PUCCINIA GRAMINIS f.sp. TRITICI, RACE C17: PHYSIOLOGY OF UREDOSPORE GERMINATION AND GERMTUBE DIFFERENTIATION By Sarah J . Hopkinson B . S c , The U n i v e r s i t y of 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 i n THE FACULTY OF GRADUATE STUDIES (Department of P l a n t Science) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © Sarah Jane Hopkinson, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P\c\rVv *"Sr.\cy,r\c v>. The University of British Columbia Vancouver, Canada Date hnw \ Q DE-6 (2/88) ABSTRACT Germinating uredospores of race C17 o f P u c c i n i a graminis f.sp. 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 (appressorium, i n f e c t i o n peg, v e s i c l e , i n f e c t i o n hypha) i n response t o a 1.5 h heat shock a t 29° C administered 2 h a f t e r germination a t 19° C. The p r o p o r t i o n of s p o r e l i n g s forming i n f e c t i o n s t r u c t u r e s was augmented by n u t r i e n t s , n-nonyl a l c o h o l and, an a p p r o p r i a t e l y timed heat shock. The heat shock temperature r e q u i r e d t o induce maximum d i f f e r e n t i a t i o n had a very p r e c i s e optimum which 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 or below t h i s optimum reduced the percent d i f f e r e n t i a t i o n by g r e a t e r than 40%. The presence of an i n h i b i t o r of p r o t e i n s y n t h e s i s , puromycin, i n the germination medium: (1) prevented uredos p o r e l i n g d i f f e r e n t i a t i o n but had no e f f e c t on germination, (2) s i g n i f i c a n t l y reduced the p r o p o r t i o n o f germtubes forming a p p r e s s o r i a , and (3) i n most cases prevented the d i v i s i o n of germtube n u c l e i . 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 from the onset of germination, throughout the formation of a p p r e s s o r i a and t o the completion of d i f f e r e n t i a t i o n . These r e s u l t s were c o n s i s t e n t w i t h the observed e f f e c t s o f heat shock on the r a t 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 net h y d r o l y s i s of p r o t e i n l e a d i n g t o an increase i n s i z e of the endogenous pool of f r e e amino a c i d s and t o an increased leakage of amino acids to the germination medium. Heat shock effectively reduced the amount of endogenous free amino acids and the extent to which amino acids were lost to the medium. It was concluded that in heat shocked sporelings protein synthesis was increased relative to protein hydrolysis by comparison with the relative rates of these two processes in germinating (non-shocked) uredosporelings. Moreover, there was no net protein synthesis during the formation of infection structures induced by heat shock. The loss of amino acids to the germination medium was selective, particularly in heat shocked sporelings. i v TABLE OF CONTENTS Page Number A b s t r a c t i i L i s t o f Tables v i i i L i s t of Figures x Acknowledgements x i i i 1. I n t r o d u c t i o n 1 2. L i t e r a t u r e Review 6 2.1 Uredospore Germination and Morphogenesis 2.1.1 Germination I n h i b i t o r s and Stimulants .... 6 2.1.2 T h i g m o d i f f e r e n t i a t i o n 7 2.1.3 T h e r m o d i f f e r e n t i a t i o n 8 2.1.4 C h e m o d i f f e r e n t i a t i o n 9 2.1.5 Mechanisms of 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 S t r u c t u r e s 10 2.2 C y t o l o g i c a l Events 2.2.1 Nuclear Changes 11 2.2.2 P r o t e i n Metabolism 13 2.3 N u t r i t i o n a l Requirements of Rust Fungi 2.3.1 Physiology of the H o s t - P a r a s i t e Complex . 17 2.3.2 Axenic C u l t u r e and Metabolism N u t r i t i o n a l Requirements 20 S y n t h e t i c Capacity 21 Sulphur Metabolism 22 M e t a b o l i t e Leakage 22 Endogenous Free Amino Ac i d s 24 Experimental Methods 26 3.1 Production and C o l l e c t i o n o f Spores 26 3.2 Spore Germination 27 3.3 Spore Germination w i t h the Induction 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 the 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 Synthesis: Puromycin 33 3.8 Nuclear 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 High 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 of Amino A c i d s as Phenylthiocarbamoyl D e r i v a t i v e s 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 V I R e s u l t s 50 4.1 Uredospore Germination 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 53 4.3 I n f l u e n c e of 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 60 4.4 E s s e n t i a l P r o t e i n Synthesis 62 4.5 Cytology of Uredosporeling Development 4.5.1 Nuclear S t a i n i n g 67 4.5.2 Nuclear Behaviour d u r i n g Germination and D i f f e r e n t i a t i o n 68 4.6 Amino A c i d A n a l y s i s 75 4.6.1 Experimental P l a n 75 4.6.2 Complications 78 4.6.3 Exogenous Free Amino A c i d s : R e s t i n g Spore Wash 81 4.6.4 Endogenous Free Amino A c i d s : E x t r a c t s of Unwashed Re s t i n g Uredospores 83 4.6.5 E f f e c t of Heat Shock on the Free Amino Ac i d s i n Germinated Uredospores and i n the Leachate A f t e r Germination 85 V l l D i s c u s s i o n 89 5.1 Temperature Requirements 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 89 5.2 The I n f l u e n c e of N u t r i e n t s on Uredospore D i f f e r e n t i a t i o n 90 5.3 The Timing of E s s e n t i a l P r o t e i n Synthesis 92 5.4 Nuclear Behaviour Accompanying Germination and D i f f e r e n t i a t i o n 95 5.4.1 S t a i n i n g w i t h DAPI 97 5.5 The E f f e c t of Heat Shock on the Amounts and Kinds of Free Amino A c i d s i n Germinating Uredospores and t h e i r Leachates 99 Summary 107 L i t e r a t u r e C i t e d 109 V l l l LIST OF TABLES Page Number I. The composition and p r e p a r a t i o n of the Ca-K-P0 4 b u f f e r and the v o l a t i l e germination s t i m u l a n t , n-nonyl a l c o h o l 28 I I . The composition 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 29 I I I . Lactophenol-Trypan 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 31 IV. Timing 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 : 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; the b u f f e r g r a d i e n t system and fl o w -r a t e 48 V I I . A summary of experiments i n v e s t i g a t i n g the i n f l u e n c e o f the components of MPG, n-nonyl a l c o h o l , and heat shock on the percentage of germlings developing i n f e c t i o n s t r u c t u r e s 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 the development of i n f e c t i o n s t r u c t u r e from germinating uredospores 63 i x IX. The e f f e c t of a p r o t e i n i n h i b i t o r , puromycin, on the development of i n f e c t i o n s t r u c t u r e from germinating uredospores. Expressed as a mean percent of the preceding s t r u c t u r e 66 X. 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 77 XI. The f r e e amino a c i d composition of the b u f f e r wash from ungerminated uredospores of P. graminis t r i t i c i ... 82 X I I . 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 uredospores, and i n non-shocked uredosporelings 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 content of unwashed r e s t i n g uredospores, and i n heat shocked uredosporelings a f t e r 8 h and 20 h 86 XIV. The f r e e amino a c i d composition of leachate from heat shocked and non-shocked uredosporelings 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 87 XV. The d i s t r i b u t i o n of the 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 spores, non-shocked and heat shocked uredosporelings 100 X LIST OF FIGURES Page Number 1. Germination and d i f f e r e n t i a t i o n of the wheat r u s t pathogen, P. graminis t r i t i c i , on the c e r e a l host.. 3 2. The chemical s t r u c t u r e of DAPI (4',6-diamidino-2-phenyl indole) 38 3. 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 RP-a 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 d e t e c t i o n 41 4. Sample p r e p a r a t i o n scheme f o r crude amino a c i d samples u s i n g Sep-Pak C 1 8 c a r t r i d g e s 44 5. Reaction scheme of amino compound by pre-column d e r i v a t i z a t i o n w i t h PITC . 47 6. The percent germination of uredospores on Ca-K b u f f e r i n both the presence and the absence of n-nonyl a l c o h o l 51 7. Timing of uredosporeling morphogenesis 52 8. Temperature requirements f o r appressorium formation from uredospore germtubes 54 9. Temperature requirements f o r the complete d i f f e r e n t i a t i o n of uredospore germlings 56 10. The r e l a t i o n s h i p between heat shock temperature and the transformed percent of a p p r e s s o r i a formed 58 11. The r e l a t i o n s h i p between heat shock temperature and the transformed percent of t o t a l s p o r e l i n g d i f f e r e n t i a t i o n 59 X I 12. The presence of a p r o t e i n 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 a p p r e s s o r i a 64 13. F o l l o w i n g 2 h germination two interphase n u c l e i . m i g r a t e d from the uredospore i n t o the e l o n g a t i n g germtube... 69 14. The germtube of non-shocked uredosporelings remained b i n u c l e a t e f o r up t o 20 h 69 15. In some cases the n u c l e i w i t h i n 20-hour-old uredosporelings were elongated i n form and had a "ragged" appearance 69 16. Four hours f o l l o w i n g germination the two n u c l e i migrated i n t o the newly formed appressorium i n i t i a l . T h e i r elongated form suggested t h a t these n u c l e i were i n conjugate telophase and p r e p a r i n g t o d i v i d e 70 17. The f i r s t round of m i t o s i s u s u a l l y occurred w i t h i n the mature appressorium 5 t o 8 h a f t e r germination 70 18. The mature appressorium contained four daughter-n u c l e i 70 19. 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 the appressorium 72 20. Ten hours f o l l o w i n g germination the 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 the appressorium by a septum.... 72 21. In most cases a nuclear p a i r d i v i d e d i n the mature v e s i c l e t o y i e l d a t o t a l o f e i g h t n u c l e i 72 X l l 22. The o r i e n t a t i o n of nuclear 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 the p o l a r i t y of the v e s i c l e 73 23. The i n f e c t i o n hypha contained e i g h t , s i x , or more commonly, four n u c l e i 73 24. In some cases a nuclear p a i r f a i l e d t o migrate w i t h other n u c l e i and remained i n the v e s i c l e 73 25. Twenty three hours f o l l o w i n g germination the i n f e c t i o n hypha most o f t e n contained f o u r expanded n u c l e i . . . . 73 26. Separation of amino a c i d standard by reverse-phase h i g h -pressure l i q u i d chromatography 76 27. Representative RP-HPLC separations of amino a c i d s i s o l a t e d from non-shocked 20-hour-old uredosporelings and the leachate of 20-hour-old heat shocked uredosporelings 79 28. Diagrammatic r e p r e s e n t a t i o n of the nu c l e a r behaviour of d i f f e r e n t i a t i n g uredospores of P. graminis t r i t i c i , race C17 96 x i i i Acknowledgements I would l i k e t o thank the people t o whom I owe my personal s a n i t y and the f i n a l completion of t h i s t h e s i s . A s p e c i a l thankyou t o Dr. Linda Boasson f o r her companionship i n the l a b and her always h e l p f u l suggestions. Thankyou t o Dr. Michael Shaw f o r the many days spent rev i e w i n g the 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 through a l l aspects of t h i s t h e s i s . T h i s work was supported by a grant from the N a t u r a l Science and Engineering Research Committee t o Dr. Micheal Shaw. 1 1. INTRODUCTION Rust f u n g i are b i o t r o p h i c pathogens on p l a n t s , causing g r e a t l o s s e s t o many c u l t i v a t e d crops. The b l a c k stem r u s t of wheat pathogen, P u c c i n i a graminis Pers. f.sp. t r i t i c i E r i k s and E. Henn. ranks among the more s e r i o u s of c e r e a l d i s e a s e s . The stem r u s t i s m a c r o c y c l i c and heteroecious; 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 spore stages on two hosts. The u r e d i a l stage, which i s the most d e s t r u c t i v e economically, perpetuates the fungus throughout the growing season by r e i n f e c t i n g the c e r e a l host. Rust 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 i d e n t i c a l but i n f e c t d i f f e r e n t host genera are c l a s s e d as formae s p e c i a l e s ( f . s p . ) . W i t h i n each forma s p e c i a l i s there are many p h y s i o l o g i c a l races which are pathogenic on only 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. P. graminis f.sp. t r i t i c i . race C17 (formerly race 56), was employed i n the c u r r e n t study. The race i s g e n e t i c a l l y s t a b l e w i t h only one r a r e subrace 56A known. I t i s h i g h l y aggressive on wheat and capable of causing huge economic l o s s e s i n North America. 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 races i s determined by complementary genes f o r r e s i s t a n c e i n the host and v i r u l e n c e genes i n the pathogen ( i . e . by gene-for-gene r e a c t i o n s ) ( F l o r 1971). In most cases 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 the i n t e r a c t i o n of a dominant gene f o r r e s i s t a n c e (R) i n the host 2 and a complimentary dominant gene for avirulence (P) in the pathogen. A l l other reactions (R-pp, rrP-, and rrpp) result in 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 readily on water. The germtube of wheat stem rust grows across the cuticular ridges of the leaf blade; upon contact with a stomatal pore germtube elongation ceases and differentiation begins. Differentiation involves the sequential development of infection structures: the appressorium (app), the infection peg (ip), the substomatal vesicle (ssv), and the infection hypha (ih). Once established, the mycelium grows primarily in an intracellular fashion, obtaining nutrients from host c e l l s through specialized structures called haustoria. Nutrients are also absorbed by intercellular mycelium. The susceptible host i s able to both induce or assist fungal morphogenesis and provide the balance of nutrients essential for 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 information regarding the biology (metabolism, nutrition, and genetics) of the rust fungi. Unfortunately, serious obstacles such as the very slow growth rates of these fungi, and their unpredictable nutritional requirements have limited the widespread use of 3 Fig. 1. Germination and differentiation 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 linear growth and begin differentiation: an appressorium (app) forms over the stomate, an infection peg (ip) grows between the guard cells and expands to form the substomatal vesicle (ssv), the vesicle gives rise to the infection hypha (ih) • 4 axenic c u l t u r e methods (Maclean 1982). Furthermore, i t seems apparent, a t l e a s t w i t h P. graminis t r i t i c i , t h a t g e n e t i c a l l y aberrant forms are encouraged t o develop, t h a t s p o r u l a t i o n i s at best e r r a t i c and t h a t fewer than h a l f the races t h a t have been t e s t e d have s u r v i v e d under the c u l t u r e c o n d i t i o n s employed (Williams 1984). According 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 necessary prelude towards o b t a i n i n g g e n e t i c a l l y normal ( h a p l o i d , d i k a r y o t i c ) pathogenic c o l o n i e s . The p r o p o r t i o n of germlings 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 , heat shock, the time of inoculum c o l l e c t i o n , and by the g e n e t i c c o n s t i t u t i o n of the spore. S p o r e l i n g d i f f e r e n t i a t i o n i s most o f t e n induced by a timed heat shock (30°C, 1.5 h) administered 2 h f o l l o w i n g germination a t 19°C. I n order t o e s t a b l i s h a given 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 requirements 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 source o f carbohydrate, reduced n i t r o g e n , and reduced sulphur (e.g. c y s t e i n e ) . Howes reported t h a t a wide range of amino compounds were excreted i n t o c u l t u r e f i l t r a t e s of P. graminis t r i t i c i a f t e r 9 days i n c u b a t i o n (see Howes and S c o t t 1973). The f u l l extent of amino a c i d leakage d u r i n g the f i r s t 20 h of germination and d i f f e r e n t i a t i o n was not assessed. T h i s study was designed t o i n v e s t i g a t e the leakage of f r e e amino a c i d s from uredospore germlings concomitant w i t h the processes o f germination and d i f f e r e n t i a t i o n (induced by heat shock). The changes i n the endogenous free amino acid pool were also assessed. 5 The objectives of the present study thus were: 1. To reexamine the temperature requirement for the process of differentiation of the wheat stem rust fungus. 2. To examine the influence of nutrients on the proportion of germlings that form infection structures. 3. To determine the timing of essential protein synthesis during differentiation. 4. To investigate nuclear behaviour accompanying germination and differentiation of P. graminis t r i t i c i , race C17: nuclear division, nuclear migration patterns, and nuclear distribution. 5. To determine the amount and kinds of free amino acids present during the germination of wheat stem rust uredospores (race C17) and the differentiation of germ tubes into infection 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 in the endogenous free amino acid pools of uredospore germlings during germination and differentiation. 5.3 To determine the amounts and kinds of amino acids leached into the medium during the process of germination and differentiation. 6 2. LITERATURE REVIEW 2.1 Uredospore Germination and Morphogenesis 2.1.1 Germination I n h i b i t o r s and Stimulants Uredospore germination and germtube d i f f e r e n t i a t i o n i s su 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 agents which resemble hormones i n t h e i r m o b i l i t y from spore t o spore, 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 b i o l o g i c a l a c t i v i t y ( A l l e n 1976). S p o r o s t a t i c l e v e l s of c i s -cinnamates are found i n uredospores. The e f f e c t o f the 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, d u r i n g h y d r a t i o n , and p r i o r t o germination, the a c t i v e cis-cinnamates are r e l e a s e d and are 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 converted t o i n a c t i v e trans-isomers 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 germination i n h i b i t o r o f wheat stem r u s t i s methyl c i s - f e r u l a t e : a methyl e s t e r of 3-methoxy-4-hydroxycinnamate (Macko e t a l . 1971). I t has an E D 5 0 of 0.2 ng/ml (Macko e t a l . 1972) and i s a c t i v e only d u r i n g germination ( m y c e l i a l growth i s not a f f e c t e d [ A l l e n 1976]). The i n h i b i t o r prevents the d i s s o l u t i o n o f the germ pore p l u g but i t s molecular s i t e o f a c t i o n i s unknown (Hess 1975). Endogenous germination s t i m u l a n t s such as n-nonyl a l c o h o l (nonanal) (French and Weintraub 1957), 6-methyl-5-hepten-2-one (Rines e t a l . 1974), coumarins, and phenols (Van Sumere e t a l . 7 1957) are not species s p e c i f i c . Macko and co-workers (1976) suggest t h a t the s t i m u l a n t s f a c i l i t a t e germination by promoting the r e l e a s e of the i n h i b i t o r s from the spore. 2.1.2 T h i g m o d i f f e r e n t i a t i o n Uredosporeling d i f f e r e n t i a t i o n may be induced i n v i t r o by p h y s i c a l s t i m u l i . I n a s e r i e s of experiments employing bean r u s t germlings and a r t i f i c i a l membranes Dickinson (1949, 1971) demonstrated t h a t the formation of 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 event t h a t i s c o r r e l a t e d w i t h s p e c i f i c t h i g m o t r o p i c s t i m u l i . D i f f e r e n t i a t i o n thus s t i m u l a t e d ( t h i g m o d i f f e r e n t i a t i o n ) r e q u i r e s f i r m attatchment of the germtube t o the 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 data suggest t h a t , i n bean r u s t , p r o t e i n s on the hyphal s u r f a c e are i n v o l v e d i n s u b s t r a t e adhesion and the t r a n s m i s s i o n of a s i g n a l t o the c y t o s k e l e t o n t o begin n u c l e a r 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) the 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 i s a 0.5 m h i g h r i d g e on the s u b s t r a t e s u r f a c e . The e l e v a t i o n of the r i d g e i s c r i t i c a l , r i d g e s g r e a t e r than 1.0 m or l e s s than 0.25 m are not e f f e c t i v e s i g n a l s and are unable t o promote maximum d i f f e r e n t i a t i o n . I t was a l s o rep o r t e d t h a t the growth of the germtubes i s o r i e n t e d by r i d g e spacings of 0.5 t o 6.7 m. Although 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 differentiate, they had l i t t l e to no effect on P. q. t r i t i c i (Maheshwari et a l . 1967b). Staples and his group (1983) found that contact stimuli are responsible for 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 origin. 2.1.3 Thermodif ferentiation Another type of induction was reported for P. q. t r i t i c i . The exposure of germinated uredospores to elevated temperatures of 30 to 31°C for 90 min followed by the return to lower temperatures promoted up to 90% differentiation (Maheshwari et a l . 1967, Dunkle and Allen 1971). The percentage of germlings that formed infection structures was influenced by the duration of heat shock, the spore to li q u i d ratio, and the composition of the medium present during the induction period (Dunkle and Allen 1971). The induction of differentiation by heat shock, as described above was repeated by Wisdom (1977). The results proved unsatisfactory, yielding differentiation percentages of less than 5%. The effect of the temperature of the heat shock was not investigated. The Ca-K buffer used by Dunkle and Allen (1968) was abandoned in favour of a medium composed of Maheshwari7s-buffer (Maheshwari et a l . 1967), peptone, and glucose (MPG). MPG supported up to 80 percent differentiation 9 (Wisdom 1977). 2.1.4 Chemodifferentiation Chemodifferentiation i s the induction of infection structures by a chemical agent. Chemical stimuli 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), certain reduced nucleotides (Staples et a l . 1983a), and v o l a t i l e leaf constituents and phenolic compounds leached or metabolized from the guard c e l l s (Grambow and Riedel 1977, Grambow 1978, Grambow and Grambow 1978). Synthetic short-chain aliphatic compounds with conjugated double bonds (e.g. acrolein) are morphogenetically active whereas saturated aliphatic aldehydes, ketones and alcohols, as well as phenylacrolein are unable to induce differentiation (Wolf 1982). The a b i l i t y of a chemical agent to stimulate differentiation opens the po s s i b i l i t y that the agent i s a normal intermediary in contact-triggered differentiation (Allen 1976). 2.1.5 Mechanisms of Differentiation The mechanisms involved in the regulation of germtube differentiation are unknown. Allen (1976) suggests that the formation of infection structures i s controlled by the uredospore genome. An appropriate stimulus activates the program. The observed inhibition of differentiation by protein and RNA inhibitors suggests that the genetic message 10 i s a c t i v a t e d by de-repressing a r e g i o n o f the 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 c y t o s k e l e t o n ( 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 the i n d u c t i o n of n u c l e a r d i v i s i o n i n bean r u s t germlings (Hoch and S t a p l e s 1985, Hoch e t a l . 1984, S t a p l e s and Hoch 1982). These authors suggest t h a t the c y t o s k e l e t o n somehow represses n u c l e a r d i v i s i o n u n t i l i t i s subjected t o heat shock or an i n d u c t i v e membrane topography. 2.1.6 The Role of I n f e c t i o n S t r u c t u r e s I n nature the 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 of important r o l e s : I n f e c t i o n s t r u c t u r e s must form before l e a f -c o l o n i z i n g hyphae can grow (Dickinson 1949, Chakravati 1966). According t o Wisdom (1977), d i f f e r e n t i a t e d germlings of the wheat stem r u s t fungus i n f e c t exposed host mesophyll; 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 are unable 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 serve t o anchor the fungus at the stomatal s i t e thereby enabling p e n e t r a t i o n . They represent the s i t e o f the t r a n s i t i o n from germtube t o hyphal growth. Th i s hypothesis i s supported by the a c t i v a t i o n of p r o t e i n , RNA and DNA 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 . 1969, Meihle 1972). And l a s t l y they enable the fungus t o complete the t r a n s i t i o n w i t h the r a p i d i t y demanded by a changing (perhaps t o a l e s s conducive) environment. Grambow (1977) found t h a t d i k a r y o t i c hyphae branching d i r e c t l y from germtubes are produced a t a h i g h r a t e but not before the t h i r d day a f t e r seeding. Dikaryotic hyphae arise from a complete set of infection structures very soon after germination. According to Williams (1971) the formation of infection structures i s an essential prelude to the establishment and maintenance of genetically normal pathogenic colonies. The transition (parasitic to saprophytic growth), i f appropriately stimulated, occurs with or without the induction of differentiated structures. However, infection structures appear to control nuclear behaviour and as a consequence increase the probability of giving rise to hyphae which are both normal and stable for the dikaryophase. Although heat shock generally appears to stimulate c e l l differentiation 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 differentiation has a negative effect on the in vitro sporulation of P. graminis t r i t i c i . race 32. 2.2 Cytological Events 2.2.1 Nuclear Changes The morphological changes leading to the formation of infection structures are accompanied by a series of precisely timed nuclear events. The resting uredospore i s dikaryotic haploid. Following germination, the two nuclei migrate with 12 the cytoplasm into the developing germtube (Heath and Heath 1978). Given the stimulus to differentiate the nuclei may divide prior to (Maheshwari et a l . 1967b), or following (Grambow and Muller 1978) the development of the appressorium. Nuclear division occurs only after the linear growth of the germtube i s arrested and i s rarely observed in nondifferentiated germlings (Maheshwari et a l . 1967, Grambow and Muller 1978, Wisdom 1977). Dickinson (1949) reported that two or three rounds of mitotic division occur during the formation of infection structures in P. graminis and P. t r i t i c i n a . The appressorium may contain four to eight nuclei which migrate into the developing vesicle. A second division in the vesicle yields eight nuclei in total. According to Wisdom (1977) the vesicle nuclei may either dissolve or coalesce leaving one to two nuclei in the infection hypha. Allen (1923) also noted that the mature appressorium of P. g. t r i t i c i contains four nuclei and that the infection hypha contains two. The infection structures induced in vitro are morphologically and cytologically similar to those induced on the host plant (Maheshwari et a l . 1967b). Staples et a l . (1984b) reported that the synthesis of nuclear DNA in U. fabae sporelings coincides with the onset of mitosis in response to the stimulus for the i n i t i a t i o n of differentiation. The replication of DNA begins between the second and fourth hour following germination. It i s not known 13 how external stimuli activate nuclear DNA polymerase (Yaniv and Staples 1974). In the absence of the stimulus to differentiate the uredosporeling nuclei are in Gl (i.e. period preceding DNA replication) (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 nuclei are known to exist in two principal but dissimilar forms. Between divisions 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 division begins, the nucleus extrudes the endosphere into the cytoplasm, contracts and rounds up to form the "unexpanded" or dividing nucleus (Savile 1939). Craigie (1959) and Wisdom (1977) observed both expanded and unexpanded nuclei in P. helianthi and P. q. t r i t i c i , respectively. 2.2.2 Protein Metabolism The changes in proteins and nucleic acids accompanying germination of uredospores and differentiation of the germtube into structures has inspired much research (Staples 1968, Dunkle et a l . 1969, Kim et a l . 1982a, Huang and Staples 1982, Shaw et a l . 1985, Wanner et a l . 1985). Rust uredospores germinate without the appreciable increase i n protein that occurs in 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 in bean and wheat rust uredospores and their capacity increases three-fold during spore hydration, prior to germination (Staples et a l . 1968). Data from studies using metabolic inhibitors 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 infection structures. The presence of puromycin allows germination and appressorial formation to occur but prevents further infection structure development (Dunkle et a l . 1969). The protein required for germination appears to be stored within the dormant uredospore. The mRNA responsible for directing the complete development of infection 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 infection structures. Although puromycin and actinomycin-D failed to prevent germination i t would be erroneous to conclude that RNA and protein synthesis does not occur during the early stages of development. Uredospores contain a complete system for protein synthesis (Yaniv and Staples 1974). Germination studies on the incorporation of [ 1 4C]-labelled amino acids, -glucose, and -valeric acid 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 indicative of protein synthesis. Although there i s an apparent synthesis of protein, germination i s not accompanied by net protein synthesis; total 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 for a wide range of organisms i t s functional significance i s unknown. Heat shock proteins (HSP) may serve to protect the c e l l against environmental stress (Ashburner 1982). Shaw et a l . (1985) have identified seven HSP in Melampsora l i n i uredospore germlings. Kim et a l . (1982a) reported on changes in detergent-soluble polypeptides from uredospores of P. cf. t r i t i c i . Dormant uredospores were found to contain more than 270 distinct polypeptides, several of which varied among physiologic races (Howes et a l . 1982). After 2 and 6 hours of germination the concentrations of at least five polypeptides decreased considerably. At 2 hours, a transient concentration increase was noted for two polypeptides, 4 hours later these concentrations had decreased again to that found in dormant uredospores. The total protein extracted from six-hour-old germlings was 86 percent of the total protein extracted from dormant spores. 16 Differentiation was accompanied by a decrease in at least five polypeptides and an increase in four others. Two new polypeptides appeared in differentiated sporelings, their molecular weights were approximately 30.0 and 20.0 kD. Differentiated sporelings were found to contain only 48 percent of the total protein extracted from dormant uredospores (Kim et a l . 1982a). Wanner et a l . (1985) reported the synthesis of differentiation-specific 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 exist for the observed decline in the total amount of extractable protein and certain polypeptides: Pulse-chase experiments show that polypeptides are continually turned over, existing protein i s broken down into i t s substituent amino acids, and the amino acids are either u t i l i z e d for resynthesis or lost into the medium (see Kim et a l . 1982a). It i s also possible that proteins bind to and form an insoluble complex with glucan and mannan residues in the c e l l wall of the germtube (Kim et a l . 1982a). Glucan-protein and mannan-protein complexes have been detected in yeasts (Ballou 1974) and i t i s possible that analogous complexes occur in the germtube walls of P. q. t r i t i c i . Finally, the decline may be due to a considerable loss of metabolites 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 translation activity i s typical immediately following a temperature shift. The normal pattern of synthesis begins soon after the c e l l s are returned to the normal temperature. 2.3 Nutritional Requirements of Rust Fungi 2.3.1 Physiology of the Host-Parasite Complex Prior to considering the nutritional requirements of rust fungi in vitr o i t i s necessary to review host-parasite relations in susceptible host tissue. The pustules of rust fungi act as foci for the accumulation of host metabolites. A compatible infection increases the rate of transpiration and respiration, decreases photosynthetic activity, alters the direction of normal phloem transport (Scott 1972) and stimulates the leakage of electrolytes from the host. These responses by the host significantly improve the ava i l a b i l i t y of nutrients to the developing fungus. With respect to structural and physiological changes associated with rust infected tissues, Bushnell (1984) recognized the juvenile and the autolytic host response. During the juvenile response most plant growth hormones increase. It 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 role i n a compatible host-pathogen relationship (Levin 1985). 18 The increase in cytokinin activity reported by Sziraki et a l . (1976) appeared to delay leaf senescence by maintaining protein synthesis (Stodart 1981). The free concentration of indole acetic acid (IAA) in plants increased two and a half fold at the infection site, 6 to 14 hr after inoculation with wheat stem rust pathogen (Artemeko et a l . 1980). It 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 infection 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 total amount of host RNA declines which indicates that the newly synthesized RNA i s rapidly degraded and turned over. Chakravorty and co-workers (1974) found that catabolic RNAse activity peaks at about 6 days at levels two to five times those of uninfected leaves. The juvenile response i s not associated with significant qualitative or quantitative changes in host proteins. Host proteins changed less in compatible host-parasite combinations than in incompatible combinations (von Broembsen and Hadwiger 1972). New isozymes have been detected on polyacrylamide gels and in most cases they appear to be of fungal origin (Johnson et a l . 1968, Staples 1965). Amino acids, amides, and carbohydrates accumulate rapidly at the infected site. During the early juvenile host response these metabolites are synthesized locally from photosynthates and ammonia, and are also translocated from distant sites on the plant. During the later autolytic stage the local degradation of protein provides a rich source of free amino acids and amides (see Bushnell 1985). The ratio of soluble to insoluble nitrogen increased at infections of stem rust on L i t t l e Club wheat (Gassner and Franke 1938). On a fresh weight basis bound amino acids increased almost twofold whereas free amino acids increased fourfold (Shaw and Colotelo 1961). Large increases in the amounts of free glutamine, Y-aminobutyric acid (ABA), threonine and several basic and aromatic acids occurred as early as two days post-inoculation. Several investigators have reported differential increases in the amounts of asparagine, arginine, phenylalanine, leucine or isoleucine, valine (Farkas and Kiraly 1961, Shaw and Colotelo 1961) and tryptophan (Kim and Rohringer 1969). The movement of host metabolites into parasitic mycelium has been followed with radiotracers. Labelled sucrose i s inverted to glucose and fructose and readily absorbed by the rust hypha. Glucose, glutamate, alanine, glycine, lysine 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 readily than others (e.g. glutamine, glutamate and aspartate). Amino acids are leached from the uredosporeling during germination and their movement into host tissues has been reported (Jones 1966, Daly et a l . 1967). The infection of the host by a rust pathogen invariably causes an accumulation of free glutamine (Shaw and Colotelo 1961). This amide, which may be derived either by an endergonic reaction from glutamate or from proteolysis i s readily translocated throughout the plant. Glutamine can serve as a source of bulk nitrogen for axenic culture (Maclean 1982) and as a precursor for the synthesis of fungal chitin (Farkas and Kiraly 1961). It i s li k e l y that glutamine plays a central role in the metabolism of rust infected leaves. 2.3.2 Axenic Culture and Metabolism Nutritional Requirements The nutritional requirements of P. q. t r i t i c i in axenic culture include inorganic salts, a source of carbohydrate, reduced nitrogen and reduced organic sulphur. Carbohydrate requirements are relatively nonspecific (Coffey and Shaw 1972) . Nitrogen may be supplied as an inorganic ammonium salt, or as one of a wide range of organic compounds, such as an amino acid. Some amino acids are better sources of nitrogen than others. In the presence of cysteine the in i t i a t i o n of saprophytic growth was poor on alanine, improved on aspartate and greatest on glutamate (Coffey and Allen 1973) . Sulphur must be provided in reduced form such as cysteine, cystine, the tripeptide glutathione, or methionine. Wheat stem rust requires a higher concentration of cysteine than methionine for 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 less 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 [ 1 4C]-sucrose synthesize at least eleven [ 1 4C]-labelled amino acids within 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 readily and the carbon appears in endogenous pools of free glucose, amino acids and phosphate esters of trehalose and glucose (Manners et a l . 1982). Reisener et a l . (1961) demonstrated that 42 percent of the label from valerate-1-[C 1 4] was incorporated into the spore as carbohydrate; organic, fatty and amino acids; amides and peptides. Glutamine, glutamate and ABA had the highest specific activity of the free amino compounds. Rust uredospores possess a l l the enzymes required for glucose catabolism and terminal oxidation (Shaw 1964), the pentose phosphate pathway, c i t r i c acid cycle, and l i p i d metabolism (Caltrider and Gottlieb 1962). Sulphur Metabolism Most rust fungi studied so far are heterotrophic for reduced, organic sulphur when grown on chemically defined medium. Although rust fungi synthesize sulphur amino acids readily from [ 3 5S]-sulphide only a limited synthesis of labelled protein can be detected (Howes and Scott 1973). More than 70 percent of the label incorporated into sulphur amino acids was lost into the medium as cysteine, S-methylcysteine, glutathione and cysteinylglycine. Labeled methionine however, appeared in mycelial protein and only negligible amounts were lost 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 for sulphur-amino-acid synthesis* These results suggest that a metabolic block exists in 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. It i s interesting to note that sulphate reduction occurs in prokaryotes, eukaryotic algae, most fungi (Scott 1972) and a l l higher plants so far 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 to the medium but offer no quantitative data. Jones and Snow (1965) reported that [ 3 5S]-labelled uredospores of P. coronata avenae lost a range of amino compounds including ethionine, ABA, methionine sulphoxide and four sulphur-containing unknowns during 12 h of germination. The amino acids detected in the culture f i l t r a t e (basal medium) of P. g. t r i t i c i after nine days were glutamate, glutamine, glycine, alanine, lysine, arginine, serine, leucine and isoleucine, phenylalanine, valine, threonine, asparagine, proline, cysteine, and methionine (Howes and Scott 1972). A considerable amount of cysteine and glycine-containing peptides, such as cysteinylglycine and glutathione accumulated in the medium. Very l i t t l e methionine and homocysteine was detected (Howes and Scott 1973). From these studies i t was evident that the loss of sulphur-containing compounds i s selective and they are not lost as rapidly nor to 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 inhibitor, methyl ferulate in the c i s or trans form are also leached into the medium. Mutual stimulation during sporeling development i s demonstrated by the positive effect 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 in axenic culture would result in a net loss of metabolites, thus imposing an excessive drain on metabolism and depleting internal metabolite pools, which in turn would result i n lesser growth rates (Maclean 1982). A poor approximation of amino acid balances may also result in detrimental effects, such as methionine toxicity (Howes and Scott 1972) and the selective leakage of amino acids from the mycelium (Maclean 1982). Endogenous Free Amino Acids Interest in the free amino acid content of rust uredospores has developed in conjunction with studies relating to self-inhibition of germination (Wilson 1958), the potential of differentiating races of rust on the basis of characteristic amino acid content (McKillican 1960), and the effects of storage on assimilative and synthetic capabilites (Wynn et a l . 1966). McKillican (1960) reported race 56 as unique in 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 original 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 later studies by Wynn et a l . (1966) i t was found that glutamic acid made up over half of the total amino acids. Other major amino acids were alanine, aspartic acid, serine, and cystine. Since no attempt was made to recover asparagine and glutamine intact, i t was probable that the levels of aspartic acid and glutamic acid included these amides. Stefayne and Bromfield (1965) noted earlier that the uredospores contain seven times the amount of glutamic acid as glutamine. Analyses of the amino acid composition of wheat rust uredospores vary widely due to sampling and analysis procedures. Accordingly, I have examined the changes in the amino acid composition of the free pool within germinating and differentiating sporelings of P. crraminis t r i t i c i at 8 and 20 h following imbibition. The results are preliminary to providing information for further studies of nitrogen metabolism and nutrition in the wheat stem rust fungus. 26 3. EXPERIMENTAL METHODS 3.1 Production and Collection of Spores The wheat stem rust uredospores, (Puccinia graminis Pers. f.sp. t r i t i c i Eriks and E. Henn., race C17), employed in this 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 in a growth chamber with a 16 h photoperiod of 9684 lux and temperatures of 25°C light, 18°C dark. After eight days of growth the plants were inoculated by l i g h t l y spreading a water-based paste of talc and uredospores (3:1) over the leaf blade. The plants were then well-misted with d i s t i l l e d water, covered with a plastic bag and placed in a dark incubator at 18°C for 24 h before they were returned to the growth chamber. Within six days the leaves showed signs of "flecking", and six days following the sori opened. Five days later the uredospores were collected by gently shaking the infected leaves into a clean test tube. The freshly harvested spores were used immediately for the amino acid analyses. In a l l other experiments the spores were stored in gelatin capsules at 4°C for up to, but not exceeding 24 h. Germination was generally between 90 and 100 percent. 27 3.2 Spore Germination Depending on the experiment, germination was carried out on either a mixed calcium and potassium phosphate (Ca-K) buffer (Table I) or MPG medium (Table II). To deplete the endogenous self-inhibitor 8 mg of uredospores were dispersed uniformly over the surface of 3 ml of Ca-K buffer in an 8-cm petri dish using an inoculation loop. After 5 min, 5 loops (5-mm diameter) of uredospores were transferred to 2 ml of the germination medium in the inner chamber of a 50-ml Conway Diffusion dish. The fi n a l spore dose was approximately 300 mm-2. The outer well contained 4 ml of 1.5 x 10 - 4 M n-nonyl alcohol (Table I). Dishes were covered with a glass plate, sealed with vaseline and incubated in the dark at 19°C. 3.3 Spore Germination with the Induction to Differentiate Inoculated dishes were incubated in the dark at 19®C for 2 h, transferred to a hot water bath set at 29.5°C for 1.5 h, then returned to 19°C. Infection structure development was complete within 20 h. 3.4 Criteria for 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 composition and p r e p a r a t i o n of the Ca-K-P0 4 b u f f e r and the v o l a t i l e germination s t i m u l a n t , n-nonyl a l c o h o l (nonanol). Ca-K-P0 4 B u f f e r 1.5 x 10- 4M Nonanol C a ( H 2 P 0 4 ) 2 x H 20 5 mg d i s s o l v e i n 100 ml H 20 S t o c k s o l u t i o n : d i l u t e 1:4 p r i o r t o use K H 2 P 0 4 27 2 mg d i s s o l v e i n 100 ml H 20 add 39 ml t o above K 2 H P 0 4 3 48 mg d i s s o l v e i n 100 ml H 20 add t o above u n t i l pH 6.8 (61 ml) N-nonyl a l c o h o l 70 u l (9.94M) H 20 1000 ml mix w i t h s t i r b a r a t h i g h speed u n t i l a l c o h o l d r o p l e t s d i s p e r s e d T o t a l volume 200 ml I n n e r w e l l o f Conway c e l l O uter w e l l o f Conway c e l l 29 Table II: The composition and preparation of the differentiation medium MPG. Ca(H 2P0 4) 2 x H 20 0. 025 g KH 2P0 4 0.449 g K 2HP0 4 1.145 g Peptone 5 g D-Glucose 30 g volume made to 1000 ml with glass d i s t i l l e d H 20 The KH2P04 and Ca(H2P04) x H20 were dissolved in 200 ml d i s t i l l e d water. K2HP04 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 st e r i l i z e d by autoclaving. swellings were counted as appressoria only after a septum delimiting the appressorium from the germtube was clearly v i s i b l e . The infection peg was recognized as a small outgrowth from the mature appressorium. Substomatal vesicles were recognized after the terminal end of the infection peg had expanded to a size equal to half that of the appressorium. As the vesicles matured they flattened out forming two lobes on either side. An infection hypha was counted when one lobe of the vesicle extended beyond the length of the other. 3 . 5 Staining and Counts Sporelings were transferred from each dish to a glass slide, stained with trypan blue in lactophenol (Table III), covered with a coverslip and sealed with nailpolish. The slides were semi-permanent and may be stored for up to one year. Counts of 100 spores were made per slide and the component infection structures were individually assessed. Differentiation was expressed as a percentage of the total spores. 3 . 6 Temperature Range Trials Temperature requirements for appressorium formation and the total differentiation of uredospore germlings was investigated by applying a heat shock of constant temperature 31 Table I I I : Lactophenol-Trypan Blue; a mounting and s t a i n i n g medium f o r semipermanent mounts of f u n g i (Boedijn, 1965). P h e n o l 20 g L a c t i c a c i d 20 g G l y c e r o l 40 g D i s t i l l e d w a t e r 20 ml Trypan B l u e 0.5% 32 (ranging from 26 to 32°C for 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 different spore lot. 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 circulated through copper tubing under one end of the brass plate while the ambient temperature cooled the other end. The temperature gradient (25 to 35°C) thus generated was allowed to stabilize for at least 2 h prior to the experiment. Following 2 h germination at 19°C the inoculated dishes were transferred to the brass plate and set at various intervals along the temperature gradient. After 1.5 h the temperature of the Ca-K buffer in each dish was measured by an electronic thermometer and a thermocouple. The dishes were then returned to 19°C for 14 h. Observations were made after a total of 17.5 h of incubation. Since the germination and differentiation capacity of spore batches differed slightly, the data within each sub-experiment were adjusted to allow for comparison between experiments. The maximum differentiation value within each sub-experiment was represented as "100"; a l l other values were calculated as a percentage of the maximum. The optimum differentiation temperature varied between experiments and ranged from 28.2 to 30.7°C. Therefore, the optimum temperatures were assigned a value of "0", a l l remaining temperatures were calculated as units deviating from this optimum. 3.7 Essential Protein Synthesis: Puromycin Uredospores were inoculated onto the Ca-K buffer and induced to differentiate under conditions previously described. A protein synthesis inhibitor, Puromycin dihydrochloride (100 ug/ml) (Sigma Co.), was dissolved in the buffer and added to, or removed from (using a 10-ml syringe), the inner well of the Conway dish at the times specified in Table IV. After each medium change, the sporelings were washed three times with fresh medium. Essential protein synthesis was measured by assessing the extent of uredosporeling differentiation after 15 h. In a l l treatments designed to examine essential protein synthesis the uredospores were k i l l e d and stained in lactophenol-trypan blue upon completion of the experiment. Each treatment had three replicate dishes, two sample counts were taken from each replicate, and 100 spores were counted per sample. The treatments were compared to the control for 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 for 2 h, exposed to a heat shock for 1.5 h, then returned to 19°C for 12 h. Stages of germination and d i f f e r e n t i a t i o n (h) Treatment at which puromycin i s added to the medium (x) 0-2 2-3 . 5 3 . 5-6 | 6-8 1 8" 1 0 1 10-15 (control) 2 - - - - - X 3 - - - - X X 4 - - - X X X 5 - - X X X X 6 X X X X X X 7 - - X - - -8 - - - X - -9 - - - - X -10 - - X X - -11 — — — X X — 35 3.8 Nuclear S t a i n i n g : DAPI Uredospores were germinated on Ca-K b u f f e r f o r 4 h i n the dark a t 19°C. The germlings were t r a n s f e r r e d t o a c l e a n s l i d e u s i n g an i n o c u l a t i o n loop and allowed t o dry. A drop of DAPI (4', 6-diamidino-2-phenylindole), 1 ug/ml g l a s s - d i s t i l l e d water, was place d d i r e c t l y on the sample, a c o v e r s l i p was a p p l i e d and the edges were sealed w i t h n a i l p o l i s h . The s l i d e was observed w i t h i n 20 min by f l u o r e s c e n t microscopy. The instrument used was a Z e i s s u n i v e r s a l microscope equipped 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 ca 300 t o 400 mu, BG38 t o absorb l i g h t and p r o t e c t sample from heat, a b a r r i e r f i l t e r w i t h a cut o f f a t 410 nm, and a Z e i s s Neofluor 40 power o b j e c t i v e . Transmitted l i g h t and fluorescence 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 obtained from u n f i x e d m a t e r i a l were unacceptable i n t h a t DAPI s t a i n e d the germtube w a l l as w e l l as the n u c l e i . S e v e r a l f i x a t i o n schedules were t e s t e d i n order t o o p t i m i z e s t a i n s p e c i f i c i t y (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 of 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 of the same c o n c e n t r a t i o n as t h a t present i n the f i x a t i v e . A f t e r washing the t i s s u e was 36 Table V: A description of the fixation schedules tested. 1. Formalin-acetic acid-alcohol (FAA)* Ethyl alcohol (50%) 90 ml Glacial acetic acid 5 ml Formalin 5 ml The material was fixed for 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 acetic acid (10%) 5.0 ml D i s t i l l e d water to 500 ml The sample was fixed for 20 h then washed three times in d i s t i l l e d water (30 min per wash). 3. Ethanol/Acetic acid (3:1) The material was fixed for 2, 24, and 48 h, dried on a slide, immersed in 200 mM KCL and rehydrated through a graded alcohol series. 4. Formaldehyde: 4% and 36% * The sample was fixed for 20 h in either a 4% solution or the vapours of 36% formaldehyde, then washed 3 times in d i s t i l l e d water (30 min per wash). 5. Glutaraldehyde: 3% * The sample was fixed for 1 h then washed three times in 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 in 70% the sample was rehydrated (10 min between washes) back to d i s t i l l e d water. * These fixatives were both used as described and in combination with a surfactant. Two surfactants were assessed individually, 0.25% Triton X-100 and 0.01% Tween-20. l e f t f o r 30 min i n each of the 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 water. The method of choice was a simple a l c o h o l dehydration and subsequent r e h y d r a t i o n of the 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 fluo r e s c e n c e was minimimal i n a l l but the very young germtubes (0 t o 2 h ) . Nuclear fluorescence 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 the dark a t 4°C. The chemical s t r u c t u r e of DAPI i s shown i n Fi g u r e 2. 3.9 Amino A c i d A n a l y s i s 3.9.1 High Performance L i q u i d Chromatography (HPLC) HPLC separations are c a r r i e d out i n high r e s o l u t i o n columns packed w i t h 3 t o 25 m p a r t i c l e s of uniform s i z e d i s t r i b u t i o n . The columns r e q u i r e the use of dedicated 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 which d e l i v e r constant flow a g a i n s t h i g h pressure. HPLC methods may be used f o r the s e p a r a t i o n of a d i v e r s e a r r a y of compounds w i t h molecular weights ranging between 50 and 20 m i l l i o n . Two approaches are 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 of amino a c i d s : a n a l y s i s u s i n g reversed-phase s e p a r a t i o n of 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 22)• Tire elution profile of a sample reflects 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 for the solid-phase emerges last. Solvents from high to intermediate polarity are used as mobile phases. Thus, water i s the solvent which gives the longest retention. The retention and selectivity of the column can be adjusted and optimised by the addition of water-miscible organic solvents (e.g. methanol, acetonitrile). Since the conditions of separation are mild the formation of artifacts i s not a problem. 3.9.2 Reagents Type I reagent grade water was prepared by running glass-d i s t i l l e d water through the Milli-Q water purification 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 Unit). Methanol and acetonitrile were HPLC-grade (BDH Chemical Co). A l l remaining components of the buffer system were ACS grade. Amino acid standards (hydrolyzate mix, No. 20088), trifluoroacetic acid (TFA), triethylamine (TEA), and the 40 derivatizing 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). The stationary phase for RP was a Waters C 1 8 Pico-Tag column for protein hydrolyzates (3.9 mm x 15 cm). RP-HPLC was performed at 41°C. When not in use the column and pumps were washed with water to remove salts and then stored in acetonitrile. 3.9.4 Sample Collection and Preparation The procedures for amino acid analysis necessitated s t r i c t control of a l l possible sources of contamination. Bacterial and fungal contamination were monitored by suspending uredospores in s t e r i l e water and plating serial dilutions onto nutrient agar. Glassware: Glassware was f i r s t washed in soap (Alconox) and water, rinsed with tap water, oven dried and then soaked in an acid bath (NoChromix, Chemonics Sci.) for at least 1 h. The glass was then rinsed three times with glass d i s t i l l e d water, rinsed once in Milli-Q water and dried at 60°C for several 41 Buffer A HPLC Pump A Buffer B HPLC Pump B WISP I RP-Column [ Column Heater Programmable Multiwavelength Detector Temp. Control Module System Interface Module Discard Bottle Data and Chromatography Control Station Component (Waters) Function Pump A (Model 510) Pump B (Model 510) WISP (Model 710B) System Interface Module 840 Data and Chromatography Control Station Amino Acid Analysis Column Temperature Control Module Programmable Multiwavelength Detector (490) delivery of elution buffer A delivery of elution buffer B automated sample i n j e c t i o n l i n k s seperate components with Control Station controls conditions for HPLC, stores and analyzes data reverse-phase separations regulation of column and c o i l temperature detection of PITC-derivatives Figure 3. Waters HPLC amino acid analysis system configuration for RP-analysis of amino acids with post-column ultraviolet detection. 42 hours. Buffer: Ca-K buffer was prepared with Milli-Q water in acid-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 unit and stored at 4°C unt i l required. Spore Germination: Procedures earlier described for inducing germination and differentiation were carried out using autoclaved, acid-washed Conway dishes and s t e r i l e buffers. Ungerminated, non-differentiated, and differentiated uredospores and their leachates were analyzed for free amino acid content. The undifferentiated and differentiated sporelings were harvested after 8 and 20 h. Each treatment consisted of six dishes, which were later combined to be analyzed as one sample. The 20 h experiment was repeated three times and the 8 h experiment twice. The extent of differentiation was monitored for each treatment using the method described in Staining and Counts (page 30) . Wash: Freshly harvested spores (50 mg) were shaken in 10 ml of buffer and 0.01% Tween-20 for 5 min, the suspension was centrifuged and the supernatant transferred to an acid-washed vessel. The above procedure was repeated three times yielding a wash volume of 30 ml which was then freeze-dried and taken up in 1 ml of Milli-Q water. The washed spores were ground and extracted as described earlier. 43 Spores: The spores were collected, rinsed with Milli-Q water, placed i n an acid-washed mortar, ground with acid-washed sand and HPLC-grade 80% ethanol, and then transferred to a centrifuge tube and stored at 20°C for 20 h. The spore extract was centrifuged at high speed for 10 min and the deproteinized supernatent decanted into an acid-washed vessel. The ethanol was evaporated to 1 ml under a stream of nitrogen gas and transferred to a 10-ml test tube. The dry weight of 50 mg spores was determined by drying the spores for 16 h at 100°C. Leachate: The leachate, together with the water used to rinse the spores, was collected from the 6 dishes with a 10-ml syringe, f i l t e r e d (0.2 m Millipore) into a 150 ml acid washed pyrex vessel, shell frozen in liquid N 2 and freeze-dried. The residue was taken up in 2 ml of Milli-Q water and transferred to a 10-ml test 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. The sample preparation scheme i s given in Figure 4. Following clean-up a l l samples, including a standard amino acid mixture, were dried down in a SpeedVac concentrator (Savant Instruments) without heat. 44 1. Activate a Sep-Pak C 1 8 cartridge with 2-10-ml volumes of methanol. 2. Wash with 2-10 ml of 0.1% trifluoroacetic acid (TFA) in Milli-Q water. 3. Wash with 10 ml of 0.1% TFA in water and methanol (80:20). 4. Mix 1 ml sample with 2 ml 0.1% TFA in water and methanol (70:30). 5. Pass the sample through the Sep-Pak. 6. Discard the f i r s t 1 ml of. effluent and collect the next 2 ml fraction which contains a l l the amino acids. Figure 4. Sample preparation scheme for crude amino acid samples using Waters Sep-Pak C 1 8 cartridges. 3.9.6 HPLC of Amino Acids as Phenylthiocarbamoyl Derivatives Buffer System Eluent A: Sodium acetate trihydrate, 19.0 gm (140 mM) was dissolved in 1000 ml of Milli-Q water and 5% TEA. The pH was adjusted to 6.4 with glacial acetic acid. The solution was vacuum f i l t e r e d and combined with 25 ml acetonitrile. Eluent B: Contained 60% acetonitrile in Milli-Q water. Degassed by sonication for 5 min. Pre-column Derivatization Derivatization Solution: An ethanol, TEA and Milli-Q water (7:1:1) solution was combined with 10% PITC. Sample Diluent: A phosphate buffer was prepared (710 mg Na2HP04 per 1000 ml titrated to pH 7.4 with 10% phosphoric acid), combined with 50 ml acetonitrile, and fi l t e r e d . Procedure: The dried sample and standard were derivatized with 20 1 of the derivatizing solution for 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 1 of sample diluent, respectively. The amino acids were derivatized in order to improve the detection sensitivity as well as overcome the inherent polarity of the free amino acids. PITC-amino acids have a broad UV spectrum with a maximum absorbance near 269 nm. PITC allows the quantitative derivatization of both primary and secondary amino acids. The reaction scheme for amino acids by pre-column derivatization with PITC i s outlined in Figure 5. Sample Preparation Prior to being loaded on the column each sample was fi 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 collected into 0.5 ml Eppendorf micro test tubes by centrifugation for 5 min in a microcentrifuge (Eppendorf Model 5413). The tubes were then placed on a spring in 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 to 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 injection generated the "junk" chromatograph and was discarded, the second injection produced the chromatograph used to calibrate the column and subsequent runs. The sample injection volume was 8 1 and the injection-to-injection cycle time was 30 min. After four injections and separations of 47 / / - N \ 4. 1 1 1 - PH 10,30 min v <fO>N = C = S + + N H - C - C - 0 Ltemperature > ^ ' R phenylisothiocyanate amino compound (in excess) y . H S H H O <(0/"N - C - N - C - C - O- > ^ ' R aqueous acid / \ A . N w 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 flow-rate designed to effectively contribute to the separation of amino compounds within a physiological sample. Time Flow Curve %A %B I n i t i a l conditions 1.0 * 95 5 10. 00 1.0 5 55 45 10.50 1.0 6 0 100 11. 50 1.0 6 0 100 12 . 00 1.5 6 0 100 12.50 1.5 6 100 0 20. 00 1.5 6 100 0 20.50 1.0 6 100 0 30.00 0.1 11 100 0 49 sample the column was recalibrated with the standard. The relationship between a specific amino acid and a known concentration was calculated i n terms of a response factor (RF). The RF i s an adjustment for compounds that do not respond equally in the detector (RF= peak area/amino acid concentration). The sample chromatograms contained a number of peaks. Most of the retention times corresponded with those of identified amino acids, the remaining peaks were cla s s i f i e d as "unknown". The RF values determined during the calibration run were used to calibrate the amount of a specific amino acid within the sample. Standard solutions of glutamine and glutathione were prepared, and 200 pM of the standard was injected onto the column. Glutamine formed a peak between serine and glycine and glutathione eluted with ammonia. Three controls were run: The Ca-K buffer contained a large sloping peak (eluting at approximately 1 to 2.2 min). The baseline was otherwise f l a t with negligible peaks. Acid washed sand ground with 80% ethanol generated a chromatogram with a f l a t baseline interrupted by two sharp peaks. The f i r s t peak eluted at 9.65 min, shortly after leucine, and the second followed phenylalanine at 11.1 min. The blank derivative contained no interfering amino compounds. 50 4. RESULTS 4.1 Uredospore Germination and D i f f e r e n t i a t i o n "In V i t r o " In the presence o f 1.5 x 1 0 - 4 M n-nonyl a l c o h o l , 90 to 100% of the uredospores germinated w i t h i n 2 h fo l lowing seeding on Ca-K buf fer ( F i g . 6) . Contrary to e a r l i e r s tudies i n which 95% d i f f e r e n t i a t i o n was obtained (Maheshwari et a l . , 1967; Dunkle et a l . . 1969), the proport ion o f germtubes forming complete i n f e c t i o n s t ruc tures on Ca-K buf fer d i d not exceed 60%. The t iming of i n f e c t i o n s t ruc ture formation i n response to a 1.5 h heat shock ( 3 0 . 5 ° C ) on Ca-K buf fer i s shown i n F i g . 7. Germtube growth was arres ted s h o r t l y a f t e r exposure to the e levated temperature. The hyphal t i p began to swel l a f t e r 3.5 h and w i t h i n 5 h the appressorium was mature ( i . e . a cross w a l l d e l i m i t e d the appressorium from the germtube). The i n f e c t i o n peg formed between 5 and 6 h and at 8 h began to extend to produce the substomatal v e s i c l e . The development of the 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 the formation of a septum between i t and the appressorium. A f t e r 10 h the i n f e c t i o n hypha began to develop on the d i s t a l end of the v e s i c l e . The cytoplasm wi th in the germtube and the appressorium began to c l e a r at 8 and 9 h , r e s p e c t i v e l y . I n f e c t i o n s t ruc ture formation was complete w i t h i n 15 to 20 h . I n i t i a l l y the induct ion o f d i f f e r e n t i a t i o n by heat 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 M n-nonyl alcohol. -4 52 Fig. 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) After 5 h a septum was v i s i b l e and the appressorium was considered mature. (D) Development of the infection peg was complete 6 h following germination. (E) After 9 h the cytoplasm began to clear within the germtube. (F) The substomatal vesicle began to expand after 8 h. (G) The cytoplasm migrated from the appressorium into the developing substomatal vesicle and a second septum become apparent (10 h). (H) The infection hypha was apparent after 15 h. A differentiated sporeling after 20 h. (592 X). 53 as described by Maheshwari et a l . (1967) yielded poor results (< 5% differentiation). By using a spore density of 300 per mm2 and controlling the temperature of the heat shock by placing the Conway dishes in a water bath, i t was possible to attain up to 60% differentiation. A l l further attempts to improve the percent differentiation; such as, changing the timing and duration of the heat shock were unsuccessful. 4.2 Temperature Range Trials The temperature required to induce the maximum proportion of germtubes to differentiate into infection structures was found to l i e within a narrow range. The optimum temperature appeared to be characteristic of a particular spore l o t and ranged from 28° to 30°C. Incubation at lower than 27.5°C or higher than 30.5°C considerably reduced the amount of appressorium formation (Fig. 8) and total differentiation (Fig. 9). The upper and lower temperature limits for a l l spore lots tested were estimated as 30.5® and 27.5°C, respectively. Heat treatment was completely ineffective when the temperature was raised 1° above the upper limit or dropped 2° below the lower limit (Figs. 8 and 9). Although the incubation temperature where the maximum total differentiation was achieved varied between spore lots, the sensitivity of the sporelings to temperature changes was similar (Figures 10 and 11). Variations 1° above or below the 54 Fig. 8. Temperature requirements for appressorium formation from uredospore germtubes. The spores were germinated at 19°C (2 h). then placed at constant temperatures (ranging from 26 to 33°C) for 1.5 h, and replaced to 19°C. The optimum temperature (><) i s the temperature at which the maximum total differentiation was attained for each spore lot. 55 80 i 60 40 20 0 60 40 20 0 60 40 20 0 60 40 20 > < • • • < • • • • • • > • • < • • • • • • Fig. 8A. Spores collected July 22,1986. Optimal differentiation temperature (><): 29.3 d C . Fig. 8B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C. Fig. 8C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C. Fig. 8D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 °C. - 2 - 1 0 + 1 +2 +3 Variation Below (-) and Above (+) Optimal Temperature (°C) 56 Fig. 9. Temperature requirements for the complete differentiation of P. graminis t r i t i c i uredospore germlings. The upper and lower limits for total differentiation were estimated to be 27.5 and 30.5°C, respectively. Germination conditions were described in Fig. 8. 57 60 40 20 • • Fig. 9A. Spores collected July 22,1986. Optimal differentiation temperature (> <): 29.3 °C. I I n 40-I J 20 p I 1 0 o Pi zn <u PH 40 20 • • • • Fig. 9B. Spores collected July 29, 1986. Optimal differentiation temperature(><): 28.2°C. Fig. 9C. Spores collected August 6,1986. Optimal differentiation temperature(><): 30.7°C. 40 20 Fig. 9D. Spores collected November 13,1986. Optimal differentiation temperature (> <): 28.8 °C. • • -1 0 +1 +2 +3 Variation Below (-) and Above (+) Optimal Temperature ( C) 58 100 -, 80 • < 60 40 20 -• • • • i • • i +2 +3 -1 +1 Variation Below (-) and Above (+) Optimal Temperature (°C) Fig. 10. The relationship between heat shock temperature and the transformed percent of appressoria formed (TPA). 5 9 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 in greater than 40% reduction in the number of sporelings forming infection structures. The formation of appressoria was less sensitive to deviations from the optimum temperature (Fig. 10) than the formation of a complete set of infection structures (Fig. 11). 4.3 The Influence of Nutrients on Sporeling Differentiation Preliminary experiments were undertaken to investigate the effect 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 infection structures (Table VII). MPG medium (peptone, glucose, and Ca-K buffer), in conjunction with both a heat shock and n-nonyl alcohol, supported up to 77% differentiation. 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 in the growth of long, matted germtubes that failed to form infection structures. Although a l l three components of MPG influenced sporeling differentiation i t was evident that their roles were not of equal importance. MPG was broken down to i t s constituent parts and each component was tested for i t s a b i l i t y to promote differentiation. The buffer alone supported 66% differentiation, peptone in d i s t i l l e d water supported 14%, and glucose in d i s t i l l e d water resulted in less 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 infection structures (PDS). The presence or absence of a treatment i s indicated by '+' or respectively. peptone MPG glucose Ca-K n-nonyl alcohol heat shock PAS PDS + + + + + 89 77 + + + + - 27 11 + + + - + 55 50 + + • + - - 0 0 + + - + + 54 44 + - + + + 71 32 - + + + + 84 39 + - - + + 36 14 - - + + + 83 66 - + - + + 5 5 * - + + + + 40 32 *supplemented with tyrosine (49 mg/l), cysteine (17 mg/l), and tryptophan (15 mg/l). 62 differentiation. The combination of peptone and glucose increased differentiation to 32%. The maximum amount of uredosporelings forming a complete set of infection structures (77%) was achieved on the complete MPG medium. These results show clearly that the response to nutrients i s non-additive. Glucose in d i s t i l l e d water promoted the growth of long germtubes which failed to differentiate. The addition of cysteine, tyrosine and tryptophan to glucose and Ca-K buffer had no significant effect on differentiation. 4.4 Essential Protein Synthesis Sporelings exposed to an inhibitor of protein synthesis before, during, and after the inductive period provide evidence for the nature and timing of protein synthesis during differentiation. It was found in the present study that the presence of puromycin (100 ppm) had a significant effect on appressorium formation and completely inhibited a l l further development (Table VIII). In most cases where the heat treatment arrested linear growth the germtube produced an apical swelling, which after 20 h failed to form a cross wall (Fig. 12a). The appressorium was earlier 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 V I I I : The e f f ec t of a p r o t e i n i n h i b i t o r , puromycin (100 ppm), on the development o f i n f e c t i o n s t ruc tures from germlings of P. graminis t r i t i c i . To induce the d i f f e r e n t i a t i o n of i n f e c t i o n s tructures a heat shock at 3 0 ° C f o r 1.5 h was administered 2 h a f t e r the s t a r t of germination a t 1 9 ° C . S p o r e l i n g development (%) Treatment A p p r e s s o r i a a* b# I n f e c t i o n peg V e s i c l e a b I n f e c t i o n hypha 69 100 61 100 53 100 40 100 ( c o n t r o l ) X 78 114 72 117 60 114 27 67 X X 73 107 69 112 50 96 1 3 X X X 72 105 58 94 17 31 0 0 —xxxx 75 109 54 88 0 0 0 0 xxxxxx 17 2 5 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 x x - 73 106 55 90 27 51 2 5 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 i s expressed as a mean percent of the t o t a l spores counted. ^development i s expressed as a percent of the c o n t r o l , underl ined 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 c o n t r o l (95% confidence - F i s h e r LSD). 64 Fig. 12. The presence of a protein inhibitor (puromycin) in the germination medium significantly reduced the number of germtubes forming appressoria. (A) The appressoria were predominantly irregular in form (bright f i e l d illumination, 592x). (B) Generally the nuclei within the germtube did not divide but remained expanded from 0 to 15 h (fluorescent micrograph, 1040x). (C) In cases where a septum was formed the nuclei divided to give rise to four daughter-nuclei. The pairing of daughter nuclei was not observed (double illumination with U.V. and highly attenuated v i s i b l e light, 1040x). 65 presence of the inhibitor however, their shape was predominantly irregular (Fig. 12a). The results found by Dunkle et 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 effect on the proportion of germtubes forming infection structures the sporelings were exposed to puromycin at specific times following heat treatment. Complete differentiation was prevented in those t r i a l s where puromycin was present from immediately after heat treatment to the completion of the experiment (20 h). During this time frame the development of the appressorium and the infection peg were unaffected but a l l further differentiation was inhibited (Tables VIII and IX). Although the sporelings appeared to recover from the effects of the inhibitor shortly after the removal of puromycin from the germination medium, the development of the infection hypha was significantly impaired in a l l cases. Although the development of vesicles did not occur before 10 h, their formation appeared to be dependent on proteins synthesized 3.5 to 8 h following germination. The formation of the infection hypha was dependent on the development of the vesicle; the latter always preceded the former. The proportion of vesicles developing infection hyphae was considerably reduced when puromycin was present at 3.5 to 8, 6 to 10, or 6 to 15 h and completely inhibited when the 66 Table IX: The effect of a protein inhibitor, puromycin (100 ppm), on the development of infection 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 development (%) Treatment Infection peg Vesicle Infection hypha a* b* a b a b 90 100 86 100 76 100 (control) X 92 103 84 98 44 59 X X 94 105 73 85 2 3 X X X 80 90 29 34 0 0 — X X X X 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 — - x - 93 104 64 75 23 31 X X — 79 88 48 56 10 11 xx- 76 85 49 57 7 10 Puromycin was present (x) and absent from (-) the germination medium (Ca-K buffer). ( ) corresponds to time intervals; 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 significantly different from the control (95% confidence - Fisher LSD) 67 inhibitor was present at 3.5 to 15 h (Tables VIII and IX). The nuclear behaviour of sporelings differentiating in 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 nuclei remained in the germtube, had a granular appearance, and were expanded in form (Fig. 12b). Occasionally the nuclei migrated into the developing appressorium and completed one round of mitotic division. The presence of an inhibitor of protein synthesis appeared to arrest germtube development at a specific phase of the c e l l cycle. The expanded form of the nuclei suggests that the c e l l was either in the last part of Gl or entering the S-phase. The four daughter nuclei did not move together but remained separate and expanded throughout the observation period (at least 20 h) (Fig. 12c). Nuclear elongation prior to mitotic division was not observed. 4.5 Cytology of Uredosporeling Development 4.5.1 Nuclear Staining The most suitable technique tested for fixation was a graded ethanol series followed by staining with 1 ug/ml DAPI. DAPI reacted rapidly with the sample, the nuclei were clearly 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 to no fluorescence. 68 Germtiibes 1.5 h old or younger fluoresced strongly making i t d i f f i c u l t to discern the nuclei. Nuclear fluorescence improved for 20 min and then remained stable for up, to 10 days when stored in the dark at 4°C. In cases where the infection hypha was developing and the nuclei had migrated into the hypha a change was observed in the vesicle. The vesicle cytoplasm became speckled, the "granules" fluoresced a bright gold. The addition of a surfactant, such as Triton X-100 or Tween-20 allowed the spores to be taken into suspension and simplified washing procedures by centrifuging the sample between changes. However, in the presence of surfactants the germtube fluoresced strongly rendering the nuclei invisible. 4.5.2 Nuclear Behaviour during Germination and Differentiation For cytological 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 differentiate as described earlier (page 27). Uredospore nuclei were stained with DAPI at various stages of germtube development. The resting uredospore was binucleate. Following germination the two nuclei usually migrated in tandem from the spore into the developing germtube and toward i t s apex (Fig. 13). These nuclei were round to oval in shape and represented the expanded or interphase nuclei described by Savile (1939). In the absence of a stimulus to differentiate 69 F i g . 13. F o l l o w i n g 2 h germination at 19°C the two interphase n u c l e i migrated from the uredospore i n t o the 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 attenuated v i s i b l e l i g h t , 1040x). F i g . 14. The germtube of non-shocked uredosporelings 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 perpetual interphase (I040x). F i g . 15. In some cases the n u c l e i w i t h i n 20-hour-old uredosporelings were elongated i n form and had a "ragged" appearance ( f l u o r e s c e n t micrograph, 1040x). Fig. 16. The linear 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 in conjugate telophase and preparing to divide (double illumination with U . V . and highly attenuated vi s i b l e light, 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 after germination (1040x). Fig. 18. The mature appressorium contained four daughter-nuclei. These nuclei moved together to form a compact tetrad (1040x. the germtube remained binucleate and appeared to be in perpetual interphase; mitotic division was not observed in 0-to 20-hour-old sporelings (Fig. 14). In some cases the nuclei of nondifferentiated 20-hour-old sporelings had a "ragged" appearance and became elongated in form (Fig. 15). The nuclear behaviour of the differentiating sporeling was complex; nuclear division was predictable and changes in form and migration patterns were observed regularly. Although nuclear division was readily observed the more subtle changes in nuclear form, which correspond to specific phases of the c e l l cycle could not be followed with certainty. During heat shock the germtube produced a bulbous appressorial i n i t i a l into which the two nuclei migrated. At 4 h the nuclei were elongated to dumbbell in shape and were situated one behind the other (Fig. 16). Their appearance suggested that these nuclei were in conjugate telophase and nearing the completion of mitosis. Usually, after 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 to 8 h following imbibition (Fig. 17). The mature appressorium contained four nuclei arranged as a compact tetrad (Fig. 18). At 7 h the four nuclei migrated toward the developing infection peg. Occasionally two nuclei divided in the appressorium (Fig. 19) but more commonly the second division occurred as the nuclei moved into the developing substomatal vesicle (7 h to 13 h). The nuclei migrated in closely 72 Fig. 19. Occasionally two nuclei divided within the appressorium. The six nuclei migrated in tandem towards the developing infection peg (double illumination with U . V . and highly attenuated v i s i b l e light, inset: fluorescent micrograph, 104Ox). Fig. 20. Ten hours following germination the six-nucleate vesicle was delimited from the appressorium by a septum (1040x). Fig. 21. In most cases a nuclear pair divided in the mature vesicle to yield a total of eight nuclei. These nuclei were distributed such that three to four nuclei aggregated on either side of the vesicle (1040x). 73 Fig. 22. The orientation of nuclear migrational patterns was found to be related to the polarity of the vesicle. Once growth of the infection hypha was initiated the nuclei moved into an arrowhead formation with the apex directed towards the developing hypha (double illumination with U.V. and highly attenuated vi s i b l e light, inset: fluorescent micrograph, 1 0 4 0 X ) . Fig. 23. The infection hypha contained eight, six, or more commonly four nuclei (1040x). Fig. 24. In some cases a nuclear pair failed to migrate with the other nuclei and remained in the vesicle (1040x). Fig. 25. Twenty three hours following germination the infection hypha most often contained four expanded nuclei (1040x). 74 associated pairs. A cross wall delimited the six-nucleate vesicle from the appressorium within 10 h (Fig. 20). In most cases a nuclear pair divided in the mature vesicle to yield a total of eight nuclei (Fig. 21). These nuclei, which were rounder and considerably smaller than those observed in the germtube, are described as dividing or unexpanded. Nuclear division was not always synchronous, five to eight nuclei were often seen within the vesicle. Within 10 to 13 h after germination six to eight nuclei were distributed such that three to four nuclei aggregated on either side of the vesicle (Fig. 21). The infection hypha of P. graminis t r i t i c i developed as a polar extension from the vesicle. Once growth of the infection hypha was initiated (ca 13 h) a nuclear migration pattern became apparent. The nuclei moved towards the developing hypha in the formation of an arrowhead (Fig. 22). The orientation of their migration was always relative to the polarity of the vesicle. The leading nucleus, which often fluoresced more strongly than the others, was directed towards the developing hypha. As growth ensued eight, six or four expanded nuclei migrated into the infection hypha (Fig. 23), occasionally a nuclear pair remained in the vesicle (Fig. 24). After 23 h four or fewer nuclei were observed in the hypha (Fig. 25). 75 4.6 Amino Acids Analysis The standard generated 18 peaks with characteristic retention times. The separation of a PITC-amino acid standard i s illustrated in Fig. 26, Table X. Good resolution was observed within 12 min. An additional 18 min was required to remove solvent peaks and reequilibrate the column between runs. Except for histidine from arginine, and glutathione from ammonia, most amino compounds were adequately separated using the RP-column . As is characteristic of RP-chromatography, the elution order was related to 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 to determine the effect 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 resting uredospores. (2) Alcohol extract from unwashed resting uredospores. (3) Alcohol extract from eight-hour-old uredosporelings: non-shocked and heat shocked. (4) Leachates from eight-hour-old uredosporelings: non-shocked 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 Aspartic acid 2.44 2 Glutamic acid 2.68 3 Serine 4.48 4 Glycine 4.69 5 Histidine / Axginine 5.22 6 Threonine 5.37 7 Alanine 5.51 8 Proline 5.66 9 N H 3 6.00 10 Tyrosine 7.06 11 Valine 7.61 12 Methionine 8.00 13 Cysteine 9.12 14 Isoleucine 9.30 15 Leucine 9.50 16 Phenylalanine 10.27 17 Lysine 11.62 18 Glutamine 4.53 19 Homoserine 4.96 20 Glutathione 6.00 * Unknown peaks — 78 (5) Alcohol extract from 20-hour-old uredosporelings: non-shocked and heat shocked. (6) Leachates from 20-hour-old uredosporelings: non-shocked and heat shocked. 4.6.2 Complications A number of problems were encountered in identifying and quantifying the amino compounds in uredospores and uredospore leachates. Representative RP-HPLC amino acid profiles from (A) the alcohol extract from non-shocked uredospores, and (B) the leachate from heat shocked uredospores, are shown in Fig. 27. Two peaks corresponding to glutamine (#18 - see Table X) and NH 3/glutathione (#9/20) are predominant in both profiles. The resolution between peaks identified as serine, glycine, and glutamine was poor. Therefore, since column selectivity 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 identified in most, but not a l l extracts. The most serious overlap occurred between glycine and glutamine and in Fig 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) for glycine and glutamine were similar in value. In order to calculate the order of magnitude of the 79 F ig . 27. Representative R P - H P L C separations of amino acids isolated from (A) non-heat shocked 20-hour-old uredosporelings and (B) the leachate of 20-hour-old heat shocked uredosporelings. For 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 in uredospores (Fig. 27A). Similar considerations apply to the analysis of the leachate (Fig. 27B), in which glutamine was also predominant. A further complication in the analysis of the leachate arose because of the presence of a very large unidentified peak (X) in the buffer (Fig. 27B) which usually overlapped aspartate (#1) and glutamate (#2). This prevented any meaningful estimates of the levels of aspartate and glutamate in the leachate. In a l l chromatograms ammonia and glutathione eluted off the column simultaneously. It was therefore impossible to obtain independent estimates of the levels of NH3 and glutathione. The NH 3/glutathione peak (#9/20) was usually 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 identified 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 transfer from the germination medium to the extraction vessel because the shorter, differentiated germtubes did not interlock to form a mat. The loss was visually estimated to 81 be not greater than 15%. Calculations of the amino acid levels per mg dry weight of uredospores were based on the dry weight of the original sample (2.78 mg) and are therefore too low by a maximum of 15%. Two unknown peaks eluted from the column shortly after the amino acid phenylalanine (#16). Based on results reported by Dwyer et a l . (1987) these peaks may be identified tentatively as tryptophan (Trp) and ornithine (Orn). These compounds appeared soon after germination occurred; they were detected in the germinating uredospores and in the leachate from 20-h-old non-shocked uredosporelings. 4.6.3 Exogenous Free Amino Acids: Resting Spore Wash The buffer in which resting uredospores were washed contained the early-eluting amino acids. These appeared to be associated with the spore wall. Although there was some overlap of aspartic and glutamic acids with the unidentified peak (X) found in 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 earlier (page 77). The results are shown in Table XI. The amino acids washed off the spores were, in order of predominance, glutamine, alanine, glutamate, aspartate, and tyrosine. An unknown compound with a retention time of 6.66 minutes was also present in the spore wash. 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 Acid nM/mg spore dry weight 3 Percent Ala 0.57 ±.07 26.0 Pro + -Tyr 0.25 ±.06 11.4 Val + -Met + -Cys ND -H e + -Leu + -Phe + -Lys ND — Glm 0.72 ±.04 32.9 HomoSer ND -Arg/His/Thr a •++ -Unknown* + — Asp 0.27 ±.03 12 . 3 Glu 0.38 ±.06 17.4 Total 2.19 ±.26 100. 0 a Each value represents the mean and range for two separate experiments. + = trace amounts, ++ = greater than trace amounts (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. asee text. *calculated assuming a RF of 4256 (average value for 14 different amino acids). 83 standard. Therefore i t was assigned an RF of 4256 which i s an average value calculated from the RF of 14 different amino acids. In addition, small peaks corresponding to the retention times of one or more of arginine, histidine, and threonine were detected (Table XI). It was impossible to t e l l which of these were actually present. Cysteine, homoserine, and lysine were not detected. 4.6.4 Endogenous Free Amino Acids: Extracts of Unwashed Resting Uredospores In order of prevalence the amino compounds in unwashed resting uredospores were alanine, the unknown, glutamic acid, and glutamine followed by smaller amounts of several other amino acids (Table XII). Alanine accounted for 30% and the unknown accounted for 22% of the total free amino acids present. The ratio of glutamic to aspartic acid was 12 (2.4/0.2). Cysteine and lysine were not detected. Comparison of the appropriate columns in Tables XI and XII shows that the total amino acids washed off resting uredospores (Table XI) account for only 16.5% (2.19 x 100 /13.3) of the total amino acids extracted from unwashed resting spores. 84 Table XII. The endogenous free amino acid level in unwashed resting uredospores (S), and in non-shocked uredosporelings of P. graminis t r i t i c i after 8 h (N8S), and 20 h (N20S) Amino Acid nM/mg spore dry weight a Ratio S N8S N20S N8S/S N20S/S Ala 4 . 04 47 4 . 99 ±. 71 2 . 50 ±. 30 1. 2 0. 6 Pro 0 .20 0 2 .20 ±. 29 1 . 10 ±. 13 11. 0 5. 5 Tyr + 2 .47 ±. 04 2 . 29 ±. 64 27 . 4 25. 4 Val 0 .43 ± 0 1 .80 ±. 43 1 .23 ±. 17 2 . 9 2 . 7 Met + 2 .54 +. 07 2 . 09 ±. 42 50. 8 41. 8 Cys ND 7 . 09 ±. 39 3 . 19 ±. 77 (>1 000) (>6 00) H e 0 .25 ±. 01 2 . 16 ±. 15 1 . 07 ±. 17 8. 6 4 . 3 Leu 0 .23 ±. 02 1 .93 ±. 33 1 . 50 ±. 54 8 . 4 6 . 5 Phe 0 .29 ±. 02 2 .93 ±. 31 3 . 10 ±. 58 10. 1 10. 7 Lys ND 1 . 17 ±. 60 1 .05 ±. 02 (>2 00) (>2 00) Glm 2 .23 ± 30 6 .99 +. 84 10 .74 ±1 . 2 3 . 1 4 . 8 HomoSer ++ ++ ++ Arg/His a ++ ++ ++ Unknown* 2 .98 ±. 75 2 .47 ±. 10 2 .95 ±. 79 1. 0 1. 2 Orn ND ++ ++ Trp ND ++ ++ Asp 0 . 2 ± 0 2 . 2 ±. 7 1 . 5 ±. 2 22 . 0 15. 0 Glu 2 .4 + . 1 10 . 0 ±2 .9 4 . 1 ±. 8 4 . 2 1. 7 Total 13 .3 ±1.7 50 .9 ±7 . 6 38 . 4 ±6 . 7 3 . 8 2 . 9 Glu:Asp 10:1 5:1 3 : 1 - -a Each value represents the mean and range for two separate experiments. + = trace amount, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. asee text. *calculated assuming a RF of 4256 (average value for 14 different amino acids. 85 4.6.5 Effect of Heat Shock on the Free Amino Acids in Germinated Uredospores and in the Leachate After Germination Unwashed uredospores were placed on Ca-K buffer at 0 h at 19° C, heat shocked at 29° C from 2 h to 3.5 h and harvested at either 8 h or 20 h. Non-shocked controls were subjected to the same protocol but were maintained at 19° C throughout the germination period. Uredosporelings and leachates were analyzed separately. The results are presented in Tables XII, XIII, and XIV. The data in Table XII show that the endogenous free amino acid level in non-shocked uredospores at 8 h was 3.8-fold, and at 20 h was 2.9-fold that in unwashed resting uredospores. Among the individual amino acids, alanine and the unknown compound showed relatively l i t t l e change on germination but a l l the other amino compounds increased markedly. Large relative increases occurred in cysteine and lysine, which were below the limit of detection in resting uredospores. The ratio of glutamate to aspartate decreased sharply on germination. Table XIII shows that, while the endogenous free amino acid level in heat shocked uredospores increased approximately 2-fold at 8 h, there was no further increase at 20 h. Among the individual amino compounds there were decreases (about 50%) in alanine and the unknown compound. Valine was relatively unchanged. A l l other amino compounds increased significantly, particularly cysteine and lysine. In general 86 Table XIII. Endogenous free amino acid content of unwashed resting uredospores (S), and heat shocked uredosporelings of P. graminis t r i t i c i after 8 h (H8S), and 20 h (H20S). Amino Acid nM/mg spore dry weight 3 Ratio S H8S H20S H8S/S H20S/S Ala Pro Tyr Val Met Cys H e Leu Phe Lys Glm HomoSer Arg/His 3 Unknown* Orn Trp Asp Glu 4.04 ±.47 0.20 ±.0 + 0.43 ±.0 + ND 0.25 ±.01 0.23 ±.02 0.29 ±.02 ND 2.23 ±.30 ++ ++ 2.98 ±.75 ND ND 0.2 ±.0 2.4 ±.1 1.80 ±.03 0.66 ±.01 0.93 ±.03 0.53 ±.13 0.56 ±.11 2.89 ±.12 0.80 ±.05 1.93 ±.19 1.41 ±.16 0.25 ±.11 8.69 ±.89 +'+ ++ 1.43 ±.18 ++ + 1.7 ±.4 4.5 ±1.0 1.40 ±.01 0.46 ±.0 1.20 ±.16 0.66 ±.03 0.84 ±.02 1.45 ±.03 0.69 ±.01 1.49 ±.02 1.44 ±.11 0.44 ±.11 8.38 ±.35 ++ ++ 1.54 ±.48 ++ ++ 1.0 ±.1 3.5 ±.0 0.5 3 . 3 10. 3 1.2 11.2 (>600) 3.2 8.4 4.9 (>60) 3 . 9 0.5 17.0 1.9 0.4 2 . 3 13 . 3 1.5 16.8 (>300) 2 . 8 6.5 5.0 (>80) 3.8 0.5 10. 0 1.5 Total 13.3 ±1.7 28.1 ±.16 24.5 ±.76 2.1 1.8 Adjusted Total 11. l b 32.3 C 28.3 C - -Glu:Asp 10:1 3 :1 4:1 - -aEach value represents the mean and range for two separate experiments. + = trace, ++ = greater than a trace (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. 3 see text. *calculated assuming a RF of 4256 (average value for 14 different amino acids, ^measured endogenous amino acids minus the amino acids in the resting spore wash. cmeasured amounts plus 15% to compensate for uredospore losses during transfer. 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 after an 8 h (H8L and N8L), and 20 h (H20L and N20L) incubation period. Amino nM/mg spore dry we i g h t a Acid N8L H8L N2 0L H2 0L Ala 0.60 ±. 21 0.70 ±. 01 4 . 52 ±. 07 0.91 ±. 11 Pro 1.78 ±. 17 2 . 99 ±. 59 2 .81 ± . 43 2 . 64 ±. 24 Tyr • 0.29 ±. 14 + 1 .21 ±. 13 0.33 +. 08 Val 0. 60 ±. 01 0.85 ±. 71 1 .91 ±. 28 + Met + + 1 .36 ±. 03 + Cys ND ND 5 . 42 ±. 13 + H e + + 1 . 61 +. 03 + Leu + + 1 . 17 ±. 03 + Phe + ND 2 . 06 ±. 05 ND Lys + + 0 . 66 +. 01 ND Glm 2 .40 ± . 20 2 . 39 ±. 33 9 .93 ±. 00 9.91 ±1 . 17 HomoSer ++ ++ ++ ++ Arg/His 3 ++ ++ ++ ++ Unknown* ND ND 3 .31 ±. 35 ND Trp ND ND ++ ND Orn ND ND ++ ND Total: Leachate 5. 67 +, 30 6.93 ±. 98 35 .97 ±. 02 13 . 79 ±. 95 (L) Wash (W)2 2 .19 ±. 26 2 .19 ±. 26 2 . 19 ±. 26 2 .19 ±. 26 L - W 3 . 48 4 . 74 33 . 78 11. 60 aEach value represents the mean and range for two separate experiments. + = trace, ++ greater than a trace, (0.04 nM/mg < t r > 0.12 nM/mg). ND - not detected. asee text. *calculated assuming a RF of 4256 (average value for 14 different amino acids. afrom Table XI. the increases in individual amino compounds were smaller than in non-shocked spores. Moreover the amino acid level in heat shocked spores was only 55% (28.1 x 100 /50.9) of that in non-shocked spores at 8 h and 64% (24.5 x 100 /38.4) at 20 h. It is thus clear that heat shock decreases protein hydrolysis in germinating uredospores. Paraphrasing the well known song, we may ask "Where have a l l the amino acids gone?". The data in Table XIV, which presents the analyses of the leachates, provides the answer. By 8 h non-shocked and shocked uredospores had lost approximately the same total amount of amino compounds to the germination medium, ca 5.7 and 7.0 nM/mg dry weight respectively. In contrast, by 20 h the non-shocked uredospores had lost nearly 36.0 and the shocked uredospores only ca 13.8 nM/mg spore dry weight respectively. Both non-shocked and heat shocked uredosporelings lost about equal amounts of glutamine. At 20 h losses in alanine, cysteine, phenylalanine, and the unknown were much greater from the non-shocked sporelings. It i s important to note that the leachate from unwashed germinating uredospores presumably includes the amino compounds which could be washed off. the resting uredospores (Table XI). Differences in the total amounts of amino compounds found in the leachates and in the spore wash are therefore also shown in Table XIV. 89 5. DISCUSSION 5.1 Temperature Retirement for the Differentiation 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 differentiation on Ca-K buffer in response to a 1.5 h heat shock of 30° C administered 2 h after seeding. Wisdom (1977) obtained only 5% differentiation using the methods described above. The effect of the heat shock temperature was not investigated. The results presented in this thesis show that : (1) Differentiation has a sharp temperature optimum. Aberrations one degree above or below the optimum heat shock temperature significantly reduced (by greater than 40%) the amount of total sporelings forming a complete set of infection structures. (2) The optimum heat shock temperature varies slightly for different spore lots. When the temperature of the heat shock was optimised up to 70% differentiation was obtained. These results indicate that Wisdom's failure to obtain better than 5% differentiation on Ca-K buffer was most li k e l y due to the use of a sub-optimal heat shock temperature. 90 5.2 The Influence of Nutrients on Uredospore Differentiation Since Wisdom (1977) obtained only 5% differentiation on Ca-K buffer she abandoned the buffer in favour of her nutrient-enriched MPG medium. Using MPG in conjunction with a 1.5 h heat shock of 30°C, Wisdom obtained up to 80% di f ferentiat ion. Williams (1971) had earlier shown that nutrients stimulate differentiation and that the nutrient stimulus i s more effective when a heat shock i s applied. The results obtained in the present study support this claim. Moreover, this response of the uredosporelings to nutrients in the germination medium was found to be non-additive. It 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 to Ca-K buffer as a differentiation medium for P. orraminis t r i t i c i uredosporelings. The precise temperature optimum for 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 differentiation of a high percentage of uredosporelings. Ca-K buffer appeared to be the most effective component of MPG. Baker et a l . (1987) reported that Ca + 2, but not K+, 91 stimulated the germination of Uromyces phaseoli uredospores. It i s also possible that calcium plays a key role in uredosporeling differentiation. One of the many functions of calcium in biological 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 for the formation of nucleic acids and phospholipids, as well as a key molecule of energy metabolism, ATP. Peptone in d i s t i l l e d water was a poor medium for differentiation. In combination with glucose and Ca-K buffer peptone significantly increased the number of sporelings that formed infection structures (up to 77%). Bacto-peptone contains a variety of simple nitrogenous compounds (including reduced organic sulphur), ions, minerals, and a negligible amount of protease and complex nitrogenous compounds (Difco Laboratories 1953). Glucose i s a potential source of energy for hyphal growth. In the presence of glucose (dissolved in glass-d i s t i l l e d water) the germtube did not appear to respond to heat shock but continued linear growth without the formation of infection structures. Although glucose alone was ineffective as a differentiation medium i t played an important role as a constituent of MPG. According to Manners and co-workers (1982) glucose i s readily absorbed and metabolized by 92 the rust hyphae. The carbon appeared in endogenous pools of free glucose, amino acids and phosphate esters of trehalose and glucose (Reisener et a l . 1961). Previous studies on the enzymatic constitution of uredospores of the wheat stem rust have indicated the presence of complete enzyme systems for the metabolism of amino acids, l i p i d s , and carbohydrates (see Shaw 1964). The presence of nutrients in the medium may provide the uredosporeling with metabolites that assist with the processes involved in germtube morphogenesis. 5.3 The Timing of Essential Protein Synthesis The timing of RNA and protein synthesis during differentiation has been described by Dunkle et a l . (1969) and Wisdom (1977). It has been shown that new polypeptides appear during the differentiation of non-shocked bean rust uredosporelings (Huang and Staples 1982), and the differentiation of heat shocked wheat stem rust uredosporelings (Kim et a l . 1982a). Although not differentiated, heat shocked flax rust uredosporelings synthesized a number of heat shock proteins (Shaw et a l . 1985). These results have been discussed in greater detail in the literature review (pages 15). The effect of puromycin on differentiation was reinvestigated under the optimum heat shock conditions 93 described earlier (page 87). Puromycin i s recognized as an effective inhibitor of protein synthesis. It i s a structural analogue of the aminoacyl adenosine, the amino-acid-bearing end of transfer-RNA. It binds readily to the aminoacyl site 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 effect 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 significant reduction in the proportion of infection pegs forming vesicles. Although the vesicles were vi s i b l e after 10 h their formation appeared to be preceded by the synthesis of essential proteins. The results suggest that these proteins are synthesized 2 h to 6.5 h prior to vesicle formation. When puromycin was present in the germination medium for the entire incubation period (0 h to 16 h) germtube differentiation was v i s i b l y affected: (1) The complete differentiation of uredosporelings was prevented. (2) The proportion of germtubes forming appressoria was significantly 94 reduced. (3) The appressoria were predominantly irregular in form. (4) The formation of a septa between the appressoria and the germtube usually failed to occur. (5) Nuclear division was rarely observed, but in the rare event of division the pairing of daughter-nuclei did not occur. The capacity of the inhibited germlings to form infection structures appeared to recover parti a l l y when puromycin was removed from the germination medium. These results suggest the existence of differentiation-specific proteins and support the claim that these proteins are stage-specific (Huang and Staples 1982). It may be concluded that essential proteins are synthesized throughout the entire process of differentiation, including the formation of the appressoria. The presence of puromycin in the germination medium throughout the incubation period usually prevented the division of germtube nuclei. Nuclear division i s generally preceded by the replication of nuclear DNA. Prior to the onset of the replication of DNA certain enzymes such as, thymidine kinase, thymidylate synthetase and others, must increase within in the c e l l . The absence of nuclear division observed in this study suggests that these enzymes are synthesized prior to the formation of the appressorium. In rare cases the enzymes appear to be present in sufficient amounts to enable replication and division to occur. 95 5.4 Nuclear Behaviour Accompanying Germination and Differentiation The two nuclei of nondifferentiated uredosporelings did not divide but appeared to remain in perpetual interphase throughout the entire observation period of 20 h. In differentiating uredosporelings nuclear division was a regular event. Its occurrence was closely associated with the timing of infection structure development. The cytological events taking place during the f i r s t 24 h of germination and differentiation are summarized in Fig. 28. A characteristic pattern of nuclear behaviour was observed in differentiating uredosporelings. The protoplasm migrated into the developing infection structures leaving an empty space in the proximal regions of the germtube. Paired daughter nuclei migrated in tandem with the cytoplasm. The 4 to 6 nuclei observed in the appressorium migrated into the vesicle as a closely associated group. This was not observed by Wisdom (1977) who reported that the movement of the 4 nuclei from the appressorium into the developing vesicle was not synchronous. She noted that one pair moved into the vesicle while the other pair remained in the appressorium for up to 10 h. Moreover, Wisdom (1977) did not report the potential for a second nuclear division in the appressorium. In the present study 6 nuclei were commonly observed moving into the developing infection peg. Migration of nuclei into 96 Two expanded nuclei migrate down the germtube toward i t s apex. The f i r s t round of mitosis is generally completed within the mature appressorium. The cytoplasm moves into the infection peg with the nuclei. A second division occurs either prior to or during nuclear migration. The mature substomatal vesicle contains six unexpanded nuclei. A third mitotic division may occur to yield eight nuclei. 13 h Eight, six, or four nuclei enter the infection hypha, occasionally a nuclear pair remains in the vesicle. Four or fewer nuclei migrate along the infection hypha. Fig. 28. Diagrammatic representation of the cytological events taking place during the differentiation of uredosporelings of P. graminis t r i t i c i , race C17. The times given are approximate. 7 h 0 = ? 97 the developing infection structures was characterized by a slight change in nuclear shape. The nuclei appeared to be "tear drop" in shape as i f they were "pulled" into the infection structures. When a break occurred in the germtube the nuclei were extruded along with the cytoplasm. In some cases, when nuclei were migrating into the infection hypha, the foremost nucleus appeared brighter than the following ones. Wisdom (1977) described an analogous situation in which a single intensely Feulgen-positive nucleus preceded the other nuclei into the infection hypha. In the rare event of a bipolar vesicle forming, two "bright" nuclei could be seen. One nucleus was situated on either side of the vesicle and adjacent to the i n i t i a l s of the infection hyphae. Finally 2 or more nuclei appeared to either break down or coalesce within the infection hypha, so that after 24 h the hypha contained up to a maximum of four distinct nuclei (Fig. 28). 5.4.1 Staining with DAPI DAPI (4', 6-diamidino-2-phenylindole) i s a DNA-specific fluorochrome. It binds relatively specifically to AT residues of double-stranded DNA and exhibits a much enhanced fluorescence in the association (Otto and Tsou 1985). This fluorescence i s not observed with RNA or protein (Coleman et al 1981). Chemically, i t i s suggested that the AT-spec i f i c i t y resides with both the guanidine group and the benzimidazole or indole ring, which may bind to the purine of 98 adenosine through base stacking (Otto and Tsou 1985). DAPI binds to DNA and fluoresces in proportion to the amount of DNA present. The relative DNA content per nucleus can be read in situ by measuring the intensity of fluorescence with a microspectrofluorometer (Coleman et a l 1981). The enhanced intensity of fluorescence observed in the leading nucleus (preceded the other nuclei into the infection hypha) may suggest that this nucleus i s not haploid but contains additional DNA. DAPI reacted rapidly with the sample and within minutes one could easily assess nuclear state and identify nuclear abnormalities. The technique appeared to be useful for determining the numbers and positions of nuclei and their division sites within the infection structures. The stain i s water-soluble and has been used successfully as a v i t a l dye in studies on red algae and pollen development (Goff and Coleman 1984, Coleman and Goff 1985). It should have further application in assessing the effects of a number of parameters (e.g. nutrition, fungicides, and host genotype manipulation) on the nuclear behaviour of the rust pathogen. 99 5.5 The Effect of Heat Shock on the Amounts and Kinds of Free Amino Acids in Germinated Uredospores and Their Leachates The results presented in Tables XI to XIV show clearly that heat shock decreases the size of the endogenous pool of free amino acids and the extent to which uredosporelings lose amino acids to the medium. The results are summarized in Table XV which presents a "balance sheet" showing the size of the endogenous and leachate amino acid pools. Under the conditions employed only endogenous reserves are available to the germinating uredospores. The amino acid data (Table XV) therefore clearly show that there i s a net hydrolysis of protein during germination, leading to an increase in the level of endogenous free amino acids. These results 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 level of total amino acids remained relatively unchanged in the heat shocked sporelings from 8 h to 20 h (39.3 to 42.0 nM/mg), whereas the level of total amino acids in non-shocked sporelings increased from 55.8 nM/mg at 8 h to 74.4 nM/mg at 20 h. The total free amino acid level (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 shocked uredosporelings at 20 h. It i s thus clear that heat shock 100 Table XV. The distribution of the free amino compounds associated with resting spores, non-shocked 8- (N8S) and 20-h-old (N20S) uredosporelings, and heat shocked 8- (H8S) and 20-h-old (H20S) uredosporelings. The results are expressed as nM amino acid per mg spore dry weight. S N8S H8S N20S H20S Endogenous 11. l b 50.1 32 . 3 C 38 . 4 28 . 2 C Leachate^ - . 5.67 6 .95 36.0 13.8 Wash 2 .19 - - -Total 13.3 55. 8 39.3 74 . 4 42.0 ^measured endogenous amino acids minus the amino acids in the resting spore wash. Measured amounts plus 15%, see Table XII. ^includes amino acids in resting spore wash (2.19 nM/mg). 101 decreases the amount of 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 net p r o t e i n s y n t h e s i s d u r i n g the formation o f i n f e c t i o n s t r u c t u r e s induced by heat shock. Nevertheless, p r o t e i n s y n t h e s i s 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 the r e l a t i v e r a t e s of these two processes i n n o n d i f f e r e n t i a t i n g (non-shocked) uredosporelings. These r e s u l t s and the c o n c l u s i o n s drawn from them are c o n s i s t e n t w i t h the e f f e c t o f puromycin on d i f f e r e n t i a t i o n . The i n h i b i t i o n of p r o t e i n s y n t h e s i s by puromycin i n h i b i t s d i f f e r e n t i a t i o n but does not appear t o decrease the l i n e a r growth of n o n d i f f e r e n t i a t e d (non-shocked) uredo s p o r e l i n g s . The r e s u l t s are a l s o c o n s i s t e n t w i t h the observed e f f e c t s of heat shock on the i n c o r p o r a t i o n of [ 3 5 S ] -methionine i n t o newly synthesized heat shock p r o t e i n s i n germinating f l a x r u s t uredosporelings reported by Shaw e t a l . (1985). A h i g h p r o p o r t i o n of the t o t a l amino a c i d pool was l o s t t o the medium du r i n g germination, p a r t i c u l a r l y i n non-shocked uredosporelings. The extent of t h i s leakage i n d i c a t e s t h a t the l o s t amino a c i d s must o r i g i n a t e from the h y d r o l y s i s of p r o t e i n . Daly and h i s colleagues (1967) a l s o found t h a t i n non-shocked uredospores of P. graminis t r i t i c i a c o n s i d e r a b l e p o r t i o n of the 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 are l o s t t o the medium and t h a t only 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 r e s y n t h e s i s of p r o t e i n s . The 102 results in this 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 to the medium increased and appeared less selective than the loss from heat shocked sporelings. In contrast, heat shock markedly decreased the loss at 20 h of most of the amino acids l i s t e d on Table XIV. Particularly striking decreases occurred in the losses of alanine, cysteine, phenylalanine and an unknown amino acid. On the other hand heat shock had no significant effect on the losses of proline and glutamine. The loss of proline and glutamine i s not surprising since they are readily formed via the conversion of many amino acids and metabolites. Proline commonly functions as a long-distance transport compound for carbon. Proline i s an ideal translocation molecule by virtue of i t s high metabolic capabilities (eg. to form glutamate, a-ketoglutarate, and pyruvate via succinate) (Miflin 1977). Glutamine, on the other hand i s the principle nitrogen donor for many biosynthetic reactions, and i t s formation i s a detoxification mechanism for the removal of ammonia. Individual amino acids were lost to the medium at different rates and in different amounts (Table XV). For example, non-shocked sporelings lost 0.6 nM/mg alanine in 8 h and 4.5 nM/mg in 20 h (ie. the loss at 20 h was 7.5 times the 103 loss at 8 h). Non-shocked sporelings also lost 2.4 nM/mg glutamine at 8 h and 9.9 nM/mg at 20 h (ie. the loss at 20 h was 4.2 times the loss at 8 h). On the other hand the loss of alanine from heat shocked sporelings at 20 h was 1.14 times the loss at 8 h and the loss of glutamine at 20 h was 4.15 times the loss at 8 h. Comparing alanine and glutamine we see that they were each lost at different rates in non-shocked and heat shocked sporelings. Comparing non-shocked and heat shocked sporelings we see that alanine was lost at different rates but glutamine at the same rate in the two sets of sporelings. Similar comparisons can be made for the other amino acids from the data in Table XV. It follows that i f amino acids are lost from uredosporelings via a membrane defect, the defect does not affect the loss of a l l amino acids to the same extent. Therefore, the loss of amino acids from the uredosporeling to the medium i s considered to 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 to the total amino acids in each pool i s : (1) different, (2) alters as germination progresses through 8 h to 20 h, and (3) i s altered by heat shock. The question which next arises i s the mechanism of the effect of the heat shock in selectively decreasing the loss of amino acids. While heat shock promotes the synthesis of certain highly conserved heat shock proteins and depresses the 104 synthesis of other proteins (Shaw et a l . 1985), the exact mechanisms involved are unknown. With respect to the leakage of amino acids into the medium i t i s possible that heat shock directly affects on the integrity and hence the permeability of the c e l l membranes. Alternatively i t may alter amino-acid transport processes across the membranes. The data in this thesis do not provide any direct evidence on which to base an answer to these questions. Irrespective of the effect of heat shock, the growth of the germtube i s accompanied by the loss of amino acids to the medium. It i s possible that exogenous amino acids arise from either the mechanical rupture or enzymatic degradation of the germtube c e l l wall. Although germtube ly 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. The break down of the germtube wall would f a c i l i t a t e the loss of amino acids as well as other c e l l metabolites and enzymes. In addition, i t i s possible that the loss of amino acids to the medium i s the result of a structural or chemical defect in the c e l l membrane. A membrane defect may lead to the loss of selective permeability and/or an alteration in amino acid transport systems. Scott and Maclean (1969) have suggested that the rust fungi resemble mammalian c e l l cultures in 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 in the hyphal membrane (Scott and Maclean 1969). If this assumption i s valid i t may explain the high uredospore density usually required for i n i t i a t i n g normal saprophytic growth. It i s well known that radioactive amino acids fed to non-shocked germinating uredospores are taken up and incorporated into protein (Shaw 1964, Shaw et a l . 1985). At high c e l l densities the leaked nutrient can reenter another c e l l in close proximity, thus maintaining an effective intercellular level by cross feeding between c e l l s . At low c e l l densities a metabolite may be lost 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 build up an extracellular concentration of the metabolite that i s in equilibrium with the minimum intracellular level, before the c e l l s die of a specific nutritional deficiency (Eagle and Piez 1962). Amino acids have important physiological roles which govern many metabolic processes (e.g. growth and enzyme acti v i t y ) . A number of interesting avenues of research have come to this authors attention during this study: the effect of exogenous amino acids on germination and differentiation; the effect of some amino acids (pathway products) on extractable enzyme ac t i v i t i e s ; the effect of exogenous amino acids on enzyme activity (derepression/repression of genes); and to explore the phenomenon of growth inhibition due to amino acid imbalances. Although research i s being pursued in 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 activation of protein synthesis during germination and the formation of infection structures has not been well documented. Given the information contained in the present study i t would be of interest to determine the amount of charged tRNA for each amino acid. To significantly affect protein synthesis the absence of a free amino acid would result in the absence of the aminoacyl-tRNA. Although some growth of several strains of wheat stem rust has been obtained axenically, only the Australian race, 126-ANZ-67, cultured by William's group (1966, 1967) has been found to grow vigorously. Analyses of the amino acid composition of the leachate may provide a useful basis for formulating the amino acid component of media for the axenic culture of stem rust. Moreover, i t would also be of interest to extract and purify the free amino acid fraction from germinating uredospores. This fraction could be used to provide the amino acid component of an axenic medium for 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 infection structures was augmented by n-nonyl alcohol. (2) A precisely timed heat shock and exogenous nutrients stimulate differentiation. This stimulus i s most effective in the presence of n-nonyl alcohol. (3) The heat shock temperature required to induce maximum differentiation has a very precise optimum. Variations one degree above or below this optimum for a given spore lot reduced the percent differentiation by greater than 40%. (4) Although the optimum temperature of the heat shock varied slightly depending on the particular spore lot the sensitivity of sporeling development to temperature changes i s remarkably constant. (5) Compared to Ca-K buffer, MPG was a superior germination and differentiation medium. (6) Ca-K buffer was the most effective component of MPG. (7) Uredosporeling differentiation occured as a series of precisely timed morphological, cytological, and physiological events. (8) DAPI staining i s a simple, rapid, and reproducible way to assess nuclear behaviour in P. graminis t r i t i c i uredosporelings. (9) Nuclear division was a regular event in differentiating uredospores. Its occurrence was closely associated with the timing of infection structure development. 108 (10) Nuclear division was a rare event in germinating (non-shocked) uredospores. (11) Essential, presumably differentiation-specific proteins, were synthesized from the onset of germination to the completion of differentiation. These proteins are required for the formation of appressoria, vesicles and, infection hyphae. (12) Spore germination was accompanied by a rapid decrease in the glutamic:aspartic acid ratio (from 20:1 to 3:1). (13) There i s a net hydrolysis of protein during germination, leading to an increase in size of the endogenous pool of free amino acids and to an increased leakage of amino acids to the germination medium. (14) Relative to non-shocked uredosporelings, heat shock decreased both the size of the endogenous pool of amino acids and the extent to which uredosporelings lose amino acids to the medium. (15) There was no net protein synthesis during the formation of infection structures induced by heat shock. (16) A high proportion of the total amino acid pool was lost to the medium during germination, particularly in non-shocked uredosporelings. (17) Free cysteine was detected in the leachate isolated from non-shocked sporelings only. (18) The loss of amino acids to the germination medium i s selective, particularly in heat shocked uredosporelings. (19). The composition of the amino acid pool in the leachate may be a useful guide in formulating media for the axenic culture of P. graminis t r i t i c i . 109 7. LITERATURE CITED Allen, R.F. 1923. A cytological study of infection of Baart and Kanred wheats by Puccinia graminis t r i t i c i . J. Agric. Res. 23: 131-152. Allen, P.J. 1972. 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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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