Biotechnology for Crop Protection - American Chemical Society

Chapter 6

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch006

Preinfection Changes in Germlings of a Rust Fungus Induced by Host Contact 1

2

Richard C. Staples and Harvey C. Hoch 1

Boyce Thompson Institute, Cornell University, Ithaca, NY 14853 New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456

2

Germlings of the bean rust fungus, Uromyces appendiculatus, invade their host plant through stomates using a set of specialized cells, the infection structures, that are stimulated to develop in response to a thigmotropic stimulus provided by the l i p of the stomatal guard c e l l . The essential thigmotropic signal is a ridge or groove in the substrate having an abrupt change in elevation of 0.5 μm. Development of the infection structures is accomplished in three important phases, i . e . a very rapid cessation of germ tube elongation, the start of DNA replication, and development of the infection structures. Development of the infection structures includes two rounds of nuclear division, the synthesis of about 15 differentiation-related proteins, and the expression of at least five differentiation-specific genes. R u s t f u n g i a r e b a s i d i o m y c e t e s which i n n a t u r e a r e o b l i g a t e p a r a s i t e s o f h i g h e r p l a n t s , i . e . r e q u i r e l i v i n g h o s t s t o complete t h e i r l i f e c y c l e . They a r e s e r i o u s p e s t s o f c r o p p l a n t s . While s e v e r a l o f t h e r u s t s have been c u l t u r e d ( 1 ) , s u r v i v a l o f a x e n i c r u s t s i s poor o u t s i d e t h e l a b o r a t o r y . T h e i r v e r y complex l i f e cycles, l o n g - c y c l e r u s t s have f i v e t y p e s o f s p o r e s w i t h t h e s e x u a l and a s e x u a l s t a g e s on d i f f e r e n t h o s t s (2) -- pose f a s c i n a t i n g q u e s t i o n s o f h o s t s e l e c t i v i t y , spore i n i t i a t i o n , e n v i r o n m e n t a l r e g u l a t i o n o f t h e l i f e c y c l e , and h o s t cues f o r f u n g a l p e n e t r a t i o n . Any o f t h e s e , were more known about them, c o u l d s e r v e as a f o c u s o f c o n t r o l s t r a t e g i e s . Uredospores a r e a s e x u a l , d i k a r y o t i c s p o r e s , many s p e c i e s o f w h i c h c o l o n i z e t h e i r h o s t s b y s e e k i n g and p e n e t r a t i n g o n l y v i a t h e stomates. T h i s r e v i e w d e s c r i b e s t h e d e v e l o p m e n t a l responses i n d u c e d i n uredospore g e r m l i n g s by h o s t c o n t a c t w h i c h g u i d e s a p p r e s s o r i u m placement over t h e s t o m a t a l opening. Many r e s e a r c h e r s have c o n t r i b u t e d t o these i d e a s b e g i n n i n g w i t h t h e i n v e n t i v e work o f Sidney D i c k i n s o n ( 3 ) . Examples o f o t h e r 0097-6156/88/0379-0082$06.00A) • 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

STAPLES AND HOCH

Preinfection Changes in Germlings

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i m p o r t a n t c o n t r i b u t i o n s t o an u n d e r s t a n d i n g o f d i f f e r e n t i a t i o n i n c l u d e r e s e a r c h by Heath and Heath ( 4 , 5 ) , Maheshwari e t a l . ( 6 ) , R o w e l l and O l i e n ( 7 ) , and Wynn ( 8 ) . The most r e c e n t t r e a t i s e on the r u s t f u n g i i s one on c e r e a l r u s t s by B u s h n e l l and R o e l f s (2.1Û)· E x t e n s i v e r e v i e w s on the d i f f e r e n t i a t i o n p r o c e s s i n c l u d e those by Emmett and P a r b e r y , ( 1 1 ) , L i t t l e f i e l d and Heath ( 2 ) , Wynn and S t a p l e s ( 1 2 ) , and Hoch and S t a p l e s ( 1 3 ) . C y t o l o g y o f I n f e c t i o n S t r u c t u r e Development Morphology. Uredospores o f many o f the r u s t s c o l o n i z e t h e i r h o s t s by s e e k i n g and p e n e t r a t i n g the stomates. The r i d g e s and i n d e n t a t i o n s o f the l e a f a r e s i g n a l s w h i c h c o n t r o l b o t h the d i r e c t i o n o f growth o f the germ tube ( o r i e n t a t i o n ) , and t h e s i t e of a p p r e s s o r i u m development f o r p e n e t r a t i o n ( d i f f e r e n t i a t i o n ) . These f u n g a l responses t o c o n t a c t s t i m u l i a r e growth changes. O r i e n t a t i o n . The o r i e n t a t i o n response by Uromvces a p p e n d i c u l a t u s g e r m l i n g s on bean l e a v e s i n an a r e a where t h e s u r f a c e i s r e g u l a r l y s t r i a t e d i s shown i n F i g . 1A. U n i f o r m l y spaced s c r a t c h e s on p l a s t i c s u r f a c e s a l s o induce germ tube o r i e n t a t i o n ( F i g . I B ) . O r i e n t a t i o n i s the f i r s t v i s u a l e f f e c t o f s e n s i n g by the u r e d o s p o r e g e r m l i n g , and o r i e n t a t i o n has been p o s t u l a t e d t o improve the chance t h a t a germ tube w i l l c o n t a c t a stomate on the h o s t l e a f by r e d u c i n g wandering ( 1 4 ) . D i f f e r e n t i a t i o n . When the germ tube c o n t a c t s a stomate, the g e r m l i n g undergoes a p r o c e s s o f d i f f e r e n t i a t i o n i n w h i c h a s e r i e s of i n f e c t i o n s t r u c t u r e s a r e formed b e g i n n i n g w i t h the a p p r e s s o r i u m w h i c h s i t s over the s t o m a t a l opening ( F i g . 2 ) . Development o f the a p p r e s s o r i u m i s f o l l o w e d by the peg, w h i c h p e n e t r a t e s between the guard c e l l s , the v e s i c l e . w h i c h f i l l s the s u b s t o m a t a l c a v i t y , and the i n f e c t i o n hvphae. which r a m i f y t h r o u g h o u t the l e a f t i s s u e . H a u s t o r i a l mother c e l l s d e v e l o p when t h e i n f e c t i o n hyphae e n c o u n t e r the m e s o p h y l l o f the h o s t . I t i s the h a u s t o r i a t h a t d e v e l o p w i t h i n the m e s o p h y l l c e l l s , and w h i c h o b t a i n nourishment f o r t h e fungus. H a u s t o r i a l mother c e l l s a r e thought t o a r i s e o n l y when the i n f e c t i o n hyphae make c o n t a c t w i t h h o s t c e l l s ; however, h a u s t o r i a l mother c e l l s do d e v e l o p o c c a s i o n a l l y i n t h e absence o f t h e h o s t (15). A l l o f the i n f e c t i o n s t r u c t u r e s p r i o r t o the h a u s t o r i a l mother c e l l s d e v e l o p as a r e s u l t o f the o r i g i n a l c o n t a c t by the g e r m l i n g w i t h the stomate. Chemicals can a l s o induce i n f e c t i o n s t r u c t u r e s , and each fungus seems t o have i t s own r e q u i r e m e n t . For example, P u c c i n i a g r a m i n i s t r i t i c i responds t o a c r o l e i n ( 1 6 ) , w h i l e U. a p p e n d i c u l a t u s responds t o K ( 1 7 ) . The t h i g m o t r o p i c response by U. a p p e n d i c u l a t u s t o the stomate, however, i s e n t i r e l y p h y s i c a l as demonstrated by Wynn ( 8 ) . +

Dimensions

o f the S i g n a l

D i f f e r e n t i a t i o n can be r e a d i l y i n d u c e d by a s i n g l e s c r a t c h ( 1 8 ) . T h i s o b s e r v a t i o n l e d t o the p r e p a r a t i o n o f t e m p l a t e s



BIOTECHNOLOGY FOR CROP PROTECTION

F i g u r e 1. Photomicrographs showing t h e development o f U. a p p e n d i c u l a t u s uredospore g e r m l i n g s and t h e s t o m a t a l pore o f the bean l e a f . (A) Growth o f U. a p p e n d i c u l a t u s g e r m l i n g s on the s u r f a c e o f t h e h o s t p l a n t (Phaseolus v u l g a r i s ) showing t h e o r i e n t a t i o n response b y t h e germ tubes t o t h e topography o f t h e l e a f . B a r s c a l e , 10 μπι. A f t e r Hoch e t a l . ( 1 9 ) . (Β) Growth o f U. a p p e n d i c u l a t u s g e r m l i n g s on t h e i n d u c t i v e s u r f a c e o f h i g h - d e n s i t y p o l y e t h y l e n e . The germ tubes a r e o r i e n t e d p e r p e n d i c u l a r t o t h e i n h e r e n t f e a t u r e s formed on t h e s u r f a c e . A p p r e s s o r i a have begun t o form on some o f t h e germ tubes (4 h a f t e r s t a r t o f g e r m i n a t i o n ) . X190. A f t e r S t a p l e s e t a l . (34)· (C) P o l y s t y r e n e r e p l i c a o f p r e c i s i o n i o n - e t c h e d s i l i c o n w a f e r t e m p l a t e s , c o n t a i n i n g r i d g e s 0.5 μπι h i g h by 4.0 μπι wide, was h i g h l y i n d u c t i v e f o r i n f e c t i o n s t r u c t u r e f o r m a t i o n i n U. appendiculatus germlings. B a r s c a l e , 10 μπι. A f t e r Hoch e t a l . (19). (D) G e r m l i n g o f U. a p p e n d i c u l a t u s s t a i n e d f o r m i c r o t u b u l e s ( l e f t ) , and F - a c t i n ( r i g h t ) . Some o f t h e m i c r o t u b u l e and F - a c t i n m i c r o f i l a m e n t p r o f i l e s (Mf) occupy a d j a c e n t s i t e s . An arrow p o i n t s t o one o f t h e two F - a c t i n c o n t a i n i n g n u c l e a r i n c l u s i o n s ( N i ) i n t h e n u c l e i (one i n each n u c l e u s ) . A c t i n - c o n t a i n i n g p e r i p h e r a l plaques (Pp) a r e a l s o shown. X1250. A f t e r S t a p l e s e t a l . ( 3 4 ) . (Ε) S c a n n i n g e l e c t r o n m i c r o g r a p h o f bean l e a f s t o m a t a l guard c e l l s (Gc) h a v i n g prominent e r e c t l i p s (Lp) t h a t s e r v e as t h e s i g n a l f o r a p p r e s s o r i u m f o r m a t i o n i n U. a p p e n d i c u l a t u s . The fungus e v e n t u a l l y e n t e r s t h e l e a f through t h e s t o m a t a l pore (Po) where i n f e c t i o n o f t h e h o s t c e l l o c c u r s . B a r s c a l e , 10 μπι. Ap, a p p r e s s o r i u m ; G t , germ tube, Sp, uredospore. A f t e r Hoch e t a l .

(1£).



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F i g u r e 2. I n f e c t i o n s t r u c t u r e s d e v e l o p e d by u r e d o s p o r e g e r m l i n g s o f U. a p p e n d i c u l a t u s . (A) Diagram o f t h e i n f e c t i o n s t r u c t u r e s d e v e l o p e d a f t e r germ tube c o n t a c t w i t h a s t o m a t a l guard c e l l . (B) SEM m i c r o g r a p h showing a p p r e s s o r i u m d e v e l o p e d o v e r a bean l e a f stomate. (C) T y p i c a l i n f e c t i o n s t r u c t u r e s o f U. a p p e n d i c u l a t u s . X400. Ap, a p p r e s s o r i u m ; Gt, germ tube; I h , i n f e c t i o n hyphae; I p , peg; Se, septum; Sp, s p o r e ; Ve, v e s i c l e .


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m i c r o f a b r i c a t e d onto s i l i c o n w a f e r s u s i n g e l e c t r o n - b e a m l i t h o g r a p h y ( 1 9 ) . The d i f f e r e n t i a t i o n s i g n a l was found t o be a s i m p l e r i d g e h a v i n g an optimum h e i g h t o f 0.5 + 0.1 μπι ( F i g . 1C). N e g a t i v e r i d g e s (grooves) a r e e q u a l l y e f f e c t i v e . O r i e n t a t i o n and d i f f e r e n t i a t i o n a r e s e p a r a t e r e s p o n s e s . W h i l e d i f f e r e n t i a t i o n o c c u r s i f t h e r i d g e i s s o l i t a r y and w e l l s e p a r a t e d from o t h e r r i d g e s , i t was found t h a t germ tubes o f t h e fungus a r e h i g h l y o r i e n t e d when m u l t i p l e r i d g e s a r e p r e s e n t w h i c h are spaced more c l o s e l y t h a n 15 μπι. D i f f e r e n t i a t i o n i s i n h i b i t e d and o r i e n t a t i o n i s enhanced as t h e s p a c i n g i s d i m i n i s h e d . The h e i g h t o f t h e d i f f e r e n t i a t i o n s i g n a l i s s m a l l compared w i t h t h e d i a m e t e r o f t h e germ tube w h i c h averages 5 t o 8 μπι ( F i g . 3). I t i s o b v i o u s t h a t t h e fungus i s a b l e t o d i s t i n g u i s h minute d i f f e r e n c e s i n l e a f s u r f a c e topography (perhaps as s m a l l as + 0.1 μπι) i n o r d e r t o t a r g e t t h e stomate. Wynn (8) h a s s u g g e s t e d t h a t on t h e bean p l a n t , t h e l i p o f t h e s t o m a t a l g u a r d c e l l was t h e 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 i g n a l t o w h i c h t h e U. a p p e n d i c u l a t u s g e r m l i n g responded. Photographs by SEM o f specimens o f t h e bean stomate w h i c h were q u i c k f r o z e n t h e n f r e e z e - d r i e d , r e v e a l e d prominent guard c e l l l i p s o r i e n t e d n e a r l y p e r p e n d i c u l a r l y t o t h e c e l l s u r f a c e ( F i g . I E ) . The l i p f r e q u e n t l y appeared t o be somewhat ragged a l o n g t h e o u t e r edge, and non u n i f o r m i n h e i g h t . Mean h e i g h t o f t h e l i p was 0.487 + 0.07 μπι, and we assume w i t h Wynn (8) t h a t t h e 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 i g n a l on t h e bean l e a f i s t h e l i p o f t h e s t o m a t a l guard c e l l . R i d g e s 0.5 μπι i n h e i g h t , so s u i t a b l e f o r i n d u c t i o n o f d i f f e r e n t i a t i o n by t h e U. a p p e n d i c u l a t u s g e r m l i n g , a r e n o t i n d u c t i v e f o r g e r m l i n g s o f P. g r a m i n i s t r i t i c i . t h e wheat stem r u s t fungus (Hoch, u n p u b l i s h e d i n f o r m a t i o n ) , and n e i t h e r were Wynn's p l a s t i c r e p l i c a s o f t h e bean l e a f stomate ( 1 2 ) . W h i l e a p p r e s s o r i a a r e i n d u c e d on g e r m l i n g s o f P. g r a m i n i s t r i t i c i by c o n t a c t w i t h t h e stomate ( 2 0 ) , t h e stomates a r e q u i t e d i f f e r e n t i n shape. U s i n g m e t h y l m e t h r a c y l a t e spheres mixed i n t o a p o l y s t y r e n e membrane, D i c k i n s o n (3) e s t i m a t e d t h a t t h e s i g n a l f o r P. r e c o n d i t a ( l e a f r u s t o f wheat) c o n s i s t s o f m u l t i p l e s m a l l r i d g e s about 0.0012 μπι i n h e i g h t and spaced about 0.12 μπι a p a r t . These e s t i m a t i o n s have n o t y e t been v e r i f i e d u s i n g e t c h e d w a f e r s , i . e . a g g r e g a t e s o f t h e g r a n u l e s ( l o n g e r d i m e n s i o n s ) may have been i n d u c t i v e ; however, D i c k i n s o n d i d demonstrate t h a t membranes c o n t a i n i n g s m a l l e r g r a n u l e s were n o t e f f e c t i v e . As t h e s e d i m e n s i o n s a r e q u i t e d i f f e r e n t from t h o s e f o r U. a p p e n d i c u l a t u s . and inasmuch as o u r w a f e r s would n o t i n d u c e P. g r a m i n i s t r i t i c i g e r m l i n g s t o d i f f e r e n t i a t e , t h e d e v e l o p m e n t a l s i g n a l s appear t o d i f f e r among t h e r u s t s . The p o s s i b i l i t y e x i s t s t h a t s m a l l changes i n t h e a r c h i t e c t u r e o f t h e l e a f would be a u s e f u l s t r a t e g y f o r r e d u c i n g t h e v i r u l e n c e o f t h e pathogen. The T h i g m o t r o p i c Response T i m e - s c a l e o f Response. A l i s t o f some o f t h e e v e n t s w h i c h o c c u r d u r i n g t h e development o f i n f e c t i o n s t r u c t u r e s , and t h e e s t i m a t e d times when t h e response o c c u r s a f t e r c o n t a c t w i t h t h e i n d u c t i v e


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r i d g e , i s shown i n T a b l e I . The f o l l o w i n g d i s c u s s i o n i s o r i e n t e d around t h e t i m e - s c a l e w h i c h was assembled from a v a r i e t y o f s o u r c e s f o r t h i s purpose.


Table I .

Time S c a l e o f D i f f e r e n t i a t i o n F o r Bean R u s t Uredospore Germlings^

Minutes 0 200 CONSTRUCTION OF VESICLE EXPRESSION OF LATE DS-GENES + SYNTHESIS OF LATE DS-PROTEINS + MIGRATION OF NUCLEI + MITOSIS I I (- 8 NUCLEI) T h e s e t i m e s a r e approximate o n l y , and were assembled from e x p e r i m e n t s c a r r i e d o u t f o r a v a r i e t y o f d i f f e r e n t p u r p o s e s . The d a t a on m i t o s i s was o b t a i n e d from Tucker e t a l . ( 1 8 ) ; DNA r e p l i c a t i o n I (15 min) from S t a p l e s e t a l . ( 2 1 ) . The morphogenetic pause and m i t o s i s I I were e s t i m a t e d from t h e b e h a v i o r o f a p o p u l a t i o n o f s p o r e s (DS, d i f f e r e n t i a t i o n - s p e c i f i c ) . 1

E a r l y Response P e r i o d . The germ tube e l o n g a t e s o v e r t h e s u r f a c e a t 1 t o 2 μπι/ιηΐη. As t h e w i d t h o f t h e s t o m a t a l o p e n i n g on a bean l e a f u s u a l l y i s n o t g r e a t e r t h a n 3 μπι, t h e germ tube must respond i n l e s s t h a n a minute i n o r d e r n o t t o o v e r s h o o t t h e opening. That the g e r m l i n g u s u a l l y does n o t o v e r s h o o t c a n be seen i n F i g . 1C, where a l l o f t h e a p p r e s s o r i a a r e r e s t i n g on top o f t h e s i g n a l r i d g e w h i c h i s 4 μπι wide. Thus, t h e i n i t i a l response t o t h e s i g n a l i s r a p i d , and as shown i n t h e f i g u r e , where a n o v e r s h o o t has o c c u r r e d , t h e fungus h a s a c o r r e c t i v e mechanism f o r r e t r a c t i n g o v e r t h e t a r g e t g u i d e d b y t h e s i g n a l . N e i t h e r t h e mechanism b y w h i c h t h e s i g n a l i s p e r c e i v e d b y t h e g e r m l i n g , n o r t h e manner b y w h i c h i t responds, i s known y e t ; however, i t seems u n l i k e l y t h a t gene e x p r e s s i o n i s i n v o l v e d . DNA R e p l i c a t i o n and M i t o s i s . One o f t h e e a r l i e s t r e s p o n s e s t o c o n t a c t i s t h e s t a r t o f DNA r e p l i c a t i o n and m i t o s i s ( 2 1 ) . The n u c l e i i n t h e germ tube a r e h a p l o i d , and r e p l i c a t i o n b e g i n s about 15 m i n a f t e r i n d u c t i o n o f d i f f e r e n t i a t i o n ( T a b l e I ) . N u c l e a r d i v i s i o n r e q u i r e s about 15 min, and i s completed 30 m i n b e f o r e t h e a p p r e s s o r i a l septum i s completed ( T a b l e I ; 1 8 ) .


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M o r p h o g e n e t i c Pause. A t the end o f t e l o p h a s e , the g e r m l i n g c o n s t r u c t s a septum a c r o s s the germ tube g u i d e d by a s e p t a l r i n g o f F - a c t i n ( 1 8 ) . When t h i s a c t i v i t y c e a s e s , the g e r m l i n g appears t o r e m a i n q u i e s c e n t f o r about an hour o r so. A c t i v i t y b e g i n s a g a i n when the v e s i c l e e l o n g a t e s out from the a p p r e s s o r i u m . P r o t e i n S y n t h e s i s and Gene E x p r e s s i o n . Development o f the i n f e c t i o n s t r u c t u r e s i s accompanied by the s y n t h e s i s o f a t l e a s t 15 d i f f e r e n t i a t i o n - r e l a t e d ( d r ) p r o t e i n s , i . e . p r o t e i n s n o t p r e s e n t i n the g e r m l i n g u n t i l d i f f e r e n t i a t i o n i s i n d u c e d F i g . 4; (22) . A d o w n s h i f t i n the s y n t h e s i s o f some p r o t e i n s a l s o o c c u r s d u r i n g i n f e c t i o n s t r u c t u r e development. To address the q u e s t i o n o f the number o f genes e x p r e s s e d d u r i n g development o f the i n f e c t i o n s t r u c t u r e s , we have s c r e e n e d a bean r u s t genomic l i b r a r y u s i n g a probe e n r i c h e d f o r the d r sequences p r e p a r e d by a m o d i f i e d cascade h y b r i d i z a t i o n procedure (23) . We have o b t a i n e d twenty d i f f e r e n t i a t i o n - s p e c i f i c c l o n e s . From r e s t r i c t i o n mapping and Southern h y b r i d i z a t i o n a n a l y s e s , t h e s e c l o n e s can be grouped i n t o s i x c l a s s e s . G l a s s I c o n s i s t s o f f o u r t e e n homologous c l o n e s , c l a s s I I c o n s i s t s o f two homologous c l o n e s , and c l a s s e s I I I t o V I c o n s i s t o f a s i n g l e c l o n e each. N o r t h e r n a n a l y s e s , used t o c o n f i r m t h a t t h e s e c l o n e s were s p e c i f i c t o the d i f f e r e n t i a t e d s t a g e o f the fungus, showed t h a t c l a s s I c l o n e s h y b r i d i z e d w i t h t r a n s c r i p t s t h a t were e q u a l l y abundant i n d i f f e r e n t i a t e d and n o n d i f f e r e n t i a t e d g e r m l i n g s . The r e m a i n i n g c l a s s e s , however, h y b r i d i z e d w i t h t r a n s c r i p t s t h a t were more abundant i n d i f f e r e n t i a t e d g e r m l i n g s . Thus, i t appears t h a t the c o n t a c t s t i m u l u s a c t i v a t e s a s m a l l gene s e t o f a t l e a s t f i v e genes t h a t a r e d e d i c a t e d t o the d i f f e r e n t i a t i o n p r o c e s s . We have a l s o o b t a i n e d c l o n e s o f genes whose e x p r e s s i o n i s d o w n s h i f t e d , b u t we have n o t c h a r a c t e r i z e d them y e t . U s i n g h y b r i d a r r e s t - t r a n s l a t i o n p r o c e d u r e s , we have i d e n t i f i e d a 23 kDa p o l y p e p t i d e w h i c h i s coded f o r by c l o n e 24 o f c l a s s I I . One o f the d r - p r o t e i n s has an approximate m o l e c u l a r w e i g h t o f 23 kDa; however, i t s i d e n t i t y w i t h t h i s p e p t i d e must a w a i t the p r e p a r a t i o n o f a s p e c i f i c a n t i b o d y . A l t h o u g h the s t u d i e s on homology a r e s t i l l r a t h e r l i m i t e d , the d i f f e r e n t i a t i o n - s p e c i f i c c l o n e s appear t o be homologous o n l y w i t h DNA from o t h e r r u s t s and not w i t h DNA from more d i s t a n t l y r e l a t e d f u n g i . So f a r , we have probed a BamHl d i g e s t o f t o t a l DNA from two r u s t s (Uromyces v i g n a e . and U. a p p e n d i c u l a t u s ) . two p l a n t p a t h o g e n i c ascomyces w h i c h develop a p p r e s s o r i a on t h e i r h o s t s ( C o c h l i o b o l o u s he terο s t r o p h u s and C o l l e t o t r i c h u m 1indemuthianum) and the s a p r o p h y t e , A s p e r g i l l u s n i d u l a n s . w h i c h does n o t d i f f e r e n t i a t e . Three c l o n e s r e p r e s e n t i n g c l a s s e s I I , IV and V I , h y b r i d i z e d t o DNA from the two r e l a t e d r u s t s b u t n o t t o the DNA from the o t h e r t h r e e more d i s t a n t l y - r e l a t e d f u n g i . A l l o f the c l o n e s i s o l a t e d so f a r h y b r i d i z e w i t h t r a n s c r i p t s w h i c h appear i n g e r m l i n g s a t the time when the v e s i c l e s form, a l t h o u g h the c l a s s V I c l o n e a l s o h y b r i d i z e s w i t h a t r a n s c r i p t w h i c h appears d u r i n g development o f the a p p r e s s o r i u m . Thus, the ds-genes appear t o have a r o l e i n c o n s t r u c t i o n o f the i n f e c t i o n s t r u c t u r e s . A more p r e c i s e t i m i n g o f e x p r e s s i o n w i l l have t o await a d d i t i o n a l s t u d i e s .



STAPLES AND HOCH


Figure 3. A developing appressorium sectioned transversely to the d i r e c t i o n of a scratch on a Mylar substrate which was previously coated with a t h i n layer of palladium/gold. X7.300. A f t e r Bourett et a l . (35).

Figure 4. Autoradiograms of proteins separated by e l e c t r o p h o r e s i s on two dimensional slab gels of 15% polyacrylamide. An ampholine-generated pH gradient was employed i n the f i r s t dimension, while a 15% SDS gel was employed i n the second dimension. The p o s i t i o n of the molecular weight markers (kDa) i s shown along the left-hand margin. Uredospore germlings on c o l l o d i o n membranes were misted with [ S]-methionine f o r 1.5 h before harvest and e x t r a c t i o n of the p r o t e i n s . (A) Nondifferentiated uredospores germinated 6 h, (B) d i f f e r e n t i a t e d uredospores germinated 6 h with appressoria and v e s i c l e s . P a r a f f i n o i l was incorporated into the c o l l o d i o n membrane to induce d i f f e r e n t i a t i o n . Underscored numbers i n d i c a t e proteins synthesized s p e c i f i c a l l y during d i f f e r e n t i a t i o n . Not a l l of the proteins have been numbered. 35


90



Elements o f the S e n s i n g Mechanism E x t r a c e l l u l a r M a t r i x . U n l e s s g e r m l i n g s o f the r u s t f u n g i a r e a t t a c h e d t o h o s t s u r f a c e s , i n f e c t i o n s t r u c t u r e s e i t h e r do n o t d e v e l o p o r a r e a b n o r m a l l y o r i e n t e d ( 1 2 ) . P r o t e i n i n the e x t r a c e l l u l a r m a t r i x o f the germ tube appears t o be an i m p o r t a n t component o f the s e n s i n g mechanism because p r o t e a s e s , e.g. Pronase E, b u t n o t o t h e r h y d r o l a s e s , l o o s e n the germ tube from the s u r f a c e and p r e v e n t t h i g m o d i f f e r e n t i a t i o n (24,25). Pronase Ε d i d n o t a f f e c t g e r m i n a t i o n o r g e r m l i n g growth, b u t a d h e s i o n t o a p o l y s t y r e n e s u r f a c e was s i g n i f i c a n t l y reduced. The s i m p l e s t e x p l a n a t i o n f o r the e f f e c t s o f the p r o t e a s e s i s t h a t e x t r a c e l l u l a r p r o t e i n s , p o s s i b l y g l y c o p r o t e i n s , a r e r e q u i r e d f o r b i n d i n g the germ tube t o the i n d u c t i v e s u r f a c e . The e x t r a c e l l u l a r m a t r i x appears t o be amorphous by TEM, and n o t h i n g i s known y e t about i t s p o s s i b l e a s s o c i a t i o n w i t h the plasmalemma o r i n t r a c e l l u l a r components. C y t o s k e l e t o n . U. a p p e n d i c u l a t u s g e r m l i n g s have an e x t e n s i v e network o f c y t o p l a s m i c m i c r o t u b u l e s o r i e n t e d p a r a l l e l w i t h the l o n g a x i s o f the hypha t h a t a r e e s p e c i a l l y abundant n e a r the h y p h a - s u b s t r a t e i n t e r f a c e F i g . ID, l e f t ; ( 2 6 ) . M i c r o t u b u l e d i s r u p t i n g agents such as c o l d , demecolcine, g r i s e o f u l v i n , and n o c o d a z o l e , e f f e c t i v e l y d e p o l y m e r i z e the m i c r o t u b u l e s i n the germ tube b u t n o t the a c t i n m i c r o f i l a m e n t s ( 2 7 ) . Enhanced m i c r o t u b u l e a r r a y s a r e o b s e r v e d i n the presence o f e i t h e r T a x o l o r D2O, agents known t o f a v o r m i c r o t u b u l e s t a b i l i z a t i o n (2j$). F - a c t i n i s observed i n t h r e e i m p o r t a n t c o n f i g u r a t i o n s : as f i l a m e n t s , a s s o c i a t e d w i t h f i l a s o m e s , and p o s i t i o n e d w i t h i n the n u c l e a r m a t r i x as an i n c l u s i o n ( F i g . ID, r i g h t ) . Treatment o f the g e r m l i n g s w i t h c y t o c h a l a s i n Ε l e a d s t o the d i s a p p e a r a n c e o f c y t o p l a s m i c f i l a m e n t s b u t has no p e r c e i v a b l e e f f e c t s on the filasomes (22). What p o s s i b l e r o l e may the c y t o s k e l e t o n have i n t r a n s m i s s i o n o f the d i f f e r e n t i a t i o n s i g n a l ? The e v i d e n c e i s o n l y c i r c u m s t a n t i a l so f a r . The l o c a t i o n o f the m i c r o t u b u l e s n e a r the c e l l - s u b s t r a t e i n t e r f a c e means t h a t the m i c r o t u b u l e s a r e l o c a t e d where they ought t o be t o have a r o l e i n r e c e p t i o n . Furthermore, nocodazole- o r g r i s e o f u l v i n - t r e a t e d g e r m l i n g s , i n w h i c h the m i c r o t u b u l e s a r e d e p o l y m e r i z e d , f a i l t o develop a p p r e s s o r i a when the germ tube c o n t a c t s a s u r f a c e s c r a t c h (Hoch, H.C, u n p u b l i s h e d ) . T h i s suggests t h a t the m i c r o t u b u l e s must be i n t a c t i n o r d e r f o r the g e r m l i n g t o respond t o an i n d u c t i v e s i g n a l . The m i c r o f i l a m e n t c y t o s k e l e t o n must a l s o be i n t a c t f o r d i f f e r e n t i a t i o n t o o c c u r , b u t i t p r o b a b l y does n o t have a d i r e c t r o l e i n s i g n a l t r a n s d u c t i o n as p o s t u l a t e d f o r the m i c r o t u b u l e s . For example, Tucker e t a l . (18) have demonstrated t h a t t r e a t m e n t s of germlings w i t h b r i e f pulses of c y t o c h a l s i n Ε which depolymerize the m i c r o f i l a m e n t s , and c o n s e q u e n t l y d i s t o r t the shape o f the germ tube, do n o t i n h i b i t the s t a r t o f m i t o s i s i n the a p p r e s s o r i u m . Even the t i m e - c o u r s e was n o t a l t e r e d . Removal o f the drug r e s t o r e s c e l l shape. However, development o f the a p p r e s s o r i a l


6.

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septum no l o n g e r o c c u r s , so t h a t c o m p l e t i o n o f a p p r e s s o r i u m c o n s t r u c t i o n p r o b a b l y does depend on the i n t e g r i t y o f the microfilaments. Transmembrane S i g n a l i n g . H i g h exogenous l e v e l s (10 mM) o f b o t h cAMP and cGMP i n d u c e DNA r e p l i c a t i o n and n u c l e a r d i v i s i o n i n U. a p p e n d i c u l a t u s g e r m l i n g s e f f i c i e n t l y , b u t the i n f e c t i o n s t r u c t u r e s are i n h i b i t e d from f o r m i n g ( 2 9 ) . However, more modest l e v e l s (10"7 Μ ) , as w e l l as exogenously a p p l i e d s t i m u l a t o r s o f a d e n y l a t e c y c l a s e and i n h i b i t o r s o f cAMP-dependent phosphatase, i n d u c e the complete sequence o f DNA r e p l i c a t i o n , n u c l e a r d i v i s i o n and development o f the i n f e c t i o n s t r u c t u r e s . The d a t a s u g g e s t t h a t U. a p p e n d i c u l a t u s g e r m l i n g s u t i l i z e a cAMP- and cGMP-dependent cascade i n m e t a b o l i s m , i n c l u d i n g a c y c l i c n u c l e o t i d e - d e p e n d e n t p r o t e i n k i n a s e w h i c h p h o s p h o r y l a t e s a 54 kDa p r o t e i n ( E p s t e i n , unpublished). We have been u n a b l e t o d i s t i n g u i s h between the n u c l e o t i d e s as the p r e f e r r e d messenger. P h o s p h a t i d i c a c i d (PA), r a p i d l y produced d u r i n g r e c e p t o r s t i m u l a t e d breakdown o f p h o s p h o i n o s i t i d e s , m o b i l i z e s C a ^ , r a i s e s the pH o f the c y t o p l a s m , and s t i m u l a t e s DNA s y n t h e s i s i n a c t i v a t e d mammalian c e l l s ( 3 0 ) . R e c e n t l y , we have found t h a t the the d i p a l m a t o y l d e r i v a t i v e o f PA, as w e l l as s e v e r a l d i a c y l g l y c e r o l s , e.g. 1 , 2 - d i p a l m i t o y l - s n - g l y c e r o l , i n d u c e b o t h m i t o s i s and i n f e c t i o n s t r u c t u r e development i n U. a p p e n d i c u l a t u s u r e d o s p o r e s (Staples, unpublished). Furthermore, e x t e r n a l C a ^ a l s o s t i m u l a t e s DNA r e p l i c a t i o n and i n f e c t i o n s t r u c t u r e development, a l t h o u g h the s t i m u l a t i o n i s more e f f e c t i v e i f low l e v e l s o f K (1 mM) are p r e s e n t ( 1 7 ) . I n a d d i t i o n , c a l m o d u l i n , the c a l c i u m r e c e p t o r p r o t e i n , has now been i s o l a t e d from u r e d o s p o r e s ( 3 1 ) , so t h a t a c a l c i u m - r e l a t e d second messenger system a l m o s t c e r t a i n l y i s present. As F a v r e and T u r i a n (3J2) have r e c e n t l y i s o l a t e d p r o t e i n k i n a s e C from m y c e l i a o f Neurospora c r a s s a . the pathway may be common i n f u n g i as w e l l as a n i m a l c e l l s . A t t h i s t i m e , t h e n , i t seems l i k e l y t h a t transmembrane s i g n a l i n g from a s u r f a c e s t i m u l u s c o u l d o c c u r i n U. a p p e n d i c u l a t u s i n c o n j u n c t i o n w i t h a second messenger system, by one o r b o t h o f the c y c l i c n u c l e o t i d e and the p h o s p h o i n o s i t i d y l pathways. G - p r o t e i n s are a f a m i l y o f guanine n u c l e o t i d e - b i n d i n g p r o t e i n s t h a t f u n c t i o n a l l y c o u p l e a wide a r r a y o f membrane r e c e p t o r s t o b i o c h e m i c a l e f f e c t o r systems t h a t r e g u l a t e second messengers ( 3 3 ) . S i n c e b o t h p h y s i c a l s t r e s s e s such as membrane d e p o l a r i z a t i o n and s t r e t c h i n g w i l l i n i t i a t e G - p r o t e i n a c t i v i t y ( 3 3 ) , a system o f G - p r o t e i n - r e l a t e d second messengers w o u l d p r o v i d e a mechanism f o r the v e r y r a p i d response t h a t u r e d o s p o r e g e r m l i n g s make t o t h i g m o t r o p i c s i g n a l s . We n o t e t h a t GTP i s as e f f e c t i v e as K as an i n d u c e r o f d i f f e r e n t i a t i o n U. appendiculatus germlings (Staples, unpublished). +

+

+

+

A

Perspective

Much r e s e a r c h i s now aimed toward an u n d e r s t a n d i n g o f h o s t r e s p o n s e s t o i n f e c t i o n . These responses t y p i c a l l y i n c l u d e v a r i o u s r e s i s t a n c e mechanisms, t o x i n e f f e c t s , i n d u c e d s u s c e p t i b i l i t y , and r a c e s p e c i f i c i t y . The p r e s e n t r e s e a r c h , and the many



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c o n t r i b u t i o n s by others which preceded i t , suggests t h a t host r e c o g n i t i o n , and a d a p t a t i o n t o h o s t morphology b y the pathogen, i s exquisite. The r u s t f u n g i are s e r i o u s t h r e a t s t o c r o p s , and have caused w i d e s p r e a d l o s s e s . Use o f r e s i s t a n t c r o p v a r i e t i e s i s a t p r e s e n t the most r e l i a b l e c o n t r o l measure f o r r u s t s where t h e s e can be d e v e l o p e d ; however, new v a r i e t i e s must be c o n t i n u a l l y d e v e l o p e d i n o r d e r t o p r o v i d e p r o t e c t i o n a g a i n s t v i r u l e n t new r a c e s o f the r u s t pathogen as t h e y a r i s e . The p r e i n f e c t i o n changes i n the r u s t f u n g i t h a t were r e v i e w e d h e r e might be e x p l o i t e d t o p r o v i d e more stable plant protection. For example, t h e r e are two p r e i n f e c t i o n s e n s i n g r e s p o n s e s t h a t might be m a n i p u l a t e d , i . e . o r i e n t a t i o n and d i f f e r e n t i a t i o n . F u r t h e r s t u d y o f how the s y n t h e s i s o f the d i f f e r e n t i a t i o n - r e s p o n s e p r o t e i n s i s c o n t r o l l e d , and o f how the e x p r e s s i o n o f t h e i r p r o t e i n s i s r e g u l a t e d , c o u l d t e l l u s much more about how a p a r a s i t e senses and responds t o i t s h o s t . Acknowledgments The r e s e a r c h b y the a u t h o r s r e v i e w e d h e r e was s u p p o r t e d i n p a r t b y g r a n t s DCB-8401409 and PCM-8315713 from the US N a t i o n a l S c i e n c e Foundation. Literature 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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Cited

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