheshwari et a l . 1967, Dunkle and A l l e n 1971). The percentage of germlings that formed i n f e c t i o n structures was influenced by the duration of heat shock, the spore to l i q u i d r a t i o , and the composition of the medium present during the induction period (Dunkle and A l l e n 1971). The induction of d i f f e r e n t i a t i o n by heat shock, as described above was repeated by Wisdom (1977). The r e s u l t s proved unsatisfactory, y i e l d i n g d i f f e r e n t i a t i o n percentages of l e s s than 5%. The e f f e c t of the temperature of the heat shock was not investigated. The Ca-K buffer used by Dunkle and A l l e n (1968) was abandoned i n favour of a medium composed of Maheshwari s-buffer 7 glucose (MPG). (Maheshwari et a l . 1967), peptone, and MPG supported up t o 80 percent d i f f e r e n t i a t i o n 9 (Wisdom 1977). 2.1.4 Chemodifferentiation Chemodifferentiation i s the induction of i n f e c t i o n structures by a chemical agent. Chemical s t i m u l i include uredospore d i s t i l l a t e s such as acrolein (2-propanal) (Macko et a l . 1978), the potassium ion (Staples et a l . 1982), c e r t a i n reduced nucleotides (Staples et a l . 1983a), and v o l a t i l e l e a f constituents and phenolic compounds leached or metabolized from the guard c e l l s (Grambow and Riedel 1977, Grambow and Grambow 1978). Grambow 1978, Synthetic short-chain a l i p h a t i c compounds with conjugated double bonds (e.g. acrolein) are morphogenetically active whereas saturated a l i p h a t i c aldehydes, ketones and alcohols, as well as phenylacrolein unable to induce d i f f e r e n t i a t i o n (Wolf 1982). are The a b i l i t y of a chemical agent to stimulate d i f f e r e n t i a t i o n opens the p o s s i b i l i t y that the agent i s a normal intermediary in contact-triggered d i f f e r e n t i a t i o n (Allen 1976). 2.1.5 Mechanisms of D i f f e r e n t i a t i o n The mechanisms involved i n the regulation of germtube d i f f e r e n t i a t i o n are unknown. Allen (1976) suggests that the formation of i n f e c t i o n structures i s controlled by the uredospore genome. program. An appropriate stimulus activates the The observed i n h i b i t i o n of d i f f e r e n t i a t i o n by protein and RNA i n h i b i t o r s suggests that the genetic message 10 i s a c t i v a t e d by d e - r e p r e s s i n g a r e g i o n o f t h e genome. A l t e r n a t i v e l y i t i s p o s s i b l e t h a t the cytoskeleton ( m i c r o f i b r i l l a r system) i s r e s p o n s i b l e f o r t h e i n d u c t i o n o f n u c l e a r d i v i s i o n i n bean r u s t g e r m l i n g s (Hoch and 1985, Hoch e t a l . 1984, S t a p l e s and Hoch 1982). s u g g e s t t h a t t h e c y t o s k e l e t o n somehow r e p r e s s e s Staples These a u t h o r s nuclear d i v i s i o n u n t i l i t i s s u b j e c t e d t o h e a t shock o r an i n d u c t i v e membrane topography. 2.1.6 The R o l e o f I n f e c t i o n S t r u c t u r e s I n n a t u r e t h e i n f e c t i o n s t r u c t u r e s p l a y a number o f important roles: I n f e c t i o n s t r u c t u r e s must form b e f o r e c o l o n i z i n g hyphae can grow ( D i c k i n s o n 1949, According Chakravati t o Wisdom (1977), d i f f e r e n t i a t e d g e r m l i n g s o f leaf1966). the wheat stem r u s t fungus i n f e c t exposed h o s t m e s o p h y l l ; u n d i f f e r e n t i a t e d s p o r e l i n g s a r e u n a b l e t o cause i n f e c t i o n . I n f e c t i o n s t r u c t u r e s s e r v e t o anchor t h e fungus a t t h e stomatal s i t e thereby enabling penetration. They represent t h e s i t e o f t h e t r a n s i t i o n from germtube t o h y p h a l growth. This hypothesis and DNA i s s u p p o r t e d by t h e a c t i v a t i o n o f p r o t e i n , s y n t h e s i s d u r i n g d i f f e r e n t i a t i o n (Dunkle e t a l . M e i h l e 1972). And RNA 1969, l a s t l y t h e y e n a b l e t h e fungus t o complete t h e t r a n s i t i o n w i t h t h e r a p i d i t y demanded by a c h a n g i n g (perhaps t o a l e s s c o n d u c i v e ) environment. found t h a t d i k a r y o t i c hyphae b r a n c h i n g Grambow (1977) d i r e c t l y from germtubes a r e p r o d u c e d a t a h i g h r a t e b u t n o t b e f o r e t h e t h i r d day a f t e r seeding. Dikaryotic hyphae a r i s e from a complete set of i n f e c t i o n structures very soon a f t e r germination. According t o Williams (1971) the formation of i n f e c t i o n structures i s an e s s e n t i a l prelude to the establishment and maintenance of g e n e t i c a l l y normal pathogenic colonies. The t r a n s i t i o n ( p a r a s i t i c to saprophytic growth), i f appropriately stimulated, occurs with or without the induction of d i f f e r e n t i a t e d structures. However, i n f e c t i o n structures appear t o control nuclear behaviour and as a consequence increase the p r o b a b i l i t y of giving r i s e to hyphae which are both normal and stable f o r the dikaryophase. Although heat shock generally appears to stimulate c e l l d i f f e r e n t i a t i o n and improve saprophytic growth of most rust fungi, Bushnell (1976) claims that heat shock does not promote growth of P. graminis f.sp. t r i t i c i , race 17. Kuck and Reisner (1985) report that d i f f e r e n t i a t i o n has a negative e f f e c t on the i n v i t r o sporulation of P. graminis t r i t i c i . race 32. 2.2 2.2.1 C y t o l o g i c a l Events Nuclear Changes The morphological changes leading to the formation of i n f e c t i o n structures are accompanied by a series of p r e c i s e l y timed nuclear events. haploid. The r e s t i n g uredospore i s d i k a r y o t i c Following germination, the two n u c l e i migrate with 12 the cytoplasm into the developing germtube (Heath and Heath 1978). Given the stimulus t o d i f f e r e n t i a t e the nuclei may d i v i d e p r i o r t o (Maheshwari et a l . 1967b), or following (Grambow and Muller 1978) the development of the appressorium. Nuclear d i v i s i o n occurs only a f t e r the l i n e a r growth of the germtube i s arrested and i s r a r e l y observed i n nondifferentiated germlings (Maheshwari et a l . 1967, Grambow and Muller 1978, Wisdom 1977). Dickinson (1949) reported that two or three rounds of m i t o t i c d i v i s i o n occur during the formation of i n f e c t i o n structures i n P. graminis and P. t r i t i c i n a . The appressorium may contain four t o eight nuclei which migrate into the developing v e s i c l e . A second d i v i s i o n i n the v e s i c l e y i e l d s eight n u c l e i i n t o t a l . According to Wisdom (1977) the v e s i c l e n u c l e i may e i t h e r dissolve or coalesce leaving one t o two n u c l e i i n the i n f e c t i o n hypha. the mature appressorium A l l e n (1923) also noted that of P. g. t r i t i c i contains four n u c l e i and that the i n f e c t i o n hypha contains two. The i n f e c t i o n structures induced i n v i t r o are morphologically and c y t o l o g i c a l l y s i m i l a r to those induced on the host plant (Maheshwari et a l . 1967b). Staples e t a l . (1984b) reported that the synthesis of nuclear DNA i n U. fabae sporelings coincides with the onset of mitosis i n response to the stimulus f o r the i n i t i a t i o n of differentiation. The r e p l i c a t i o n of DNA begins between the second and fourth hour following germination. I t i s not known 13 how external s t i m u l i activate nuclear DNA and Staples 1974). polymerase (Yaniv In the absence of the stimulus to d i f f e r e n t i a t e the uredosporeling n u c l e i are i n Gl ( i . e . period preceding DNA r e p l i c a t i o n ) (Staples et a l . 1984b). Mitochondrial DNA synthesis occurs during germination but i s not detected following the appearance of the appressoria (Staples 1974). Uredospore n u c l e i are known to e x i s t i n two p r i n c i p a l but d i s s i m i l a r forms. Between d i v i s i o n s the interphase nucleus i s described as "expanded". The expanded nucleus i s composed of two parts, the "ectosphere", containing a l l the chromatin and the "endosphere", containing the nucleolus. As the process of d i v i s i o n begins, the nucleus extrudes the endosphere i n t o the cytoplasm, contracts and rounds up to form the "unexpanded" or d i v i d i n g nucleus (Savile 1939). Craigie (1959) and Wisdom (1977) observed both expanded and unexpanded n u c l e i i n P. h e l i a n t h i and P. q. t r i t i c i , respectively. 2.2.2 Protein Metabolism The changes i n proteins and nucleic acids accompanying germination of uredospores and d i f f e r e n t i a t i o n of the germtube into structures has inspired much research Dunkle et a l . 1969, Shaw et a l . 1985, (Staples 1968, Kim et a l . 1982a, Huang and Staples 1982, Wanner et a l . 1985). Rust uredospores germinate without the appreciable increase i n protein that occurs i n most saprophytes, such as 14 Fusarium (Cochrane et a l . 1971) and Aspergillus niqer (Staples et a l . 1962). Even so, f u l l y functional ribosomes are present i n bean and wheat rust uredospores and t h e i r capacity increases three-fold during spore hydration, p r i o r t o germination (Staples e t a l . 1968). Data from studies using metabolic i n h i b i t o r s of RNA (actinomycin-D) and protein (puromycin) synthesis indicate that new protein synthesis i s essential for differentiation. The presence of actinomycin-D during the heat shock prevents the i n i t i a t i o n of i n f e c t i o n structures. The presence of puromycin allows germination and appressorial formation to occur but prevents further i n f e c t i o n structure development (Dunkle et a l . 1969). The protein required f o r germination appears to be stored within the dormant uredospore. The mRNA responsible f o r d i r e c t i n g the complete development of i n f e c t i o n structures i s synthesized at the time of heat shock. Essential protein synthesis i s then programmed by the new mRNA during elaboration of the i n f e c t i o n structures. Although puromycin and actinomycin-D f a i l e d t o prevent germination i t would be erroneous t o conclude that RNA and protein synthesis does not occur during the early stages of development. Uredospores contain a complete system f o r protein synthesis (Yaniv and Staples 1974). Germination studies on the incorporation of [ C ] - l a b e l l e d amino acids, -glucose, and 14 - v a l e r i c a c i d into protein of wheat stem rust (Reisener 1967) and bean rust (Staples and Bedigan 1967), and the appearance 15 of new isozymes of glucose,dehydrogenase, cytochrome oxidase, and acid phosphatase (Staples and Stahmann 1964) are i n d i c a t i v e of protein synthesis. Although there i s an apparent synthesis of protein, germination i s not by net protein synthesis; accompanied t o t a l free and bound amino acids remain unchanged or decrease (Staples et a l . 1962). Heat shock promotes the synthesis of new types of RNA and protein (Kim et a l . 1982a, Huang and Staples 1982, Shaw et a l . 1985). Although the heat shock response has been reported f o r a wide range of organisms i t s functional s i g n i f i c a n c e i s unknown. Heat shock proteins (HSP) may serve to protect the c e l l against environmental stress (Ashburner 1982). al. Shaw et (1985) have i d e n t i f i e d seven HSP i n Melampsora l i n i uredospore germlings. Kim et a l . (1982a) reported on changes i n detergentsoluble polypeptides from uredospores of P. cf. t r i t i c i . Dormant uredospores were found t o contain more than 270 d i s t i n c t polypeptides, several of which varied among physiologic races (Howes et a l . 1982). A f t e r 2 and 6 hours of germination the concentrations of at l e a s t f i v e polypeptides decreased considerably. At 2 hours, a transient concentration increase was noted f o r two polypeptides, 4 hours l a t e r these concentrations had decreased again to that found i n dormant uredospores. The t o t a l protein extracted from six-hour-old germlings was 86 percent of the t o t a l protein extracted from dormant spores. 16 D i f f e r e n t i a t i o n was f i v e polypeptides accompanied by a decrease i n at l e a s t and an increase i n four others. Two new polypeptides appeared i n d i f f e r e n t i a t e d sporelings, t h e i r molecular weights were approximately 30.0 and 20.0 kD. D i f f e r e n t i a t e d sporelings were found to contain only 48 percent of the t o t a l protein extracted from dormant uredospores (Kim et a l . 1982a). Wanner et a l . (1985) reported the synthesis of d i f f e r e n t i a t i o n - s p e c i f i c proteins (DSP) at a time corresponding to the appearance of the substomatal vesicle. Several p o s s i b i l i t i e s e x i s t f o r the observed decline i n the t o t a l amount of extractable protein and c e r t a i n polypeptides: Pulse-chase experiments show that polypeptides are continually turned over, e x i s t i n g protein i s broken down into i t s substituent amino acids, and the amino acids are e i t h e r u t i l i z e d f o r resynthesis or l o s t into the medium (see Kim et a l . 1982a). I t i s also possible that proteins bind to and form an insoluble complex with glucan and mannan residues i n the c e l l wall of the germtube (Kim et a l . 1982a). Glucan- protein and mannan-protein complexes have been detected i n yeasts (Ballou 1974) and i t i s possible that analogous complexes occur i n the germtube walls of P. q. t r i t i c i . F i n a l l y , the decline may metabolites be due to a considerable loss of and enzymes into the medium during germination and c e l l wall degeneration. Pfesofsky-Vig and Brambl (1985) report that the 17 appearance of HSP and the depression of general t r a n s l a t i o n a c t i v i t y i s t y p i c a l immediately following a temperature shift. The normal pattern of synthesis begins soon a f t e r the c e l l s are returned t o the normal 2.3 2.3.1 temperature. N u t r i t i o n a l Requirements of Rust Fungi Physiology of the Host-Parasite Complex P r i o r to considering the n u t r i t i o n a l requirements of rust fungi i n v i t r o i t i s necessary to review host-parasite r e l a t i o n s i n susceptible host t i s s u e . The pustules of rust fungi act as f o c i f o r the accumulation of host metabolites. A compatible i n f e c t i o n increases the rate of t r a n s p i r a t i o n and r e s p i r a t i o n , decreases photosynthetic a c t i v i t y , a l t e r s the d i r e c t i o n of normal phloem transport (Scott 1972) and stimulates the leakage of e l e c t r o l y t e s from the host. These responses by the host s i g n i f i c a n t l y improve the a v a i l a b i l i t y of nutrients t o the developing fungus. With respect to s t r u c t u r a l and physiological changes associated with rust infected tissues, Bushnell (1984) recognized the juvenile and the a u t o l y t i c host response. During the juvenile response most plant growth hormones increase. I t i s unclear however whether the observed increase i s caused by pathogen or host-produced hormones. There i s evidence that plant derived hormones do not play an active r o l e i n a compatible host-pathogen r e l a t i o n s h i p (Levin 1985). 18 The increase i n cytokinin a c t i v i t y reported by S z i r a k i et a l . (1976) appeared to delay l e a f senescence by maintaining protein synthesis (Stodart 1981). The free concentration of indole a c e t i c acid (IAA) i n plants increased two and a h a l f f o l d at the i n f e c t i o n s i t e , 6 to 14 hr a f t e r inoculation with wheat stem rust pathogen (Artemeko et a l . 1980). I t i s l i k e l y that IAA acts with cytokinin to control the metabolic state of the host c e l l . The host nucleus and nucleolus generally enlarge during rust i n f e c t i o n and the synthesis of nucleolar and extranucleolar RNA synthesis i s enhanced (Manocha and Shaw 1966, Whitney et a l . 1962, Bhattacharya et a l . 1965). However, the t o t a l amount of host RNA that the newly synthesized RNA over. declines which indicates i s rapidly degraded and turned Chakravorty and co-workers (1974) found that c a t a b o l i c RNAse a c t i v i t y peaks at about 6 days at l e v e l s two to f i v e times those of uninfected leaves. The j u v e n i l e response i s not associated with s i g n i f i c a n t q u a l i t a t i v e or quantitative changes i n host proteins. Host proteins changed l e s s i n compatible host-parasite combinations than i n incompatible combinations 1972). New (von Broembsen and Hadwiger isozymes have been detected on polyacrylamide gels and i n most cases they appear to be of fungal o r i g i n et a l . 1968, (Johnson Staples 1965). Amino acids, amides, and carbohydrates accumulate rapidly at the infected s i t e . During the early juvenile host response these metabolites are synthesized l o c a l l y from photosynthates and ammonia, and are also translocated from distant s i t e s on the plant. During the l a t e r a u t o l y t i c stage the l o c a l degradation of protein provides a r i c h source of free amino acids and amides (see Bushnell 1985). The r a t i o of soluble to insoluble nitrogen increased at i n f e c t i o n s of stem rust on Franke 1938). increased L i t t l e Club wheat (Gassner and On a fresh weight basis bound amino acids almost twofold whereas free amino acids increased f o u r f o l d (Shaw and Colotelo 1961). Large increases i n the amounts of free glutamine, Y-aminobutyric a c i d (ABA), threonine and several basic and aromatic acids occurred e a r l y as two days post-inoculation. as Several investigators have reported d i f f e r e n t i a l increases i n the amounts of asparagine, arginine, phenylalanine, v a l i n e (Farkas and K i r a l y 1961, leucine or isoleucine, Shaw and Colotelo 1961) and tryptophan (Kim and Rohringer 1969). The movement of host metabolites has been followed with radiotracers. into p a r a s i t i c mycelium Labelled sucrose i s inverted to glucose and fructose and r e a d i l y absorbed by the rust hypha. Glucose, glutamate, alanine, glycine, l y s i n e and arginine are taken up; the f i r s t four compounds are metabolized by the fungus. Some amino acids (e.g. serine and alanine) are absorbed from the host by the fungus more r e a d i l y than others (e.g. glutamine, glutamate and aspartate). Amino acids are leached from the uredosporeling during germination and t h e i r movement i n t o host tissues has been reported 1966, (Jones Daly e t a l . 1967). The i n f e c t i o n of the host by a rust pathogen i n v a r i a b l y causes an accumulation of free glutamine (Shaw and Colotelo 1961). This amide, which may be derived e i t h e r by an endergonic reaction from glutamate or from p r o t e o l y s i s i s r e a d i l y translocated throughout the plant. Glutamine can serve as a source of bulk nitrogen f o r axenic culture (Maclean 1982) and as a precursor f o r the synthesis of fungal c h i t i n (Farkas and K i r a l y 1961). I t i s l i k e l y that glutamine plays a central r o l e i n the metabolism of rust infected leaves. 2.3.2 Axenic Culture and Metabolism N u t r i t i o n a l Requirements The n u t r i t i o n a l requirements of P. q. t r i t i c i i n axenic culture include inorganic s a l t s , a source of carbohydrate, reduced nitrogen and reduced organic sulphur. Carbohydrate requirements are r e l a t i v e l y nonspecific (Coffey and Shaw 1972) . Nitrogen may be supplied as an inorganic ammonium s a l t , or as one of a wide range of organic compounds, such as an amino a c i d . Some amino acids are better sources of nitrogen than others. In the presence of cysteine the i n i t i a t i o n of saprophytic growth was poor on alanine, improved on aspartate and greatest on glutamate (Coffey and A l l e n 1973) . Sulphur must be provided i n reduced form such as cysteine, cystine, the t r i p e p t i d e glutathione, or methionine. Wheat stem rust requires a higher concentration of cysteine than methionine f o r optimal growth (Howes and Scott 1973). The amount and the combination of sulphur containing amino acids i s c r i t i c a l and may vary between races of rust (Singh and Sethi 1982). The nutrient requirements appear l e s s exacting once the rust fungus becomes established on axenic medium. Synthetic Capacity Germinating stem rust uredospores slowly absorb, metabolize and synthesize a wide range of compounds. Uredospores germinated on a medium containing [ C]-sucrose 14 synthesize at l e a s t eleven [ C ] - l a b e l l e d amino acids within 14 two hours (Kasting et a l . 1959). Among the f i r s t amino compounds to be synthesized were glutamine, glutamate, and aspartate (Reisener et a l . 1961). Glucose i s taken up r e a d i l y and the carbon appears i n endogenous pools of free glucose, amino acids and phosphate esters of trehalose and (Manners et a l . 1982). glucose Reisener et a l . (1961) demonstrated that 42 percent of the label from v a l e r a t e - 1 - [ C ] 14 was incorporated i n t o the spore as carbohydrate; organic, f a t t y and amino acids; amides and peptides. Glutamine, glutamate and ABA had the highest s p e c i f i c a c t i v i t y of the free amino compounds. Rust uredospores possess a l l the enzymes required f o r glucose catabolism and terminal oxidation (Shaw 1964), the pentose phosphate pathway, c i t r i c acid cycle, and lipid metabolism (Caltrider and Gottlieb 1962). Sulphur Metabolism Most rust fungi studied so f a r are heterotrophic f o r reduced, organic sulphur when grown on chemically defined medium. Although rust fungi synthesize sulphur amino acids r e a d i l y from [ S ] - s u l p h i d e 35 only a l i m i t e d synthesis of l a b e l l e d protein can be detected (Howes and Scott 1973). More than 70 percent of the label incorporated into sulphur amino acids was l o s t i n t o the medium as cysteine, glutathione and cysteinylglycine. S-methylcysteine, Labeled methionine however, appeared i n mycelial protein and only n e g l i g i b l e amounts were l o s t to the f i l t r a t e . P. q. t r i t i c i was unable to reduce inorganic sulphate f o r sulphur-amino-acid synthesis* These r e s u l t s suggest that a metabolic block e x i s t s i n the pathway of inorganic sulphur metabolism. Howes and Scott (1973) propose that rust fungi are unable to reduce 3'- phosphoadenosine-5'-phosphosulphate to thiosulphate or thiosulphate to sulphide. I t i s i n t e r e s t i n g to note that sulphate reduction occurs i n prokaryotes, most fungi (Scott 1972) eukaryotic algae, and a l l higher plants so f a r rested (Schiff and Hodson 1973). Metabolite Leakage In axenic culture the germtube and mycelium leak metabolic intermediates. Staples and Wynn (1965), and Tulloch (1962) mention possible losses of free amino acids, sulphur- containing compounds and glycine-containing peptides t o the medium but o f f e r no quantitative data. Jones and Snow (1965) reported that [ S ] - l a b e l l e d uredospores of P. coronata avenae 35 l o s t a range of amino compounds including ethionine, ABA, methionine sulphoxide and four sulphur-containing during 12 h of germination. unknowns The amino acids detected i n the culture f i l t r a t e (basal medium) of P. g. t r i t i c i a f t e r nine days were glutamate, glutamine, glycine, alanine, l y s i n e , arginine, serine, leucine and isoleucine, phenylalanine, v a l i n e , threonine, asparagine, proline, cysteine, and methionine (Howes and Scott 1972). A considerable amount of cysteine and glycine-containing peptides, such as c y s t e i n y l g l y c i n e and glutathione accumulated i n the medium. Very l i t t l e methionine and homocysteine was detected and Scott 1973). (Howes From these studies i t was evident that the loss of sulphur-containing compounds i s s e l e c t i v e and they are not l o s t as r a p i d l y nor t o the same extent as other free amino acids. ABA, Y-glutamylglutamate (Howes and Scott 1972), carbohydrate, protein, such as ribonuclease (Chakravorty and Shaw 1974) and the germination i n h i b i t o r , methyl f e r u l a t e i n the c i s or trans form are also leached into the medium. Mutual stimulation during sporeling development i s demonstrated by the p o s i t i v e e f f e c t of increasing inoculum density (Kuhl et a l . 1971), and the success of "nurse culture" 24 techniques, "conditioned agar" (Scott 1976) and coculture experiments (Hartley and Williams 1971a, 1971b). A minimal or unbalanced medium i n axenic culture would r e s u l t i n a net loss of metabolites, thus imposing an excessive drain on metabolism and depleting internal metabolite pools, which i n turn would r e s u l t i n l e s s e r growth rates (Maclean 1982). A poor approximation of amino acid balances may also r e s u l t i n detrimental e f f e c t s , such as methionine t o x i c i t y (Howes and Scott 1972) and the s e l e c t i v e leakage of amino acids from the mycelium (Maclean 1982). Endogenous Free Amino Acids Interest i n the free amino acid content of rust uredospores has developed i n conjunction with studies r e l a t i n g to s e l f - i n h i b i t i o n of germination (Wilson 1958), the p o t e n t i a l of d i f f e r e n t i a t i n g races of rust on the basis of c h a r a c t e r i s t i c amino acid content (McKillican 1960), and the e f f e c t s of storage on assimilative and synthetic c a p a b i l i t e s (Wynn et a l . 1966). McKillican (1960) reported race 56 as unique i n that the dormant uredospore lacked glutamic acid and contained large amounts of aspartic acid and a-alanine. Free amino acids represented approximately 0.5 percent of the o r i g i n a l spore weight. Using the same race Stefayne and Bromfield (1965) found that the major ninhydrin-positive compounds were glutamine, glutathione, glutamic acid and ammonia. Approximately 1.2 percent of the spore weight was 25 composed of free amino acids. In l a t e r studies by Wynn e t a l . (1966) i t was found that glutamic acid made up over h a l f of the t o t a l amino acids. Other major amino acids were alanine, aspartic acid, serine, and cystine. Since no attempt was made to recover asparagine and glutamine i n t a c t , i t was probable that the l e v e l s of aspartic acid and glutamic acid these amides. Stefayne and Bromfield included (1965) noted e a r l i e r that the uredospores contain seven times the amount of glutamic a c i d as glutamine. Analyses of the amino a c i d composition of wheat rust uredospores vary widely due to sampling and analysis procedures. Accordingly, I have examined the changes i n the amino acid composition of the free pool within germinating and d i f f e r e n t i a t i n g sporelings of P. crraminis t r i t i c i at 8 and 20 h following imbibition. providing information The r e s u l t s are preliminary t o f o r further studies of nitrogen metabolism and n u t r i t i o n i n the wheat stem rust fungus. 26 3. EXPERIMENTAL METHODS 3.1 Production and C o l l e c t i o n of Spores The wheat stem rust uredospores, (Puccinia graminis Pers. f.sp. t r i t i c i E r i k s and E. Henn., race C17), employed i n t h i s study were obtained through the courtesy of Dr. D. Samborski (Agricultural Research Station, Winnipeg) and were increased on Tritium aestivum L., c.v. L i t t l e Club. Plants were grown i n a growth chamber with a 16 h photoperiod of 9684 lux and temperatures dark. of 25°C l i g h t , 18°C A f t e r eight days of growth the plants were inoculated by l i g h t l y spreading a water-based paste of t a l c and uredospores (3:1) over the l e a f blade. The plants were then well-misted with d i s t i l l e d water, covered with a p l a s t i c bag and placed i n a dark incubator a t 18°C f o r 24 h before they were returned to the growth chamber. Within s i x days the leaves showed signs of "flecking", and s i x days following the s o r i opened. Five days l a t e r the uredospores were c o l l e c t e d by gently shaking the infected leaves into a clean t e s t tube. The f r e s h l y harvested spores were used immediately f o r the amino acid analyses. In a l l other experiments the spores were stored i n g e l a t i n capsules at 4°C f o r up to, but not exceeding 24 h. percent. Germination was generally between 90 and 100 27 3.2 Spore Germination Depending on the experiment, germination was c a r r i e d out on e i t h e r a mixed calcium and potassium phosphate (Ca-K) buffer (Table I) or MPG medium (Table I I ) . To deplete the endogenous s e l f - i n h i b i t o r 8 mg of uredospores were dispersed uniformly over the surface of 3 ml of Ca-K b u f f e r i n an p e t r i dish using an inoculation loop. (5-mm 8-cm A f t e r 5 min, 5 loops diameter) of uredospores were transferred to 2 ml of the germination medium i n the inner chamber of a 50-ml Conway D i f f u s i o n dish. mm . -2 The f i n a l spore dose was approximately The outer well contained 4 ml of 1.5 x 1 0 alcohol (Table I ) . -4 300 M n-nonyl Dishes were covered with a glass plate, sealed with vaseline and incubated i n the dark at 19°C. 3.3 Spore Germination with the Induction to D i f f e r e n t i a t e Inoculated dishes were incubated i n the dark at 19®C f o r 2 h, transferred to a hot water bath set at 29.5°C f o r 1.5 h, then returned to 19°C. Infection structure development was complete within 20 h. 3.4 C r i t e r i a f o r the Assessment of Sporelincr Development A spore was considered germinated when the germtube length was equal to, or exceeded the spore diameter. Terminal 28 Table I : The c o m p o s i t i o n and p r e p a r a t i o n o f t h e Ca-K-P0 b u f f e r and t h e v o l a t i l e g e r m i n a t i o n s t i m u l a n t , n - n o n y l a l c o h o l (nonanol). 4 Ca-K-P0 4 Buffer 1.5 x 1 0 - M N o n a n o l 4 Ca(H P0 )2 x H 0 5 mg d i s s o l v e i n 100 m l H 0 2 4 2 2 KH P0 27 2 mg d i s s o l v e i n 100 m l H 0 add 39 m l t o a b o v e 2 4 Stock s o l u t i o n : d i l u t e 1:4 p r i o r t o u s e N-nonyl a l c o h o l (9.94M) 70 u l 2 K HP0 3 48 mg d i s s o l v e i n 100 m l H 0 add t o a b o v e u n t i l pH 6.8 ( 6 1 m l ) 2 4 2 Total volume H 0 2 1000 m l mix w i t h s t i r b a r a t h i g h speed u n t i l alcohol droplets dispersed 200 m l I n n e r w e l l o f Conway cell O u t e r w e l l o f Conway cell 29 Table I I : The composition and preparation of the d i f f e r e n t i a t i o n medium MPG. Ca(H P0 ) KH P0 K HP0 Peptone D-Glucose 2 2 4 2 x H0 0. 025 g 0.449 g 1.145 g 5 g 30 g 2 4 2 4 volume made t o 1000 ml with g l a s s d i s t i l l e d H 0 2 The KH P0 and Ca(H P0 ) x H 0 were dissolved i n 200 ml d i s t i l l e d water. K HP0 was added u n t i l pH 6.8 was reached. Peptone and glucose were added and the pH was adjusted as necessary with phosphoric acid or KOH. The medium was s t e r i l i z e d by autoclaving. 2 4 2 2 4 2 4 swellings were counted as appressoria only a f t e r a septum d e l i m i t i n g the appressorium from the germtube was c l e a r l y visible. The i n f e c t i o n peg was recognized as a small outgrowth from the mature appressorium. Substomatal v e s i c l e s were recognized a f t e r the terminal end of the i n f e c t i o n peg had expanded t o a s i z e equal t o h a l f that of the appressorium. As the v e s i c l e s matured they flattened out forming two lobes on e i t h e r side. An i n f e c t i o n hypha was counted when one lobe of the v e s i c l e extended beyond the length of the other. 3.5 Staining and Counts Sporelings were transferred from each dish to a glass s l i d e , stained with trypan blue i n lactophenol (Table I I I ) , covered with a coverslip and sealed with n a i l p o l i s h . The s l i d e s were semi-permanent and may be stored f o r up to one year. Counts of 100 spores were made per s l i d e and the component i n f e c t i o n structures were i n d i v i d u a l l y assessed. D i f f e r e n t i a t i o n was expressed as a percentage of the t o t a l spores. 3.6 Temperature Range T r i a l s Temperature requirements f o r appressorium formation and the t o t a l d i f f e r e n t i a t i o n of uredospore germlings was investigated by applying a heat shock of constant temperature 31 T a b l e I I I : L a c t o p h e n o l - T r y p a n B l u e ; a mounting and s t a i n i n g medium f o r semipermanent mounts o f f u n g i ( B o e d i j n , 1965). Phenol Lactic acid Glycerol D i s t i l l e d water Trypan Blue 20 20 40 20 0.5% g g g ml 32 (ranging from 26 t o 32°C f o r 1.5 h) 2 h following germination at 19°C. The temperature range was tested over four sub- experiments, each of which u t i l i z e d a d i f f e r e n t spore l o t . The heat shock applied during the temperature range t r i a l s was administered by placing the Conway dishes on a temperature regulated brass plate. Warm water (35°C) from a hot water bath was c i r c u l a t e d through copper tubing under one end of the brass plate while the ambient temperature cooled the other end. The temperature gradient (25 t o 35°C) thus generated was allowed to s t a b i l i z e f o r a t least 2 h p r i o r to the experiment. Following 2 h germination at 19°C the inoculated dishes were transferred to the brass plate and set a t various i n t e r v a l s along the temperature gradient. A f t e r 1.5 h the temperature of the Ca-K buffer i n each dish was measured by an e l e c t r o n i c thermometer and a thermocouple. to 19°C f o r 14 h. The dishes were then returned Observations were made a f t e r a t o t a l of 17.5 h of incubation. Since the germination and d i f f e r e n t i a t i o n capacity of spore batches d i f f e r e d s l i g h t l y , the data within each subexperiment were adjusted t o allow f o r comparison between experiments. The maximum d i f f e r e n t i a t i o n value within each sub-experiment was represented as "100"; a l l other values were calculated as a percentage of the maximum. The optimum d i f f e r e n t i a t i o n temperature varied between experiments and ranged from 28.2 t o 30.7°C. Therefore, the optimum temperatures were assigned a value of "0", a l l remaining temperatures were calculated as u n i t s deviating from t h i s optimum. 3.7 E s s e n t i a l Protein Synthesis: Puromycin Uredospores were inoculated onto the Ca-K buffer and induced to d i f f e r e n t i a t e under conditions previously described. A protein synthesis i n h i b i t o r , Puromycin dihydrochloride (100 ug/ml) (Sigma Co.), was dissolved i n the buffer and added to, or removed from (using a 10-ml syringe), the inner well of the Conway dish at the times s p e c i f i e d i n Table IV. A f t e r each medium change, the sporelings were washed three times with fresh medium. E s s e n t i a l protein synthesis was measured by assessing the extent of uredosporeling d i f f e r e n t i a t i o n a f t e r 15 h. In a l l treatments designed to examine e s s e n t i a l protein synthesis the uredospores were k i l l e d and stained i n lactophenol-trypan blue upon completion of the experiment. Each treatment had three r e p l i c a t e dishes, two sample counts were taken from each r e p l i c a t e , and 100 spores were counted per sample. The treatments were compared to the control f o r each morphogenic group by one-factor analysis of variance. The complete experiment was repeated twice. 34 Table IV: The timing of essential protein synthesis: Experimental design. The uredosporelings were germinated at 19^C f o r 2 h, exposed to a heat shock f o r 1.5 h, then returned to 19°C f o r 12 h. Treatment (control) 2 3 4 5 6 7 8 9 10 11 Stages of germination and d i f f e r e n t i a t i o n (h) a t which puromycin i s added t o the medium (x) 0-2 2-3 . 5 3 . 5-6 - - - X X - - — — | 6-8 1 " 1 8 10 10-15 - - X X X X X X X X X X X X X X - X - X X — - X X X - X - — 35 3.8 N u c l e a r S t a i n i n g : DAPI U r e d o s p o r e s were g e r m i n a t e d on Ca-K b u f f e r f o r 4 h i n t h e d a r k a t 19°C. The g e r m l i n g s were t r a n s f e r r e d t o a c l e a n slide u s i n g an i n o c u l a t i o n l o o p and a l l o w e d t o d r y . A d r o p o f DAPI (4', 6 - d i a m i d i n o - 2 - p h e n y l i n d o l e ) , 1 ug/ml g l a s s - d i s t i l l e d w a t e r , was p l a c e d d i r e c t l y on t h e sample, a c o v e r s l i p was a p p l i e d and t h e edges were s e a l e d w i t h n a i l p o l i s h . The s l i d e was o b s e r v e d w i t h i n 20 min by f l u o r e s c e n t m i c r o s c o p y . The i n s t r u m e n t used was a Z e i s s u n i v e r s a l m i c r o s c o p e e q u i p p e d w i t h a 100 W mercury lamp t o d e l i v e r e x c i t a t i o n l i g h t by e p i f l u o r e s c e n t mode, a Z e i s s UG1 e x c i t e r f i l t e r w i t h a passband from c a 300 t o 400 mu, BG38 t o absorb l i g h t and p r o t e c t sample from h e a t , a b a r r i e r f i l t e r w i t h a c u t o f f a t 410 nm, and a Z e i s s N e o f l u o r 40 power o b j e c t i v e . Transmitted l i g h t and f l u o r e s c e n c e photographs were made w i t h F u j i c o l o r 400 ASA d a y l i g h t f i l m . The r e s u l t s o b t a i n e d from u n f i x e d m a t e r i a l were u n a c c e p t a b l e i n t h a t DAPI s t a i n e d t h e germtube w a l l as w e l l as the n u c l e i . to optimize S e v e r a l f i x a t i o n s c h e d u l e s were t e s t e d i n o r d e r stain specificity (Table V ) . A l l f i x e d samples were p l a c e d on a g l a s s s l i d e and s t a i n e d i n a drop o f 1 ug/ml DAPI. A l l t i s s u e s t h a t were f i x e d i n a l c o h o l - c o n t a i n i n g f i x a t i v e s were washed i n a l c o h o l o f t h e same c o n c e n t r a t i o n a s that present i n the f i x a t i v e . A f t e r w a s h i n g t h e t i s s u e was 36 Table V: 1. A description of the f i x a t i o n schedules tested. Formalin-acetic acid-alcohol (FAA)* Ethyl alcohol (50%) 90 ml G l a c i a l acetic acid 5 ml Formalin 5 ml The material was fixed f o r 2, 24, and 48 h, then rehydrated through an alcohol series to water. 2. Chrom-acetic solution (Johansen, 1940) Aqueous chromic acid (10%) 2.5 ml Aqueous a c e t i c acid (10%) 5.0 ml D i s t i l l e d water to 500 ml The sample was fixed f o r 20 h then washed three times i n d i s t i l l e d water (30 min per wash). 3. Ethanol/Acetic acid (3:1) The material was fixed f o r 2, 24, and 48 h, dried on a s l i d e , immersed i n 200 mM KCL and rehydrated through a graded alcohol s e r i e s . 4. Formaldehyde: 4% and 36% * The sample was fixed f o r 20 h i n either a 4% solution or the vapours of 36% formaldehyde, then washed 3 times i n d i s t i l l e d water (30 min per wash). 5. Glutaraldehyde: 3% * The sample was fixed f o r 1 h then washed three times i n d i s t i l l e d water (30 min per wash). 6. Ethanol series The material was dehydrated through an ethanol series (15%, 30%, 40%, 60%, 70%) allowing 5 min between changes. After at least 4 h i n 70% the sample was rehydrated (10 min between washes) back to d i s t i l l e d water. * These f i x a t i v e s were both used as described and i n combination with a surfactant. Two surfactants were assessed i n d i v i d u a l l y , 0.25% T r i t o n X-100 and 0.01% Tween-20. l e f t f o r 30 min i n each o f t h e f o l l o w i n g : 50%, 30%, 15% e t h y l a l c o h o l , and d i s t i l l e d w a t e r . The method o f c h o i c e was a s i m p l e a l c o h o l d e h y d r a t i o n and subsequent r e h y d r a t i o n o f t h e sample p r i o r t o s t a i n i n g w i t h DAPI. The n u c l e i were c l e a r l y v i s i b l e and background f l u o r e s c e n c e was m i n i m i m a l i n a l l b u t t h e v e r y young (0 t o 2 h ) . germtubes N u c l e a r f l u o r e s c e n c e improved f o r 20 min and remained s t a b l e f o r up t o 10 days when s t o r e d i n t h e d a r k a t 4°C. 3.9 The c h e m i c a l s t r u c t u r e o f DAPI i s shown i n F i g u r e 2. Amino A c i d A n a l y s i s 3.9.1 H i g h Performance L i q u i d Chromatography (HPLC) HPLC s e p a r a t i o n s a r e c a r r i e d o u t i n h i g h r e s o l u t i o n columns packed w i t h 3 t o 25 distribution. m p a r t i c l e s o f uniform size The columns r e q u i r e t h e u s e o f d e d i c a t e d i n j e c t o r s f o r sample i n t r o d u c t i o n , s e n s i t i v e d e t e c t o r s , and s p e c i a l pumps w h i c h d e l i v e r c o n s t a n t f l o w a g a i n s t h i g h pressure. HPLC methods may be u s e d f o r t h e s e p a r a t i o n o f a d i v e r s e a r r a y o f compounds w i t h m o l e c u l a r w e i g h t s r a n g i n g between 50 and 20 m i l l i o n . Two approaches a r e c u r r e n t l y a v a i l a b l e f o r HPLC a n a l y s i s o f amino a c i d s : a n a l y s i s u s i n g r e v e r s e d - p h a s e s e p a r a t i o n o f p r e d e r i v a t i z e d amino a c i d s , and a n a l y s i s u s i n g ion-exchange methods w i t h p o s t - d e r i v a t i z e d amino a c i d s . 38 H Fig. 2. DAPI (4\6-diamidmo-2-phenylindole) binds specifically to AT residues of double-stranded DNA. The AT-specificity resides with both the guanidine group and the indole ring, which may bind to the purine of adenosine through base stacking (Otto and Tsou, 1985). V 39 Reverse-Phase (RP) Bonded-Phase Chromatography The stationary phase of a RP column i s non-polar, consisting of s i l i c a gel with covalently bound hydrocarbon chains (lengths ranging from C^ to C 2)• 2 Tire e l u t i o n p r o f i l e of a sample r e f l e c t s the degree of hydrophobicity inherently unique to each of i t s components. The compound with the highest a f f i n i t y f o r the solid-phase emerges l a s t . Solvents from high to intermediate p o l a r i t y are used as mobile phases. Thus, water i s the solvent which gives the longest retention. The retention and s e l e c t i v i t y of the column can be adjusted and optimised by the addition of water-miscible organic solvents (e.g. methanol, a c e t o n i t r i l e ) . Since the conditions of separation are mild the formation of a r t i f a c t s i s not a problem. 3.9.2 Reagents Type I reagent grade water was prepared by running g l a s s - d i s t i l l e d water through the M i l l i - Q water p u r i f i c a t i o n system (Millipore Co). This system combines activated carbon adsorption, mixed-bed deionization, an organics-scavenging cartridge (Organex-Q), and 0.22 m s t e r i l i z i n g membrane f i l t r a t i o n (Millipak F i l t e r U n i t ) . Methanol and a c e t o n i t r i l e were HPLC-grade (BDH Co). grade. Chemical A l l remaining components of the buffer system were ACS Amino acid standards (hydrolyzate mix, No. 20088), t r i f l u o r o a c e t i c acid (TFA), triethylamine (TEA), and the 40 d e r i v a t i z i n g agent phenylisothiocyanate (PITC) (No. 26922) were obtained from the Pierce Chemical Company. 3.9.3 Instrument The equipment used was designed by Waters and consisted of two Model 510 pumps, a Model 710B WISP, a System Interface Module, an 840 Data and Chromatography Control Station, a Temperature Control Module, and a 490 Programmable Multiwavelength Detector (Figure 3). RP was a Waters C 1 8 The stationary phase f o r Pico-Tag column f o r protein hydrolyzates (3.9 mm x 15 cm). RP-HPLC was performed a t 41°C. When not i n use the column and pumps were washed with water t o remove s a l t s and then stored i n a c e t o n i t r i l e . 3.9.4 Sample C o l l e c t i o n and Preparation The procedures f o r amino acid analysis necessitated s t r i c t control of a l l possible sources of contamination. B a c t e r i a l and fungal contamination were monitored by suspending uredospores i n s t e r i l e water and p l a t i n g s e r i a l d i l u t i o n s onto nutrient agar. Glassware: Glassware was f i r s t washed i n soap (Alconox) and water, rinsed with tap water, oven dried and then soaked i n an acid bath (NoChromix, Chemonics Sci.) f o r at l e a s t 1 h. The glass was then rinsed three times with glass d i s t i l l e d water, rinsed once i n M i l l i - Q water and dried a t 60°C f o r several 41 Buffer A HPLC Pump A I RP-Column [ Column Heater WISP HPLC Pump B Buffer B Programmable Multiwavelength Detector Temp. Control Module Discard Bottle System Interface Module Data and Chromatography Control Station Component Pump A (Waters) Function (Model 510) d e l i v e r y of e l u t i o n b u f f e r A Pump B (Model 510) d e l i v e r y of e l u t i o n b u f f e r B WISP (Model System 710B) I n t e r f a c e Module automated sample injection l i n k s seperate components with C o n t r o l S t a t i o n 840 Data and Chromatography Control Station controls conditions for HPLC, s t o r e s and analyzes data Amino A c i d A n a l y s i s Column reverse-phase Temperature r e g u l a t i o n of column c o i l temperature C o n t r o l Module Programmable Multiwavelength D e t e c t o r (490) d e t e c t i o n of separations and PITC-derivatives Figure 3. Waters HPLC amino a c i d analysis system configuration f o r RP-analysis of amino acids with post-column u l t r a v i o l e t detection. 42 hours. Buffer: Ca-K buffer was prepared with M i l l i - Q water i n a c i d - washed glassware, s t e r i l i z e d through a 0.2 m Millipore f i l t r a t i o n u n i t and stored at 4°C u n t i l required. Spore Germination: Procedures e a r l i e r described f o r inducing germination and d i f f e r e n t i a t i o n were c a r r i e d out using autoclaved, acid-washed Conway dishes and s t e r i l e buffers. Ungerminated, non-differentiated, and d i f f e r e n t i a t e d uredospores and t h e i r leachates were analyzed f o r free amino acid content. The undifferentiated and d i f f e r e n t i a t e d sporelings were harvested a f t e r 8 and 20 h. Each treatment consisted of s i x dishes, which were l a t e r combined t o be analyzed as one sample. The 20 h experiment was repeated three times and the 8 h experiment twice. The extent of d i f f e r e n t i a t i o n was monitored f o r each treatment using the method described i n Staining and Counts (page 30) . Wash: Freshly harvested spores (50 mg) were shaken i n 10 ml of b u f f e r and 0.01% Tween-20 f o r 5 min, the suspension was centrifuged and the supernatant transferred to an vessel. acid-washed The above procedure was repeated three times y i e l d i n g a wash volume of 30 ml which was then freeze-dried and taken up i n 1 ml of M i l l i - Q water. The washed spores were ground and extracted as described e a r l i e r . 43 Spores: The spores were c o l l e c t e d , rinsed with M i l l i - Q water, placed i n an acid-washed mortar, ground with acid-washed sand and HPLC-grade 80% ethanol, and then transferred t o a centrifuge tube and stored at 20°C f o r 20 h. The spore extract was centrifuged at high speed f o r 10 min and the deproteinized supernatent decanted into an acid-washed vessel. The ethanol was evaporated t o 1 ml under a stream of nitrogen gas and transferred to a 10-ml t e s t tube. The dry weight of 50 mg spores was determined by drying the spores f o r 16 h at 100°C. Leachate: The leachate, together with the water used to rinse the spores, was c o l l e c t e d from the 6 dishes with a syringe, f i l t e r e d (0.2 10-ml m M i l l i p o r e ) into a 150 ml acid washed pyrex vessel, s h e l l frozen i n l i q u i d N 2 and freeze-dried. The residue was taken up i n 2 ml of M i l l i - Q water and transferred to a 10-ml t e s t tube. 3.9.5 Sample Clean-up A C 1 8 Sep-Pak cartridge (designed by Waters) was used to remove l i p i d , pigment, residual protein, and other hydrophobic materials from a l l samples. given i n Figure 4. The sample preparation scheme i s Following clean-up a l l samples, including a standard amino acid mixture, were dried down i n a SpeedVac concentrator (Savant Instruments) without heat. 44 1. Activate a Sep-Pak C methanol. 2. Wash with 2-10 ml of 0.1% t r i f l u o r o a c e t i c a c i d (TFA) i n M i l l i - Q water. 3. Wash with 10 ml of 0.1% TFA i n water and methanol (80:20). 4. Mix 1 ml sample with 2 ml 0.1% TFA i n water and methanol (70:30). 5. Pass the sample through the Sep-Pak. 6. Discard the f i r s t 1 ml of. e f f l u e n t and c o l l e c t the next 2 ml f r a c t i o n which contains a l l the amino acids. 1 8 cartridge with 2-10-ml volumes of Figure 4. Sample preparation scheme f o r crude amino a c i d samples using Waters Sep-Pak C cartridges. 1 8 3.9.6 HPLC of Amino Acids as Phenylthiocarbamoyl Derivatives Buffer System Eluent A: Sodium acetate trihydrate, 19.0 gm (140 mM) dissolved i n 1000 ml of M i l l i - Q water and 5% TEA. adjusted t o 6.4 with g l a c i a l a c e t i c acid. was The pH was The s o l u t i o n was vacuum f i l t e r e d and combined with 25 ml a c e t o n i t r i l e . Eluent B: Contained 60% a c e t o n i t r i l e i n M i l l i - Q water. Degassed by sonication f o r 5 min. Pre-column Derivatization Derivatization Solution: An ethanol, TEA and M i l l i - Q water (7:1:1) solution was combined with 10% PITC. Sample Diluent: A phosphate buffer was prepared (710 mg Na HP0 per 1000 ml t i t r a t e d t o pH 7.4 with 10% phosphoric 2 4 acid), combined with 50 ml a c e t o n i t r i l e , and f i l t e r e d . Procedure: with 20 The dried sample and standard were derivatized 1 of the d e r i v a t i z i n g solution f o r 30 min at room temperature. The solvents were removed under vacuum with the SpeedVac centrifuge. The sample and the standards were reconstituted with 25 and 500 respectively. 1 of sample diluent, The amino acids were derivatized i n order t o improve the detection s e n s i t i v i t y as well as overcome the inherent p o l a r i t y of the free amino acids. PITC-amino acids have a broad UV spectrum with a maximum absorbance near 269 nm. PITC allows the quantitative d e r i v a t i z a t i o n of both primary and secondary amino acids. The reaction scheme f o r amino acids by pre-column d e r i v a t i z a t i o n with PITC i s outlined i n Figure 5. Sample Preparation P r i o r t o being loaded on the column each sample was f i l t e r e d (MSI, Cameo dispensable nylon f i l t e r s , 3 mm membrane, 0.45 m pore size) and c o l l e c t e d into 0.5 ml Eppendorf micro t e s t tubes by centrifugation f o r 5 min i n a microcentrifuge (Eppendorf Model 5413). The tubes were then placed on a spring i n 1.5 ml glass v i a l s (Pierce Chemical Co.), covered with a membrane, sealed with a cap, and placed on the autosampler tray. The pump table was programmed t o generate the appropriate buffer gradient system and flow-rate (Table VI) . Chromatography Four 1 of standard were injected per run (200 pM), the f i r s t i n j e c t i o n generated the "junk" chromatograph and was discarded, the second i n j e c t i o n produced the chromatograph used t o c a l i b r a t e the column and subsequent runs. i n j e c t i o n volume was 8 The sample 1 and the i n j e c t i o n - t o - i n j e c t i o n cycle time was 30 min. A f t e r four i n j e c t i o n s and separations of 47 <fO> = = / - N \ / ^ N C H S - L PH 10,30 min + NH-C-C-0 R ' . 11 + phenylisothiocyanate (in excess) y 1 4. S ' > amino compound H H O <(0/"N - C - N - C - C - O^ v temperature > R aqueous acid / \ w A . N O R phenylthiocarbamyl amino acids Fig. 5. Reaction scheme of amino compound by pre-column derivatization with PITC. 48 Table VT: Pump method; the buffer gradient system and flowrate designed t o e f f e c t i v e l y contribute to the separation of amino compounds within a p h y s i o l o g i c a l sample. Time I n i t i a l conditions 10. 00 10.50 11. 50 12 . 00 12.50 20. 00 20.50 30.00 Flow Curve %A %B 1.0 1.0 1.0 1.0 1.5 1.5 1.5 1.0 0.1 * 95 55 0 0 0 100 100 100 100 5 45 100 100 100 0 0 0 0 5 6 6 6 6 6 6 11 49 sample the column was recalibrated with the standard. The r e l a t i o n s h i p between a s p e c i f i c amino a c i d and a known concentration was factor (RF). calculated i n terms of a response The RF i s an adjustment f o r compounds that do not respond equally i n the detector concentration). of peaks. (RF= peak area/amino acid The sample chromatograms contained a number Most of the retention times corresponded with those of i d e n t i f i e d amino acids, the remaining peaks were c l a s s i f i e d as "unknown". The RF values determined during the c a l i b r a t i o n run were used to c a l i b r a t e the amount of a s p e c i f i c amino acid within the sample. Standard solutions of glutamine and glutathione were prepared, and 200 pM of the standard injected onto the column. was Glutamine formed a peak between serine and glycine and glutathione eluted with ammonia. Three controls were run: The Ca-K buffer contained large sloping peak (eluting at approximately 1 to 2.2 The baseline was otherwise f l a t with n e g l i g i b l e peaks. a min). Acid washed sand ground with 80% ethanol generated a chromatogram with a f l a t baseline interrupted by two sharp peaks. f i r s t peak eluted at 9.65 min, shortly a f t e r leucine, and second followed phenylalanine at 11.1 min. d e r i v a t i v e contained The The blank no i n t e r f e r i n g amino compounds. the 50 4. RESULTS 4.1 Uredospore G e r m i n a t i o n and D i f f e r e n t i a t i o n "In V i t r o " In t h e presence o f 1.5 x 10 - 4 M n-nonyl a l c o h o l , 100% o f t h e uredospores germinated w i t h i n 2 h s e e d i n g on C a - K b u f f e r (Fig. 90 to following 6). C o n t r a r y t o e a r l i e r s t u d i e s i n which 95% d i f f e r e n t i a t i o n was o b t a i n e d (Maheshwari e t a l . , t h e p r o p o r t i o n o f germtubes structures infection shock 1967; Dunkle e t a l . . forming complete infection on Ca-K b u f f e r d i d not exceed 60%. The t i m i n g o f s t r u c t u r e formation i n response t o a 1.5 (30.5°C) 1969), on Ca-K b u f f e r i s shown i n F i g . 7. h heat Germtube growth was a r r e s t e d s h o r t l y a f t e r exposure t o t h e elevated temperature. 3.5 h and The hyphal t i p began t o s w e l l after w i t h i n 5 h t h e appressorium was mature ( i . e . a cross wall d e l i m i t e d t h e appressorium from the germtube). The infection peg formed between 5 and 6 h and a t 8 h began t o extend produce the substomatal v e s i c l e . The development v e s i c l e was u s u a l l y completed w i t h i n 10 h by t h e a septum between i t and the appressorium. of A f t e r 10 h the the The cytoplasm w i t h i n t h e germtube and t h e appressorium began t o c l e a r a t 8 and 9 h , Infection the formation of i n f e c t i o n hypha began t o develop on t h e d i s t a l end o f vesicle. to respectively. s t r u c t u r e f o r m a t i o n was complete w i t h i n 15 t o 20 h . I n i t i a l l y t h e i n d u c t i o n o f d i f f e r e n t i a t i o n by h e a t shock 51 Time (hr) Fig. 6. The percent germination of uredospores on Ca-K buffer in both the presence ( • ) and absence ( O ) of 1.5 x 10 -4 M n-nonyl alcohol. 52 F i g . 7. Timing of sporeling morphogenesis. (A) A sporeling 1 h following germination. (B) A sporeling 3.5 h following germination, the appressorium i n i t i a l has formed. (C) A f t e r 5 h a septum was v i s i b l e and the appressorium was considered mature. (D) Development of the i n f e c t i o n peg was complete 6 h following germination. (E) After 9 h the cytoplasm began to c l e a r within the germtube. (F) The substomatal v e s i c l e began to expand a f t e r 8 h. (G) The cytoplasm migrated from the appressorium into the developing substomatal v e s i c l e and a second septum become apparent (10 h). (H) The i n f e c t i o n hypha was apparent a f t e r 15 h. A d i f f e r e n t i a t e d sporeling a f t e r 20 h. (592 X). 53 as described by Maheshwari e t a l . (1967) yielded poor r e s u l t s (< 5% d i f f e r e n t i a t i o n ) . mm 2 By using a spore density of 300 per and c o n t r o l l i n g the temperature of the heat shock by placing the Conway dishes i n a water bath, i t was possible t o a t t a i n up t o 60% d i f f e r e n t i a t i o n . A l l further attempts t o improve the percent d i f f e r e n t i a t i o n ; such as, changing the timing and duration of the heat shock were unsuccessful. 4.2 Temperature Range T r i a l s The temperature required t o induce the maximum proportion of germtubes t o d i f f e r e n t i a t e into i n f e c t i o n structures was found t o l i e within a narrow range. The optimum temperature appeared t o be c h a r a c t e r i s t i c of a p a r t i c u l a r spore l o t and ranged from 28° t o 30°C. Incubation a t lower than 27.5°C or higher than 30.5°C considerably reduced the amount of appressorium formation (Fig. 8) and t o t a l d i f f e r e n t i a t i o n (Fig. 9). The upper and lower temperature l i m i t s f o r a l l spore l o t s tested were estimated as 30.5® and 27.5°C, respectively. Heat treatment was completely i n e f f e c t i v e when the temperature was raised 1° above the upper l i m i t or dropped 2° below the lower l i m i t (Figs. 8 and 9). Although the incubation temperature where the maximum t o t a l d i f f e r e n t i a t i o n was achieved varied between spore l o t s , the s e n s i t i v i t y of the sporelings t o temperature changes was similar (Figures 10 and 11). Variations 1° above or below the 54 F i g . 8. Temperature requirements f o r appressorium formation from uredospore germtubes. The spores were germinated a t 19°C (2 h ) . then placed a t constant temperatures (ranging from 26 to 33°C) f o r 1.5 h, and replaced t o 19°C. The optimum temperature (><) i s the temperature at which the maximum t o t a l d i f f e r e n t i a t i o n was attained f o r each spore l o t . 55 80 i < > 60 40 Fig. 8A. Spores collected July 22,1986. Optimal differentiation temperature (><): 29.3 C . • d • 20 • 0 • < 60 • 40 Fig. 8B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C. • • 20 • • 0 60 Fig. 8C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C. > 40 • 20 • 0 < 60 • Fig. 8D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 ° C . • 40 • • 20 - 2 - 1 0 + 1 • • +2 +3 Variation Below (-) and Above (+) Optimal Temperature (°C) 56 F i g . 9. Temperature requirements f o r the complete d i f f e r e n t i a t i o n of P. graminis t r i t i c i uredospore germlings. The upper and lower l i m i t s f o r t o t a l d i f f e r e n t i a t i o n were estimated to be 27.5 and 30.5°C, respectively. Germination conditions were described i n F i g . 8. 57 60 • 40 Fig. 9A. Spores collected July 22,1986. Optimal differentiation temperature (> <): 29.3 °C. • 20 I I Fig. 9B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C. n 40- I J 20 p I1 o 0 Pi zn Fig. 9C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C. 40 <u 20 • • PH • • 40 Fig. 9D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 °C. 20 • • -1 0 +1 +2 +3 Variation Below (-) and Above (+) Optimal Temperature ( C) 58 100 -, 80 • 60 < 40 20 • i -1 +1 • • • • • +2 Variation Below (-) and Above (+) Optimal Temperature (°C) Fig. 10. The relationship between heat shock temperature and the transformed percent of appressoria formed (TPA). i +3 59 Variation Below (-) and Above (+) Optimal Temperature (°C) Fig. 11. The relationship between heat shock temperature and the transformed percent of total sporeling differentiation (TPD). 60 optimum temperature resulted i n greater than 40% reduction i n the number of sporelings forming i n f e c t i o n structures. The formation of appressoria was l e s s s e n s i t i v e to deviations from the optimum temperature (Fig. 10) than the formation of a complete set of i n f e c t i o n structures (Fig. 11). 4.3 The Influence of Nutrients on Sporeling D i f f e r e n t i a t i o n Preliminary experiments were undertaken to investigate the e f f e c t of nutrients (peptone and glucose), a germination stimulant (n-nonyl alcohol), and a heat shock (30°C, 1.5 h), on the percentage of sporelings developing i n f e c t i o n structures (Table VII). MPG medium (peptone, glucose, and Ca- K b u f f e r ) , i n conjunction with both a heat shock and n-nonyl alcohol, supported up to 77% d i f f e r e n t i a t i o n . When the heat shock was not administered, less than 11% of the sporelings differentiated. The absence of both, n-nonyl alcohol and the heat shock resulted i n the growth of long, matted germtubes that f a i l e d to form i n f e c t i o n structures. Although a l l three components of MPG influenced sporeling d i f f e r e n t i a t i o n i t was evident that t h e i r roles were not of equal importance. MPG was broken down t o i t s constituent parts and each component was tested f o r i t s a b i l i t y to promote differentiation. The buffer alone supported 66% d i f f e r e n t i a t i o n , peptone i n d i s t i l l e d water supported 14%, and glucose i n d i s t i l l e d water resulted i n l e s s than 5% sporeling 61 Table VII: A summary of experiments investigating the influence of the components of MPG, n-nonyl alcohol, and a 29.5°C (1.5 h) heat shock on the percent of germtubes forming appressoria (PAS) and the percent of sporelings forming a complete set of i n f e c t i o n structures (PDS). The presence or absence of a treatment i s indicated by '+' or respectively. MPG peptone g l u c o s e Ca-K + + + + + + + * - + + + + + + - + + • + + + + - + + + + n-nonyl alcohol heat shock + + + + + + + + + + + + + + + + + + - - *supplemented with tyrosine (49 mg/l), cysteine tryptophan (15 mg/l). PAS PDS 89 27 55 0 54 71 84 36 83 5 40 (17 mg/l), and 77 11 50 0 44 32 39 14 66 5 32 62 differentiation. The combination of peptone and glucose increased d i f f e r e n t i a t i o n to 32%. uredosporelings (77%) was The maximum amount of forming a complete set of i n f e c t i o n achieved on the complete MPG medium. structures These r e s u l t s show c l e a r l y that the response to nutrients i s non-additive. Glucose i n d i s t i l l e d water promoted the growth of long germtubes which f a i l e d to d i f f e r e n t i a t e . The addition of cysteine, tyrosine and tryptophan to glucose and Ca-K buffer had no s i g n i f i c a n t e f f e c t on d i f f e r e n t i a t i o n . 4.4 E s s e n t i a l Protein Synthesis Sporelings exposed to an i n h i b i t o r of protein synthesis before, during, and a f t e r the inductive period provide evidence f o r the nature and timing of protein synthesis during differentiation. I t was found i n the present study that the presence of puromycin (100 ppm) had a s i g n i f i c a n t e f f e c t on appressorium formation and completely i n h i b i t e d a l l further development (Table VIII). In most cases where the heat treatment arrested l i n e a r growth the germtube produced an apical swelling, which a f t e r 20 h f a i l e d to form a cross wall (Fig. 12a). The appressorium was e a r l i e r defined by Staples et al.(1983c) as a swelling of the hyphal t i p that i s separated from the germtube by a septum. In the absence of puromycin the appressoria were oval to roundish. In the 63 Table VIII: The e f f e c t o f a p r o t e i n i n h i b i t o r , puromycin (100 ppm), on t h e development o f i n f e c t i o n s t r u c t u r e s from g e r m l i n g s o f P. g r a m i n i s t r i t i c i . To induce t h e d i f f e r e n t i a t i o n o f i n f e c t i o n s t r u c t u r e s a heat shock a t 3 0 ° C f o r 1.5 h was a d m i n i s t e r e d 2 h a f t e r t h e s t a r t o f germination at 1 9 ° C . S p o r e l i n g development Treatment Appressoria a* b# Infection peg Vesicle a b (%) Infection hypha 69 100 61 100 53 100 40 100 X 78 114 72 117 60 114 27 67 XX 73 107 69 112 50 96 1 3 XXX 72 105 58 94 17 31 0 0 —xxxx 75 109 54 88 0 0 0 0 xxxxxx 17 2 3 0 0 0 0 - x — 78 114 71 115 49 93 21 53 — x - 76 111 67 109 43 81 12 30 —-x- 78 113 72 118 46 88 11 27 —xx— 72 105 57 93 27 52 3 7 xx- 73 106 55 90 27 51 2 5 (control) 25 Puromycin was p r e s e n t (x) and absent from (-) t h e g e r m i n a t i o n medium (Ca-K b u f f e r ) . ( ~) c o r r e s p o n d s t o t i m e i n t e r v a l s ; 0-2 h , 2-3.5 h , 3.5-6 h , 6-8 h , 8-10 h , and 10-15 h . *development i s expressed as a mean p e r c e n t o f t h e t o t a l spores counted. ^development i s expressed as a percent of the c o n t r o l , underlined values are s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l (95% c o n f i d e n c e - F i s h e r L S D ) . 64 Fig. 12. The presence of a protein i n h i b i t o r (puromycin) i n the germination medium s i g n i f i c a n t l y reduced the number of germtubes forming appressoria. (A) The appressoria were predominantly i r r e g u l a r i n form (bright f i e l d illumination, 592x). (B) Generally the nuclei within the germtube d i d not divide but remained expanded from 0 to 15 h (fluorescent micrograph, 1040x). (C) In cases where a septum was formed the nuclei divided t o give r i s e to four daughter-nuclei. The p a i r i n g of daughter nuclei was not observed (double illumination with U.V. and highly attenuated v i s i b l e l i g h t , 1040x). 65 presence of the i n h i b i t o r however, t h e i r shape was predominantly i r r e g u l a r (Fig. 12a). The r e s u l t s found by Dunkle e t a l . were confirmed (with the exception of appressorium formation) by a series of preliminary experiments. To further c l a r i f y the timing of protein synthesis and i t s e f f e c t on the proportion of germtubes forming i n f e c t i o n structures the sporelings were exposed to puromycin at s p e c i f i c times following heat treatment. Complete d i f f e r e n t i a t i o n was prevented i n those t r i a l s where puromycin was present from immediately a f t e r heat treatment to the completion of the experiment (20 h). During t h i s time frame the development of the appressorium and the i n f e c t i o n peg were unaffected but a l l further d i f f e r e n t i a t i o n was i n h i b i t e d (Tables VIII and IX). Although the sporelings appeared t o recover from the e f f e c t s of the i n h i b i t o r shortly a f t e r the removal of puromycin from the germination medium, the development of the i n f e c t i o n hypha was s i g n i f i c a n t l y impaired i n a l l cases. Although the development of v e s i c l e s d i d not occur before 10 h, t h e i r formation appeared t o be dependent on proteins synthesized 3.5 to 8 h following germination. The formation of the i n f e c t i o n hypha was dependent on the development of the v e s i c l e ; the l a t t e r always preceded the former. The proportion of v e s i c l e s developing i n f e c t i o n hyphae was considerably reduced when puromycin was present at 3.5 t o 8, 6 to 10, or 6 to 15 h and completely i n h i b i t e d when the 66 Table IX: The e f f e c t of a protein i n h i b i t o r , puromycin (100 ppm), on the development of i n f e c t i o n structures from germlings of P. graminis t r i t i c i . Development i s expressed as a mean percent of the preceding structure. Sporeling Treatment I n f e c t i o n peg a* b* development (%) a I n f e c t i o n hypha a b Vesicle b 90 100 86 100 76 100 X 92 103 84 98 44 59 XX 94 105 73 85 2 3 XXX 80 90 29 34 0 0 —XXXX 72 80 0 0 0 0 xxxxxx 10 11 0 0 0 0 —x 91 101 69 81 43 57 88 98 64 74 28 37 93 104 64 75 23 31 79 88 48 56 10 11 76 85 49 57 7 10 (control) —-xXX— xx- Puromycin was present (x) and absent from (-) the germination medium (Ca-K b u f f e r ) . ( ) corresponds to time i n t e r v a l s ; 0-2 h, 2-3.5 h, 3.5-6 h, 6-8 h, 8-10 h, and 10-15 h. *development as a mean percent of the preceding structure. ^development as a mean percent of the preceding structure, expressed as a percent of the control, underlined values are s i g n i f i c a n t l y d i f f e r e n t from the control (95% confidence - Fisher LSD) 67 i n h i b i t o r was present at 3.5 t o 15 h (Tables VIII and IX). The nuclear behaviour of sporelings d i f f e r e n t i a t i n g i n the presence of puromycin was interesting. Following heat treatment the 2 nuclei migrated from the spore, into the germtube and towards the appressorial i n i t i a l . In most cases the n u c l e i remained i n the germtube, had a granular appearance, and were expanded i n form (Fig. 12b). Occasionally the nuclei migrated into the developing appressorium and completed one round of m i t o t i c d i v i s i o n . The presence of an i n h i b i t o r of protein synthesis appeared to arrest germtube development at a s p e c i f i c phase of the c e l l cycle. The expanded form of the n u c l e i suggests that the c e l l was e i t h e r i n the l a s t part of Gl or entering the S-phase. The four daughter nuclei d i d not move together but remained separate and expanded throughout the observation period (at l e a s t 20 h) (Fig. 12c). Nuclear elongation p r i o r to m i t o t i c d i v i s i o n was not observed. 4.5 4.5.1 Cytology of Uredosporeling Development Nuclear Staining The most suitable technique tested f o r f i x a t i o n was a graded ethanol series followed by staining with 1 ug/ml DAPI. DAPI reacted r a p i d l y with the sample, the n u c l e i were c l e a r l y v i s i b l e and fluoresced a bright blue. Most of the germtubes remained transparent with l i t t l e t o no fluorescence. 68 Germtiibes 1.5 h o l d or younger fluoresced strongly making i t d i f f i c u l t to discern the n u c l e i . Nuclear fluorescence improved f o r 20 min and then remained stable f o r up, to 10 days when stored i n the dark at 4°C. In cases where the i n f e c t i o n hypha was developing and the n u c l e i had migrated into the hypha a change was observed i n the v e s i c l e . The v e s i c l e cytoplasm became speckled, the "granules" fluoresced a bright gold. The addition of a surfactant, such as T r i t o n X-100 or Tween-20 allowed the spores to be taken into suspension and s i m p l i f i e d washing procedures by centrifuging the sample between changes. However, i n the presence of surfactants the germtube fluoresced strongly rendering the n u c l e i i n v i s i b l e . 4.5.2 Nuclear Behaviour during Germination and Differentiation For c y t o l o g i c a l studies the uredospores of P. graminis t r i t i c i were germinated on a Ca-K buffer. The sporelings were induced to d i f f e r e n t i a t e as described e a r l i e r (page 27). Uredospore n u c l e i were stained with DAPI at various stages of germtube development. The r e s t i n g uredospore was binucleate. Following germination the two nuclei usually migrated i n tandem from the spore into the developing germtube and toward i t s apex (Fig. 13). These n u c l e i were round to oval i n shape and represented the expanded or interphase n u c l e i described by Savile (1939). In the absence of a stimulus to d i f f e r e n t i a t e 69 F i g . 13. F o l l o w i n g 2 h g e r m i n a t i o n a t 19°C t h e two i n t e r p h a s e n u c l e i m i g r a t e d from t h e uredospore i n t o t h e e l o n g a t i n g germtube (Double i l l u m i n a t i o n w i t h U.V. and h i g h l y a t t e n u a t e d v i s i b l e l i g h t , 1040x). F i g . 14. The germtube o f non-shocked u r e d o s p o r e l i n g s remained b i n u c l e a t e f o r up t o 20 h. The n u c l e i u s u a l l y appeared t o remain i n p e r p e t u a l i n t e r p h a s e ( I 0 4 0 x ) . F i g . 15. I n some c a s e s t h e n u c l e i w i t h i n 2 0 - h o u r - o l d u r e d o s p o r e l i n g s were e l o n g a t e d i n form and had a "ragged" appearance ( f l u o r e s c e n t m i c r o g r a p h , 1040x). Fig. 16. The l i n e a r growth of the germtube was arrested during the 1.5 h heat shock period. Four hours following germination the two nuclei had migrated into the newly formed appressorium i n i t i a l . Their elongated form suggested that these nuclei were i n conjugate telophase and preparing to divide (double illumination with U . V . and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 1040x). Fig. 17. The f i r s t round of mitosis usually occurred within the mature appressorium 5 and 8 h a f t e r germination (1040x). Fig. 18. The mature appressorium contained four daughtern u c l e i . These n u c l e i moved together to form a compact tetrad (1040x. the germtube remained binucleate and appeared to be i n perpetual interphase; m i t o t i c d i v i s i o n was not observed i n 0to 20-hour-old sporelings (Fig. 14). In some cases the nuclei of nondifferentiated 20-hour-old sporelings had a "ragged" appearance and became elongated i n form (Fig. 15). The nuclear behaviour of the d i f f e r e n t i a t i n g sporeling was complex; nuclear d i v i s i o n was predictable and changes i n form and migration patterns were observed regularly. Although nuclear d i v i s i o n was r e a d i l y observed the more subtle changes i n nuclear form, which correspond to s p e c i f i c phases of the c e l l cycle could not be followed with certainty. shock the germtube produced a bulbous appressorial into which the two nuclei migrated. During heat initial At 4 h the n u c l e i were elongated to dumbbell i n shape and were situated one behind the other (Fig. 16). Their appearance suggested that these n u c l e i were i n conjugate telophase and nearing the completion of mitosis. Usually, a f t e r 4.5 h a cross wall formed and delimited the appressorium from the germtube. The f i r s t round of mitosis was usually complete within 5 t o 8 h following imbibition (Fig. 17). The mature appressorium contained four n u c l e i arranged as a compact tetrad (Fig. 18). At 7 h the four nuclei migrated toward the developing i n f e c t i o n peg. Occasionally two n u c l e i divided i n the appressorium (Fig. 19) but more commonly the second d i v i s i o n occurred as the nuclei moved into the developing substomatal v e s i c l e (7 h to 13 h). The nuclei migrated i n c l o s e l y 72 Fig. 19. Occasionally two nuclei divided within the appressorium. The s i x nuclei migrated i n tandem towards the developing i n f e c t i o n peg (double i l l u m i n a t i o n with U . V . and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 104Ox). Fig. 20. Ten hours following germination the six-nucleate v e s i c l e was delimited from the appressorium by a septum (1040x). Fig. 21. In most cases a nuclear p a i r divided i n the mature v e s i c l e to y i e l d a t o t a l of eight n u c l e i . These n u c l e i were d i s t r i b u t e d such that three to four nuclei aggregated on either side of the v e s i c l e (1040x). 73 Fig. 22. The orientation of nuclear migrational patterns was found to be related to the p o l a r i t y of the v e s i c l e . Once growth of the i n f e c t i o n hypha was i n i t i a t e d the nuclei moved into an arrowhead formation with the apex directed towards the developing hypha (double illumination with U.V. and highly attenuated v i s i b l e l i g h t , inset: fluorescent micrograph, 1040X). Fig. 23. The i n f e c t i o n hypha contained eight, six, or more commonly four nuclei (1040x). Fig. 24. In some cases a nuclear p a i r f a i l e d to migrate with the other nuclei and remained i n the v e s i c l e (1040x). Fig. 25. Twenty three hours following germination the i n f e c t i o n hypha most often contained four expanded nuclei (1040x). 74 associated p a i r s . A cross wall delimited the six-nucleate v e s i c l e from the appressorium within 10 h (Fig. 20). In most cases a nuclear p a i r divided i n the mature v e s i c l e to y i e l d a t o t a l of eight n u c l e i (Fig. 21). These n u c l e i , which were rounder and considerably smaller than those observed i n the germtube, are described as d i v i d i n g or unexpanded. Nuclear d i v i s i o n was not always synchronous, f i v e to eight n u c l e i were often seen within the v e s i c l e . Within 10 to 13 h a f t e r germination s i x to eight n u c l e i were d i s t r i b u t e d such that three to four n u c l e i aggregated on e i t h e r side of the v e s i c l e (Fig. 21). The i n f e c t i o n hypha of P. graminis t r i t i c i developed as a polar extension from the v e s i c l e . i n f e c t i o n hypha was Once growth of the i n i t i a t e d (ca 13 h) a nuclear pattern became apparent. migration The n u c l e i moved towards the developing hypha i n the formation of an arrowhead (Fig. 22). The o r i e n t a t i o n of t h e i r migration was p o l a r i t y of the v e s i c l e . always r e l a t i v e to the The leading nucleus, which often fluoresced more strongly than the others, was directed towards the developing hypha. As growth ensued eight, s i x or four expanded n u c l e i migrated into the i n f e c t i o n hypha (Fig. 23), occasionally a nuclear p a i r remained i n the v e s i c l e (Fig. 24). A f t e r 23 h four or fewer n u c l e i were observed i n the hypha (Fig. 25). 75 4.6 Amino Acids Analysis The standard generated 18 peaks with c h a r a c t e r i s t i c retention times. The separation of a PITC-amino acid standard i s i l l u s t r a t e d i n F i g . 26, Table X. was observed within 12 min. Good resolution An additional 18 min was required to remove solvent peaks and r e e q u i l i b r a t e the column between runs. Except f o r h i s t i d i n e from arginine, and glutathione from ammonia, most amino compounds were adequately separated using the RP-column . As i s c h a r a c t e r i s t i c of RPchromatography, the e l u t i o n order was related t o the increasing hydrophobic nature of the solute ( i . e . the more water soluble the compound, the faster i t was eluted). 4.6.1 Experimental Plan The objective was t o determine the e f f e c t of the heat shock on (A) the endogenous free amino acid pool and (B) the exogenous free amino acids leaching from the germinating uredospore. The following fractions were therefore analyzed: (1) Buffer wash from r e s t i n g uredospores. (2) Alcohol extract from unwashed r e s t i n g uredospores. (3) Alcohol extract from eight-hour-old uredosporelings: non-shocked and heat shocked. (4) Leachates from eight-hour-old uredosporelings: nonshocked and heat shocked. Retention Time (min) Fig. 26. Separation of amino acid standards (Pierce H). Eluent A: 140 mM sodium acetate, 5% TEA, pH 6.4; eluent B: 60% acetonitrile in water; gradient: 5% B to 45% B in 10 min on curve 5; flow-rate: 1 mVrnin for 12 min, 1.5 ml/min for 8 min: column: Pico-Tag RP-column for protein hydrolyzates; detector: ultraviolet (254 nm) at 0.1 a.u.f.s. For peak identification see Table X. Table X. PITC-arnino acid peaks identified by number and retention time as separated by RP-HPLC under the conditions shown in figure 26. Number Amino Acid Retention Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Aspartic acid Glutamic acid Serine Glycine Histidine / Axginine Threonine Alanine Proline NH Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Lysine 2.44 2.68 4.48 4.69 5.22 5.37 5.51 5.66 6.00 7.06 7.61 8.00 9.12 9.30 9.50 10.27 11.62 18 19 20 Glutamine Homoserine Glutathione 4.53 4.96 6.00 * 3 Unknown peaks — 78 (5) Alcohol extract from 20-hour-old uredosporelings: non-shocked and heat shocked. (6) Leachates from 20-hour-old uredosporelings: nonshocked and heat shocked. 4.6.2 Complications A number of problems were encountered i n i d e n t i f y i n g and quantifying the amino compounds i n uredospores and uredospore leachates. Representative RP-HPLC amino acid p r o f i l e s from (A) the alcohol extract from non-shocked uredospores, and (B) the leachate from heat shocked uredospores, are shown i n F i g . 27. Two peaks corresponding to glutamine (#18 - see Table X) and NH /glutathione (#9/20) are predominant 3 i n both p r o f i l e s . The r e s o l u t i o n between peaks i d e n t i f i e d as serine, glycine, and glutamine was poor. Therefore, since column s e l e c t i v i t y could not be improved, s t r i c t l y quantitative estimates of the amounts of these amino compounds per mg spores could not be obtained. Despite the overlap, peaks corresponding to serine, glycine, and glutamine were i d e n t i f i e d i n most, but not a l l extracts. The most serious overlap occurred between glycine and glutamine and i n F i g 27 glycine was completely obscured by glutamine (#18). In those runs where the separation between glycine and glutamine was achieved the glycine present was always only about 1/3 the height of the glutamine peak. The response factors (RFs) f o r glycine and glutamine were s i m i l a r i n value. In order to calculate the order of magnitude of the 79 F i g . 27. Representative R P - H P L C separations of amino acids isolated f r o m ( A ) non-heat shocked 20-hour-old uredosporelings and (B) the leachate of 20-hour-old heat shocked uredosporelings. F o r peak identification see Table X . 80 concentration of glutamine estimates based on the area of the glutamine peak have been reduced by 30%. Such estimates leave no doubt that glutamine i s predominant among the amino compounds i n uredospores (Fig. 27A). Similar considerations apply to the analysis of the leachate (Fig. 27B), i n which glutamine was also predominant. A further complication i n the analysis of the leachate arose because of the presence of a very large u n i d e n t i f i e d peak (X) i n the buffer (Fig. 27B) which usually overlapped aspartate (#1) and glutamate (#2). This prevented any meaningful estimates of the l e v e l s of aspartate and glutamate i n the leachate. In a l l chromatograms ammonia and glutathione eluted o f f the column simultaneously. I t was therefore impossible to obtain independent estimates of the l e v e l s of NH glutathione. 3 and The NH /glutathione peak (#9/20) was usually 3 larger than a l l other amino compounds except glutamine (Fig. 27). Ten amino acids (alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, and lysine) were i d e n t i f i e d and quantified with confidence. Where possible (see above) the amount of each amino acid was expressed as nM per mg spore dry weight. There was some unavoidable loss of heat shocked uredosporelings during t r a n s f e r from the germination medium t o the extraction vessel because the shorter, d i f f e r e n t i a t e d germtubes d i d not i n t e r l o c k to form a mat. The loss was v i s u a l l y estimated to 81 be not greater than 15%. Calculations of the amino acid l e v e l s per mg dry weight of uredospores were based on the dry weight of the o r i g i n a l sample (2.78 mg) and are therefore too low by a maximum of 15%. Two unknown peaks eluted from the column shortly a f t e r the amino acid phenylalanine (#16). by Dwyer et a l . Based on r e s u l t s reported (1987) these peaks may be i d e n t i f i e d t e n t a t i v e l y as tryptophan (Trp) and ornithine (Orn). These compounds appeared soon a f t e r germination occurred; they were detected i n the germinating uredospores and i n the leachate from 20-h-old non-shocked uredosporelings. 4.6.3 Exogenous Free Amino Acids: Resting Spore Wash The buffer i n which resting uredospores were washed contained the e a r l y - e l u t i n g amino acids. associated with the spore wall. These appeared to be Although there was some overlap of aspartic and glutamic acids with the u n i d e n t i f i e d peak (X) found i n the buffer, i t was possible to estimate the amounts of these amino acids by approximating the area of the peak. An estimate of glutamine was obtained as described e a r l i e r (page 77). The r e s u l t s are shown i n Table XI. The amino acids washed o f f the spores were, i n order of predominance, glutamine, alanine, glutamate, aspartate, and tyrosine. An unknown compound with a retention time of minutes was also present i n the spore wash. 6.66 Since i t was unknown i t could not be quantified by i t s comparison with a 82 Table XI. Free amino acid composition of the buffer wash from ungerminated uredospores of P. graminis t r i t i c i . Amino A c i d nM/mg spore dry w e i g h t 3 Percent Ala Pro Tyr Val Met Cys He Leu Phe Lys 0.57 ±.07 26.0 0.25 ±.06 11.4 Glm HomoSer Arg/His/Thr Unknown* 0.72 ±.04 ND + + + ND + + + ND a •++ + Asp Glu 0.27 ±.03 0.38 ±.06 Total 2.19 ±.26 - — 32.9 — 12 . 3 17.4 100. 0 Each value represents the mean and range f o r two separate experiments. + = trace amounts, ++ = greater than trace amounts (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids). a a 83 standard. Therefore i t was assigned an RF of 4256 which i s an average value calculated from the RF of 14 d i f f e r e n t amino acids. In addition, small peaks corresponding t o the retention times of one or more of arginine, h i s t i d i n e , and threonine were detected (Table XI). which of these were actually present. I t was impossible t o t e l l Cysteine, homoserine, and l y s i n e were not detected. 4.6.4 Endogenous Free Amino Acids: Extracts of Unwashed Resting Uredospores In order of prevalence the amino compounds i n unwashed r e s t i n g uredospores were alanine, the unknown, glutamic acid, and glutamine followed by smaller amounts of several other amino acids (Table XII). Alanine accounted f o r 30% and the unknown accounted f o r 22% of the t o t a l free amino acids present. (2.4/0.2). The r a t i o of glutamic to a s p a r t i c acid was 12 Cysteine and l y s i n e were not detected. Comparison of the appropriate columns i n Tables XI and XII shows that the t o t a l amino acids washed o f f r e s t i n g uredospores (Table XI) account f o r only 16.5% (2.19 x 100 /13.3) of the t o t a l amino acids extracted from unwashed r e s t i n g spores. 84 Table XII. The endogenous free amino acid l e v e l i n unwashed r e s t i n g uredospores (S), and i n non-shocked uredosporelings of P. graminis t r i t i c i a f t e r 8 h (N8S), and 20 h (N20S) nM/mg spore dry Amino Acid S Ala Pro Tyr Val Met Cys He Leu Phe Lys 4 . 04 0.20 + 0.43 + ND 0.25 0.23 0.29 ND Glm HomoSer Arg/His Unknown* Orn Trp a Asp Glu Total Glu:Asp N8S 47 0 ± 0 ±. 01 ±. 02 ±. 02 2 .23 ± 30 ++ ++ 2 .98 ±. 75 ND ND 0. 2 ± 0 2 .4 + .1 13 .3 10:1 ±1.7 4. 99 2 .20 2 .47 1 .80 2 .54 7 . 09 2 . 16 1 .93 2 .93 1 . 17 weight Ratio a N20S ±. 71 ±. 29 ±. 04 ±. 43 +. 07 ±. 39 ±. 15 ±. 33 ±. 31 ±. 60 2 . 50 1 . 10 2 . 29 1 .23 2 . 09 3 . 19 1 . 07 1 . 50 3 . 10 1 .05 N8S/S ±. 30 ±. 13 ±. 64 ±. 17 ±. 42 ±. 77 ±. 17 ±. 54 ±. 58 ±. 02 6.99 +. 84 10 .74 ±1 . 2 ++ ++ ++ ++ 2 .47 ±. 10 2 .95 ±. 79 ++ ++ ++ ++ 2 . 2 ±. 7 10 . 0 ±2 .9 50 .9 5:1 1 . 5 ±. 2 4. 1 ±. 8 ±7 . 6 38 . 4 ±6 . 7 3: 1 N20S/S 1. 2 11. 0 27 .4 2 .9 50. 8 (>1 000) 8. 6 8 .4 10. 1 (>2 00) 0. 6 5. 5 25. 4 2 .7 41. 8 (>6 00) 4 .3 6 .5 10. 7 (>2 00) 3 .1 4 .8 1. 0 1. 2 22 .0 4 .2 15. 0 1. 7 3 .8 2 .9 - - Each value represents the mean and range f o r two separate experiments. + = trace amount, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids. a a 85 4.6.5 E f f e c t of Heat Shock on the Free Amino Acids i n Germinated Uredospores and i n the Leachate A f t e r Germination Unwashed uredospores were placed on Ca-K buffer a t 0 h at 19° C, heat shocked at 29° C from 2 h t o 3.5 h and harvested at e i t h e r 8 h or 20 h. Non-shocked controls were subjected to the same protocol but were maintained at 19° C throughout the germination period. analyzed separately. Uredosporelings and leachates were The r e s u l t s are presented i n Tables XII, XIII, and XIV. The data i n Table XII show that the endogenous free amino acid l e v e l i n non-shocked uredospores at 8 h was 3.8-fold, and at 20 h was 2.9-fold that i n unwashed r e s t i n g uredospores. Among the i n d i v i d u a l amino acids, alanine and the unknown compound showed r e l a t i v e l y l i t t l e change on germination but a l l the other amino compounds increased markedly. Large r e l a t i v e increases occurred i n cysteine and lysine, which were below the l i m i t of detection i n r e s t i n g uredospores. The r a t i o of glutamate t o aspartate decreased sharply on germination. Table XIII shows that, while the endogenous free amino acid l e v e l i n heat shocked uredospores increased approximately 2-fold a t 8 h, there was no further increase at 20 h. the i n d i v i d u a l amino compounds there were decreases 50%) i n alanine and the unknown compound. Among (about Valine was r e l a t i v e l y unchanged. A l l other amino compounds increased s i g n i f i c a n t l y , p a r t i c u l a r l y cysteine and l y s i n e . In general 86 Table XIII. Endogenous free amino acid content of unwashed r e s t i n g uredospores (S), and heat shocked uredosporelings of P. graminis t r i t i c i a f t e r 8 h (H8S), and 20 h (H20S). Amino Acid nM/mg spore dry w e i g h t S H8S 4.04 ±.47 0.20 ±.0 Ala Pro Tyr Val Met Cys He Leu Phe Lys 2.23 ±.30 8.69 ±.89 8.38 ±.35 2.98 ±.75 ND ND 1.43 ±.18 1.54 ±.48 0.2 2.4 1.7 ±.4 4.5 ±1.0 + Asp Glu Total ++ ++ ±.0 ±.1 ±.03 ±.01 ±.03 ±.13 ±.11 ±.12 ±.05 ±.19 ±.16 ±.11 +'+ ++ ++ + 1.40 0.46 1.20 0.66 0.84 1.45 0.69 1.49 1.44 0.44 H8S/S ND 0.25 ±.01 0.23 ±.02 0.29 ±.02 ND + 3 H20S 1.80 0.66 0.93 0.53 0.56 2.89 0.80 1.93 1.41 0.25 0.43 ±.0 Glm HomoSer Arg/His Unknown* Orn Trp Ratio 3 ±.01 ±.0 ±.16 ±.03 ±.02 ±.03 ±.01 ±.02 ±.11 ±.11 ++ ++ ++ ++ 1.0 3.5 ±.1 ±.0 13.3 ±1.7 28.1 ±.16 24.5 ±.76 Adjusted Total 11. l Glu:Asp 10:1 b 32.3 3 :1 C 28.3 C 4:1 0.5 3. 3 10. 3 1.2 11.2 (>600) 3.2 8.4 4.9 (>60) H20S/S 0.4 2. 3 13 . 3 1.5 16.8 (>300) 2. 8 6.5 5.0 (>80) 3. 9 3.8 0.5 0.5 17.0 1.9 10. 0 1.5 2.1 1.8 - - - - E a c h value represents the mean and range f o r two separate experiments. + = trace, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids, ^measured endogenous amino acids minus the amino acids i n the resting spore wash. measured amounts plus 15% t o compensate f o r uredospore losses during transfer. a 3 c 87 Table XIV. The free amino acid composition of leachate emanating from heat shocked (H) and non-shocked (N) uredosporelings of P. graminis t r i t i c i a f t e r an 8 h (H8L and N8L), and 20 h (H20L and N20L) incubation period. nM/mg spore dry wei g h t Amino Acid N8L Ala Pro Tyr • Val Met Cys He Leu Phe Lys 3 Total: Leachate (L) Wash (W) L -W 2 N2 0L H2 0L ±. 21 ±. 17 ±. 14 ±. 01 0.70 ±. 01 2 . 99 ±. 59 + 0.85 ±. 71 + ND + + ND + 4. 52 2 .81 1 .21 1 .91 1 .36 5 . 42 1 . 61 1 . 17 2 . 06 0. 66 2 .40 ± . 20 ++ ++ ND ND ND 2 . 39 ±. 33 ++ ++ ND ND ND 9.93 ±. 00 ++ ++ 3 .31 ±. 35 ++ ++ 5. 67 +,30 6.93 ±. 98 35 .97 ±. 02 13 . 79 ±. 95 2 .19 ±. 26 2 .19 ±. 26 2 . 19 ±. 26 2 .19 ±. 26 0.60 1.78 0.29 0. 60 + ND + + + + Glm HomoSer Arg/His Unknown* Trp Orn H8L a 3 .48 4 .74 ±. 07 ± .43 ±. 13 ±. 28 ±. 03 ±. 13 +.03 ±. 03 ±. 05 +.01 33 .78 0.91 ±. 11 2 . 64 ±. 24 0.33 +.08 + + + + + ND ND 9.91 ±1 . 17 ++ ++ ND ND ND 11. 60 E a c h value represents the mean and range f o r two separate experiments. + = trace, ++ greater than a trace, (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. see text. *calculated assuming a RF of 4256 (average value f o r 14 d i f f e r e n t amino acids. from Table XI. a a a the increases i n i n d i v i d u a l amino compounds were smaller than i n non-shocked spores. Moreover the amino a c i d l e v e l i n heat shocked spores was only 55% (28.1 x 100 /50.9) o f that i n nonshocked spores a t 8 h and 64% (24.5 x 100 /38.4) a t 20 h. I t i s thus c l e a r that heat shock decreases protein hydrolysis i n germinating uredospores. Paraphrasing the well known song, we may ask "Where have a l l the amino acids gone?". The data i n Table XIV, which presents the analyses of the leachates, provides the answer. By 8 h non-shocked and shocked uredospores had l o s t approximately the same t o t a l amount of amino compounds t o the germination medium, ca 5.7 and 7.0 nM/mg dry weight respectively. In contrast, by 20 h the non-shocked uredospores had l o s t nearly 36.0 and the shocked uredospores only ca 13.8 nM/mg spore dry weight respectively. Both non- shocked and heat shocked uredosporelings l o s t about equal amounts of glutamine. At 20 h losses i n alanine, cysteine, phenylalanine, and the unknown were much greater from the nonshocked sporelings. I t i s important t o note that the leachate from unwashed germinating uredospores presumably includes the amino compounds which could be washed off. the r e s t i n g uredospores (Table XI). Differences i n the t o t a l amounts of amino compounds found i n the leachates and i n the spore wash are therefore also shown i n Table XIV. 89 5. 5.1 DISCUSSION Temperature Retirement f o r the D i f f e r e n t i a t i o n of P. graminis t r i t i c i Uredosporelings Maheshwari et a l . (1967a) demonstrated that greater than 90% of P. graminis t r i t i c i uredosporelings undergo complete d i f f e r e n t i a t i o n on Ca-K buffer i n response to a 1.5 h heat shock of 30° C administered 2 h a f t e r seeding. Wisdom (1977) obtained only 5% d i f f e r e n t i a t i o n using the methods described above. The e f f e c t of the heat shock temperature was not investigated. The r e s u l t s presented i n t h i s t h e s i s show that : (1) D i f f e r e n t i a t i o n has a sharp temperature optimum. Aberrations one degree above or below the optimum heat shock temperature s i g n i f i c a n t l y reduced (by greater than 40%) the amount of t o t a l sporelings forming a complete set of i n f e c t i o n structures. (2) The optimum heat shock temperature v a r i e s s l i g h t l y f o r d i f f e r e n t spore l o t s . When the temperature of the heat shock was optimised up to 70% d i f f e r e n t i a t i o n was obtained. These r e s u l t s indicate that Wisdom's f a i l u r e to obtain better than 5% d i f f e r e n t i a t i o n on Ca-K buffer was most l i k e l y due t o the use of a sub-optimal heat shock temperature. 90 5.2 The Influence of Nutrients on Uredospore D i f f e r e n t i a t i o n Since Wisdom (1977) obtained only 5% d i f f e r e n t i a t i o n on Ca-K b u f f e r she abandoned the buffer i n favour of her nutrient-enriched MPG medium. Using MPG i n conjunction with a 1.5 h heat shock of 30°C, Wisdom obtained up t o 80% d i f f e r e n t i a t ion. Williams (1971) had e a r l i e r shown that nutrients stimulate d i f f e r e n t i a t i o n and that the nutrient stimulus i s more e f f e c t i v e when a heat shock i s applied. The r e s u l t s obtained i n the present study support t h i s claim. Moreover, t h i s response of the uredosporelings t o nutrients i n the germination medium was found t o be non-additive. I t was also found that n-nonyl alcohol not only stimulates germination but also increases the proportion of germtubes forming infection structures. Results from preliminary experiments indicated that MPG was superior t o Ca-K buffer as a d i f f e r e n t i a t i o n medium f o r P. orraminis t r i t i c i uredosporelings. The precise temperature optimum f o r the heat shock observed with sporelings germinated on Ca-K buffer (page 87) was not observed with sporelings germinated on MPG. On MPG, a single given temperature (30° C) consistently induced the d i f f e r e n t i a t i o n of a high percentage of uredosporelings. Ca-K buffer appeared t o be the most e f f e c t i v e component of MPG. Baker et a l . (1987) reported that C a , but not K+, + 2 91 stimulated the germination of Uromyces phaseoli uredospores. I t i s also possible that calcium plays a key r o l e i n uredosporeling d i f f e r e n t i a t i o n . One of the many functions of calcium i n b i o l o g i c a l systems i s the regulation of c e l l membrane permeability to water and ions; low calcium generally increases permeability whereas high calcium decreases i t . The potassium phosphates supply the hyphal c e l l with phosphorus. Phosphorus i s required f o r the formation of nucleic acids and phospholipids, as well as a key molecule of energy metabolism, ATP. Peptone i n d i s t i l l e d water was a poor medium f o r differentiation. In combination with glucose and Ca-K b u f f e r peptone s i g n i f i c a n t l y increased the number of sporelings that formed i n f e c t i o n structures (up to 77%). contains Bacto-peptone a v a r i e t y of simple nitrogenous compounds (including reduced organic sulphur), ions, minerals, and a n e g l i g i b l e amount of protease and complex nitrogenous compounds (Difco Laboratories 1953). Glucose i s a potential source of energy f o r hyphal growth. In the presence of glucose (dissolved i n glass- d i s t i l l e d water) the germtube d i d not appear t o respond to heat shock but continued l i n e a r growth without the formation of i n f e c t i o n structures. Although glucose alone was i n e f f e c t i v e as a d i f f e r e n t i a t i o n medium i t played an important r o l e as a constituent of MPG. According t o Manners and co- workers (1982) glucose i s r e a d i l y absorbed and metabolized by 92 the rust hyphae. The carbon appeared i n endogenous pools of free glucose, amino acids and phosphate esters of trehalose and glucose (Reisener et a l . 1961). Previous studies on the enzymatic c o n s t i t u t i o n of uredospores of the wheat stem rust have indicated the presence of complete enzyme systems f o r the metabolism of amino acids, l i p i d s , and carbohydrates (see Shaw 1964). The presence of nutrients i n the medium may provide the uredosporeling with metabolites that a s s i s t with the processes involved i n germtube morphogenesis. 5.3 The Timing of Essential Protein Synthesis The timing of RNA and protein synthesis during d i f f e r e n t i a t i o n has been described by Dunkle et a l . Wisdom (1977). (1969) and I t has been shown that new polypeptides appear during the d i f f e r e n t i a t i o n of non-shocked bean rust uredosporelings (Huang and Staples 1982), and the d i f f e r e n t i a t i o n of heat shocked wheat stem rust uredosporelings (Kim et a l . 1982a). Although not d i f f e r e n t i a t e d , heat shocked f l a x rust uredosporelings synthesized a number of heat shock proteins (Shaw et a l . 1985). These r e s u l t s have been discussed i n greater d e t a i l i n the l i t e r a t u r e review (pages 15). The e f f e c t of puromycin on d i f f e r e n t i a t i o n was reinvestigated under the optimum heat shock conditions 93 described e a r l i e r (page 87). Puromycin i s recognized as an e f f e c t i v e i n h i b i t o r of protein synthesis. I t i s a structural analogue of the aminoacyl adenosine, the amino-acid-bearing end of transfer-RNA. I t binds r e a d i l y t o the aminoacyl s i t e of the large ribosomal subunit and forms a complex with the elongating peptide chain. The peptidyl-puromycin complex prevents further amino acid incorporation. The complex dissociates from the ribosome and chain elongation i s terminated (Gale et a l . 1972). During the present study puromycin was added to, or removed from, the germination medium (Ca-K buffer) at various stages during sporeling development. The e f f e c t of puromycin on germtube morphogenesis was dependent on the timing and duration of the puromycin treatment. The addition of puromycin to Ca-K buffer at 3.5 h followed by i t s removal at 8 h caused a s i g n i f i c a n t reduction i n the proportion of i n f e c t i o n pegs forming v e s i c l e s . Although the v e s i c l e s were v i s i b l e a f t e r 10 h t h e i r formation appeared to be preceded by the synthesis of e s s e n t i a l proteins. The r e s u l t s suggest that these proteins are synthesized 2 h t o 6.5 h p r i o r to v e s i c l e formation. When puromycin was present i n the germination medium f o r the e n t i r e incubation period (0 h to 16 h) germtube d i f f e r e n t i a t i o n was v i s i b l y affected: (1) The complete d i f f e r e n t i a t i o n of uredosporelings was prevented. proportion of germtubes forming appressoria was (2) The significantly 94 reduced. form. (4) (3) The appressoria were predominantly i r r e g u l a r i n The formation of a septa between the appressoria and the germtube usually f a i l e d to occur. (5) Nuclear d i v i s i o n was r a r e l y observed, but i n the rare event of d i v i s i o n the p a i r i n g of daughter-nuclei d i d not occur. The capacity of the i n h i b i t e d germlings to form i n f e c t i o n structures appeared to recover p a r t i a l l y when puromycin was removed from the germination medium. These r e s u l t s suggest the existence of d i f f e r e n t i a t i o n s p e c i f i c proteins and support the claim that these proteins are stage-specific (Huang and Staples 1982). I t may be concluded that essential proteins are synthesized throughout the entire process of d i f f e r e n t i a t i o n , including the formation of the appressoria. The presence of puromycin i n the germination medium throughout the incubation period usually prevented the d i v i s i o n of germtube n u c l e i . Nuclear d i v i s i o n i s generally preceded by the r e p l i c a t i o n of nuclear DNA. P r i o r t o the onset of the r e p l i c a t i o n of DNA certain enzymes such as, thymidine kinase, thymidylate synthetase and others, must increase within i n the c e l l . The absence of nuclear d i v i s i o n observed i n t h i s study suggests that these enzymes are synthesized p r i o r to the formation of the appressorium. In rare cases the enzymes appear to be present i n s u f f i c i e n t amounts t o enable r e p l i c a t i o n and d i v i s i o n t o occur. 95 5.4 Nuclear Behaviour Accompanying Germination and Differentiation The two n u c l e i of nondifferentiated uredosporelings d i d not divide but appeared to remain i n perpetual interphase throughout the e n t i r e observation period of 20 h. In d i f f e r e n t i a t i n g uredosporelings nuclear d i v i s i o n was a regular event. I t s occurrence was c l o s e l y associated with the timing of i n f e c t i o n structure development. The c y t o l o g i c a l events taking place during the f i r s t 24 h of germination and d i f f e r e n t i a t i o n are summarized i n F i g . 28. A c h a r a c t e r i s t i c pattern of nuclear behaviour was observed i n d i f f e r e n t i a t i n g uredosporelings. The protoplasm migrated into the developing i n f e c t i o n structures leaving an empty space i n the proximal regions of the germtube. Paired daughter n u c l e i migrated i n tandem with the cytoplasm. The 4 to 6 n u c l e i observed i n the appressorium migrated into the v e s i c l e as a c l o s e l y associated group. This was not observed by Wisdom (1977) who reported that the movement of the 4 n u c l e i from the appressorium into the developing v e s i c l e was not synchronous. She noted that one p a i r moved into the v e s i c l e while the other p a i r remained i n the appressorium f o r up to 10 h. Moreover, Wisdom (1977) d i d not report the p o t e n t i a l f o r a second nuclear d i v i s i o n i n the appressorium. In the present study 6 n u c l e i were commonly observed moving into the developing i n f e c t i o n peg. Migration of n u c l e i into 96 Two expanded nuclei migrate down the germtube toward i t s apex. The f i r s t round of mitosis i s generally completed within the mature appressorium. 7 h 0=? The cytoplasm moves into the i n f e c t i o n peg with the nuclei. A second d i v i s i o n occurs either p r i o r to or during nuclear migration. The mature substomatal v e s i c l e contains six unexpanded nuclei. A t h i r d m i t o t i c d i v i s i o n may occur to y i e l d eight n u c l e i . 13 h Eight, s i x , or four nuclei enter the i n f e c t i o n hypha, occasionally a nuclear p a i r remains i n the v e s i c l e . Four or fewer nuclei migrate along the infection hypha. F i g . 28. Diagrammatic representation of the c y t o l o g i c a l events taking place during the d i f f e r e n t i a t i o n of uredosporelings of P. graminis t r i t i c i , race C17. The times given are approximate. 97 the developing i n f e c t i o n structures was characterized by a s l i g h t change i n nuclear shape. The n u c l e i appeared t o be "tear drop" i n shape as i f they were "pulled" into the i n f e c t i o n structures. When a break occurred i n the germtube the n u c l e i were extruded along with the cytoplasm. In some cases, when n u c l e i were migrating into the i n f e c t i o n hypha, the foremost nucleus appeared brighter than the following ones. Wisdom (1977) described an analogous s i t u a t i o n i n which a single intensely Feulgen-positive nucleus preceded the other n u c l e i into the i n f e c t i o n hypha. In the rare event of a b i p o l a r v e s i c l e forming, two "bright" nuclei could be seen. One nucleus was situated on either side of the v e s i c l e and adjacent t o the i n i t i a l s of the i n f e c t i o n hyphae. Finally 2 or more n u c l e i appeared to either break down or coalesce within the i n f e c t i o n hypha, so that a f t e r 24 h the hypha contained up to a maximum of four d i s t i n c t n u c l e i (Fig. 28). 5.4.1 Staining with DAPI DAPI (4', 6-diamidino-2-phenylindole) i s a DNA-specific fluorochrome. I t binds r e l a t i v e l y s p e c i f i c a l l y to AT residues of double-stranded DNA and exhibits a much enhanced fluorescence i n the association (Otto and Tsou 1985). This fluorescence i s not observed with RNA or protein (Coleman et a l 1981). Chemically, i t i s suggested that the AT- s p e c i f i c i t y resides with both the guanidine group and the benzimidazole or indole ring, which may bind t o the purine of 98 adenosine through base stacking (Otto and Tsou 1985). DAPI binds to DNA and fluoresces i n proportion to the amount of DNA present. The r e l a t i v e DNA content per nucleus can be read i n s i t u by measuring the i n t e n s i t y of fluorescence with a microspectrofluorometer (Coleman et a l 1981). The enhanced i n t e n s i t y of fluorescence observed i n the leading nucleus (preceded the other nuclei into the i n f e c t i o n hypha) may suggest that t h i s nucleus i s not haploid but contains additional DNA. DAPI reacted rapidly with the sample and within minutes one could e a s i l y assess nuclear state and i d e n t i f y nuclear abnormalities. The technique appeared to be useful f o r determining the numbers and positions of n u c l e i and t h e i r d i v i s i o n s i t e s within the i n f e c t i o n structures. The s t a i n i s water-soluble and has been used successfully as a v i t a l dye i n studies on red algae and pollen development (Goff and Coleman 1984, Coleman and Goff 1985). I t should have further a p p l i c a t i o n i n assessing the e f f e c t s of a number of parameters (e.g. n u t r i t i o n , fungicides, and host genotype manipulation) on the nuclear behaviour of the rust pathogen. 99 5.5 The E f f e c t of Heat Shock on the Amounts and Kinds of Free Amino Acids i n Germinated Uredospores and Their Leachates The r e s u l t s presented i n Tables XI t o XIV show c l e a r l y that heat shock decreases the s i z e of the endogenous pool of free amino acids and the extent to which uredosporelings lose amino acids t o the medium. The r e s u l t s are summarized i n Table XV which presents a "balance sheet" showing the s i z e of the endogenous and leachate amino acid pools. Under the conditions employed only endogenous reserves are a v a i l a b l e t o the germinating uredospores. The amino a c i d data (Table XV) therefore c l e a r l y show that there i s a net hydrolysis o f protein during germination, leading t o an increase i n the l e v e l of endogenous free amino acids. These r e s u l t s are consistent with data obtained from pulse chase experiments (Kim et a l . 1982a) which demonstrated that the majority of uredospore proteins are turned over during germination. The l e v e l of t o t a l amino acids remained r e l a t i v e l y unchanged i n the heat shocked sporelings from 8 h to 20 h (39.3 t o 42.0 nM/mg), whereas the l e v e l of t o t a l amino acids i n non-shocked sporelings increased from 55.8 nM/mg a t 8 h t o 74.4 nM/mg at 20 h. The t o t a l free amino acid l e v e l (endogenous and leachate) associated with non-shocked uredosporelings was thus 1.8-fold (74.4/42.0) higher than the amount of amino acids associated with heat uredosporelings a t 20 h. shocked I t i s thus c l e a r that heat shock 100 Table XV. The d i s t r i b u t i o n of the free amino compounds associated with r e s t i n g spores, non-shocked 8- (N8S) and 20h-old (N20S) uredosporelings, and heat shocked 8- (H8S) and 20-h-old (H20S) uredosporelings. The r e s u l t s are expressed as nM amino acid per mg spore dry weight. S Endogenous Leachate^ Wash Total 11. l b 2 .19 13.3 N8S H8S 50.1 32 . 3 . 5.67 6 .95 55. 8 C N20S H20S 38 . 4 28 . 2 36.0 13.8 39.3 74 . 4 42.0 ^measured endogenous amino acids minus the amino acids i n the r e s t i n g spore wash. Measured amounts plus 15%, see Table XII. ^includes amino acids i n resting spore wash (2.19 nM/mg). C 101 d e c r e a s e s t h e amount o f p r o t e i n h y d r o l y s i s i n germinating uredospores. The r e s u l t s show f u r t h e r t h a t t h e r e i s no n e t p r o t e i n s y n t h e s i s during the formation of i n f e c t i o n s t r u c t u r e s induced by h e a t shock. Nevertheless, protein synthesis i s increased r e l a t i v e t o p r o t e i n h y d r o l y s i s by comparison w i t h t h e relative r a t e s o f t h e s e two p r o c e s s e s i n n o n d i f f e r e n t i a t i n g (nonshocked) u r e d o s p o r e l i n g s . These r e s u l t s and t h e conclusions drawn from them a r e c o n s i s t e n t w i t h t h e e f f e c t o f puromycin on differentiation. The i n h i b i t i o n of p r o t e i n synthesis by p u r o m y c i n i n h i b i t s d i f f e r e n t i a t i o n b u t does n o t appear t o d e c r e a s e t h e l i n e a r growth o f n o n d i f f e r e n t i a t e d (non-shocked) uredosporelings. The r e s u l t s are a l s o c o n s i s t e n t with the o b s e r v e d e f f e c t s o f h e a t shock on t h e i n c o r p o r a t i o n o f [ 3 5 S]- m e t h i o n i n e i n t o newly s y n t h e s i z e d h e a t shock p r o t e i n s i n germinating f l a x r u s t uredosporelings r e p o r t e d by Shaw e t a l . (1985). A h i g h p r o p o r t i o n o f t h e t o t a l amino a c i d p o o l was t o t h e medium d u r i n g g e r m i n a t i o n , uredosporelings. The lost p a r t i c u l a r l y i n non-shocked extent of t h i s leakage i n d i c a t e s t h a t t h e l o s t amino a c i d s must o r i g i n a t e from t h e h y d r o l y s i s o f protein. D a l y and h i s c o l l e a g u e s (1967) a l s o found t h a t i n non-shocked u r e d o s p o r e s o f P. g r a m i n i s t r i t i c i a considerable p o r t i o n o f t h e f r e e amino a c i d s a r i s i n g from p r o t e i n degradation a r e l o s t t o t h e medium and t h a t o n l y a s m a l l p o r t i o n are u t i l i z e d f o r the resynthesis of p r o t e i n s . The 102 r e s u l t s i n t h i s thesis show that the loss of free amino acids from non-shocked sporelings to the medium i s 2.6 (36.0/13.8) times greater than the loss from heat shocked sporelings. As spore germination progressed (from 8 h to 20 h) the loss of amino acids from non-shocked uredosporelings t o the medium increased and appeared l e s s s e l e c t i v e than the l o s s from heat shocked sporelings. In contrast, heat shock markedly decreased the loss a t 20 h of most of the amino acids l i s t e d on Table XIV. P a r t i c u l a r l y s t r i k i n g decreases occurred i n the losses o f alanine, cysteine, phenylalanine and an unknown amino acid. On the other hand heat shock had no s i g n i f i c a n t e f f e c t on the losses o f p r o l i n e and glutamine. The loss of p r o l i n e and glutamine i s not s u r p r i s i n g since they are r e a d i l y formed v i a the conversion of many amino acids and metabolites. Proline commonly functions as a long-distance transport compound f o r carbon. Proline i s an ideal translocation molecule by v i r t u e of i t s high metabolic c a p a b i l i t i e s (eg. t o form glutamate, aketoglutarate, and pyruvate v i a succinate) ( M i f l i n 1977). Glutamine, on the other hand i s the p r i n c i p l e nitrogen donor f o r many biosynthetic reactions, and i t s formation is a d e t o x i f i c a t i o n mechanism f o r the removal of ammonia. Individual amino acids were l o s t t o the medium at d i f f e r e n t rates and i n d i f f e r e n t amounts (Table XV). For example, non-shocked sporelings l o s t 0.6 nM/mg alanine i n 8 h and 4.5 nM/mg i n 20 h (ie. the loss at 20 h was 7.5 times the 103 loss a t 8 h). Non-shocked sporelings also l o s t 2.4 nM/mg glutamine a t 8 h and 9.9 nM/mg a t 20 h ( i e . the loss a t 20 h was 4.2 times the loss a t 8 h). On the other hand the l o s s of alanine from heat shocked sporelings a t 20 h was 1.14 times the loss a t 8 h and the loss of glutamine a t 20 h was 4.15 times the loss a t 8 h. Comparing alanine and glutamine we see that they were each l o s t a t d i f f e r e n t rates i n non-shocked and heat shocked sporelings. Comparing non-shocked and heat shocked sporelings we see that alanine was l o s t a t d i f f e r e n t rates but glutamine a t the same rate i n the two sets of sporelings. Similar comparisons can be made f o r the other amino acids from the data i n Table XV. I t follows that i f amino acids are l o s t from uredosporelings v i a a membrane defect, the defect does not a f f e c t the l o s s of a l l amino acids to the same extent. Therefore, the loss o f amino acids from the uredosporeling t o the medium i s considered t o be selective. In summary, the composition of each amino acid pool (exogenous and leachate pools) i s unique. The percent composition of each amino acid t o the t o t a l amino acids i n each pool i s : (1) d i f f e r e n t , (2) a l t e r s as germination progresses through 8 h t o 20 h, and (3) i s a l t e r e d by heat shock. The question which next a r i s e s i s the mechanism of the e f f e c t of the heat shock i n s e l e c t i v e l y decreasing the loss of amino acids. While heat shock promotes the synthesis of c e r t a i n highly conserved heat shock proteins and depresses the 104 synthesis of other proteins (Shaw et a l . 1985), the mechanisms involved are unknown. exact With respect to the leakage of amino acids into the medium i t i s possible that heat shock d i r e c t l y a f f e c t s on the i n t e g r i t y and hence the permeability of the c e l l membranes. A l t e r n a t i v e l y i t may transport processes across the membranes. a l t e r amino-acid The data i n t h i s t h e s i s do not provide any d i r e c t evidence on which to base an answer to these questions. Irrespective of the e f f e c t of heat shock, the growth of the germtube i s accompanied by the loss of amino acids to the medium. I t i s possible that exogenous amino acids a r i s e from e i t h e r the mechanical rupture or enzymatic degradation of the germtube c e l l w a l l . Although germtube l y s i s was not observed i t i s l i k e l y that some degradation of the hyphal wall occurs during germination. facilitate metabolites The break down of the germtube wall would the loss of amino acids as well as other and enzymes. cell In addition, i t i s possible that the loss of amino acids to the medium i s the r e s u l t of a s t r u c t u r a l or chemical defect i n the c e l l membrane. membrane defect may A lead to the loss of s e l e c t i v e permeability and/or an a l t e r a t i o n i n amino acid transport systems. Scott and Maclean (1969) have suggested that the rust fungi resemble mammalian c e l l cultures i n that both c e l l lines appear to have a membrane defect allowing the loss of newly synthesized metabolites into the medium. The appearance of free amino acids within the leachate implies the absence of 105 regulation of permeability i n the hyphal membrane (Scott and Maclean 1969). I f t h i s assumption i s v a l i d i t may explain the high uredospore density usually required f o r i n i t i a t i n g normal saprophytic growth. I t i s well known that radioactive amino acids fed to non-shocked germinating uredospores are taken up and incorporated i n t o protein (Shaw 1964, Shaw et a l . 1985). At high c e l l d e n s i t i e s the leaked nutrient can reenter another c e l l i n close proximity, thus maintaining an e f f e c t i v e i n t e r c e l l u l a r l e v e l by cross feeding between c e l l s . At low c e l l d e n s i t i e s a metabolite may be l o s t into the medium at a rate equal to i t s rate of synthesis. A c r i t i c a l population density i s able to b u i l d up an e x t r a c e l l u l a r concentration of the metabolite that i s i n equilibrium with the minimum i n t r a c e l l u l a r l e v e l , before the c e l l s die of a s p e c i f i c n u t r i t i o n a l deficiency (Eagle and Piez 1962). Amino acids have important p h y s i o l o g i c a l r o l e s which govern many metabolic processes activity). (e.g. growth and enzyme A number of i n t e r e s t i n g avenues of research have come to t h i s authors attention during t h i s study: the e f f e c t of exogenous amino acids on germination and d i f f e r e n t i a t i o n ; the e f f e c t of some amino acids (pathway products) on extractable enzyme a c t i v i t i e s ; the e f f e c t of exogenous amino acids on enzyme a c t i v i t y (derepression/repression of genes); and to explore the phenomenon of growth i n h i b i t i o n due to amino a c i d imbalances. Although research i s being pursued i n these areas most of the work has focussed on organisms other 106 than the rust fungi. Most of the recent work published i s concerned with the timing and the products of protein synthesis; however, the a c t i v a t i o n of protein synthesis during germination and the formation of i n f e c t i o n structures has not been well documented. Given the information contained i n the present study i t would be of i n t e r e s t to determine the amount of charged tRNA f o r each amino acid. To s i g n i f i c a n t l y a f f e c t protein synthesis the absence of a free amino acid would r e s u l t i n the absence of the aminoacyl-tRNA. Although some growth of several s t r a i n s of wheat stem rust has been obtained axenically, only the Australian 126-ANZ-67, cultured by William's group (1966, 1967) found to grow vigorously. has been Analyses of the amino a c i d composition of the leachate may formulating race, provide a useful basis f o r the amino acid component of media f o r the axenic culture of stem rust. Moreover, i t would also be of i n t e r e s t to extract and p u r i f y the free amino a c i d f r a c t i o n from germinating uredospores. This f r a c t i o n could be used to provide the amino acid component of an axenic medium f o r rust culture. 107 6. SUMMARY (1) The percent germination of P. graminis t r i t i c i , race C17, uredospores and the proportion of germtubes forming complete i n f e c t i o n structures was augmented by n-nonyl alcohol. (2) A p r e c i s e l y timed heat shock and exogenous nutrients stimulate d i f f e r e n t i a t i o n . This stimulus i s most e f f e c t i v e i n the presence of n-nonyl alcohol. (3) The heat shock temperature required to induce maximum d i f f e r e n t i a t i o n has a very precise optimum. Variations one degree above or below t h i s optimum f o r a given spore l o t reduced the percent d i f f e r e n t i a t i o n by greater than 40%. (4) Although the optimum temperature of the heat shock varied s l i g h t l y depending on the p a r t i c u l a r spore l o t the s e n s i t i v i t y of sporeling development to temperature changes i s remarkably constant. (5) Compared t o Ca-K buffer, MPG was a superior germination and d i f f e r e n t i a t i o n medium. (6) Ca-K b u f f e r was the most e f f e c t i v e component of (7) Uredosporeling d i f f e r e n t i a t i o n occured as a series of p r e c i s e l y timed morphological, c y t o l o g i c a l , and physiological events. (8) DAPI s t a i n i n g i s a simple, rapid, and reproducible way to assess nuclear behaviour i n P. graminis t r i t i c i uredosporelings. (9) Nuclear d i v i s i o n was a regular event i n d i f f e r e n t i a t i n g uredospores. I t s occurrence was c l o s e l y associated with the timing of i n f e c t i o n structure development. MPG. 108 (10) Nuclear d i v i s i o n was a rare event i n germinating (nonshocked) uredospores. (11) E s s e n t i a l , presumably d i f f e r e n t i a t i o n - s p e c i f i c proteins, were synthesized from the onset of germination t o the completion of d i f f e r e n t i a t i o n . These proteins are required f o r the formation of appressoria, v e s i c l e s and, i n f e c t i o n hyphae. (12) Spore germination was accompanied by a rapid decrease i n the glutamic:aspartic acid r a t i o (from 20:1 to 3:1). (13) There i s a net hydrolysis of protein during germination, leading t o an increase i n s i z e of the endogenous pool of free amino acids and t o an increased leakage of amino acids to the germination medium. (14) Relative t o non-shocked uredosporelings, heat shock decreased both the s i z e of the endogenous pool of amino acids and the extent t o which uredosporelings lose amino acids t o the medium. (15) There was no net protein synthesis during the formation of i n f e c t i o n structures induced by heat shock. (16) A high proportion of the t o t a l amino a c i d pool was l o s t to the medium during germination, p a r t i c u l a r l y i n nonshocked uredosporelings. (17) Free cysteine was detected i n the leachate i s o l a t e d from non-shocked sporelings only. (18) The loss of amino acids to the germination medium i s s e l e c t i v e , p a r t i c u l a r l y i n heat shocked uredosporelings. (19). The composition of the amino a c i d pool i n the leachate may be a useful guide i n formulating media f o r the axenic culture of P. graminis t r i t i c i . 109 7. LITERATURE CITED A l l e n , R.F. 1923. A c y t o l o g i c a l study of i n f e c t i o n of Baart and Kanred wheats by Puccinia graminis t r i t i c i . J . Agric. Res. 23: 131-152. A l l e n , P.J. 1972. S p e c i f i c i t y of the cis-isomers of i n h i b i t o r s of uredospore germination i n the rust fungi. Natl. Acad. S c i . USA. 69: 3497-3500. Proc. . 1976. 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Puccinia graminis f.sp. tritici, race c17 : physiology of uredospore germination and germtube differentiation Hopkinson, Sarah J. 1988
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