Phytoalexins and Their Elicitors

Chapter 6

Phytoalexins and Their Elicitors Ν. T. Keen

Microbes and Microbial Products as Herbicides Downloaded from pubs.acs.org by AUBURN UNIV on 04/28/19. For personal use only.

Department of Plant Pathology, University of California—Riverside, Riverside, CA 92521

Phytoalexins are an important component of the plant disease defense reaction called the hypersensitive response. Successful pathogens have evolved methods for dealing with plant phytoalexins, including suppressors of their production, detoxification of the phytoalexins and in some cases avoiding elaboration of substances, called elicitors, that would otherwise initiate the defense reaction. Elicitors obtained from pathogens are of considerable utility for study of various aspects of plant biology because of their interaction with the products of plant disease resistance genes. Substantial information has been obtained on how elicitors are perceived by plant cells and how they function, but much remains to be done. Finally, elicitors may prove of value for the economic production of exotic plant secondary metabolites and as specific herbicides. P h y t o a l e x i n s and e l l d t o r s t h a t s t i m u l a t e t h e i r p r o d u c t i o n by p l a n t t i s s u e s have r e c e i v e d i n c r e a s i n g a t t e n t i o n as a consequence o f several recent developments. F i r s t , a f t e r years of controversy, p h y t o a l e x i n s a r e now g e n e r a l l y a c c e p t e d a s a bona fide mechanism o f d i s e a s e d e f e n s e 1n h i g h e r p l a n t s . Second, race s p e c i f i c e l l d t o r s have been I s o l a t e d t h a t c o n f e r t h e p l a n t s p e c i f i c i t y o f t h e pathogen race w h i c h produces them. T h i s s p e c i f i c i t y I m p l i e s t h a t t h e e l l d t o r s a r e d e t e r m i n a t i v e o f t h e I n t e r a c t i o n s and t h a t t h e y s h o u l d be u s e f u l f o r t h e e l u c i d a t i o n o f r e c o g n i t i o n and s i g n a l t r a n s d u c t i o n mechanisms in plants. T h i r d , r e c e n t e v i d e n c e has i l l u m i n a t e d t h e mechanisms whereby e l l d t o r s l e a d t o d e r e p r e s s i o n o f genes encoding p h y t o a l e x i n b l o s y n t h e t i c enzymes and o t h e r defense response g e n e s . Fourth, p r o g r e s s has o c c u r r e d i n t h e use o f e l i c i t o r s t o produce e x o t i c n a t u r a l p r o d u c t s by p l a n t c e l l c u l t u r e s .

0097-6156/90Λ)439-Ό114$06.00Α) © 1990 American Chemical Society

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D i s e a s e R e s i s t a n c e 1n P l a n t s D i s e a s e d e f e n s e 1n p l a n t s 1s a complex o f mechanisms, I n c l u d i n g a n a t o m i c a l and m o r p h o l o g i c a l f e a t u r e s as w e l l as preformed chemical substances. P h y t o a l e x i n p r o d u c t i o n 1s u s u a l l y a s s o c i a t e d w i t h a p l a n t d e f e n s e system c a l l e d the h y p e r s e n s i t i v e response (HR), which 1s o n l y Invoked f o l l o w i n g pathogen I n f e c t i o n . The I n d u c t i o n o f h y p e r s e n s i t i v e r e a c t i o n s 1n p l a n t s 1s under s e v e r a l c o n t r o l c i r c u i t s and t h u s 1s I n v o l v e d a t s e v e r a l l e v e l s o f pathogen r e c o g n i t i o n and exclusion. Two such l e v e l s a r e c l a s s e d as g e n e r a l r e s i s t a n c e ( I n which an e n t i r e p l a n t s p e c i e s r e a c t s a g a i n s t a l l members o f an e n t i r e pathogen taxon) o r as s p e c i f i c r e s i s t a n c e (1n which c e r t a i n c u l t i v a r s of a p l a n t species are r e s i s t a n t to only c e r t a i n biotypes o r r a c e s o f a pathogen s p e c i e s ) . Incompatible plant-pathogen c o m b i n a t i o n s r e s u l t from the o c c u r r e n c e o f the h o s t d e f e n s e r e a c t i o n and p l a n t r e s i s t a n c e , which a r e 1n t u r n presumed t o r e s u l t from r e c o g n i t i o n o f the I n f e c t i n g p a t h o g e n . Compatible host-pathogen c o m b i n a t i o n s , on the o t h e r hand, a r e those 1n which r e c o g n i t i o n and h o s t d e f e n s e do not o c c u r , the pathogen d e v e l o p s t o a c o n s i d e r a b l e e x t e n t 1n the p l a n t t i s s u e and d i s e a s e o c c u r s . P h y t o a l e x i n p r o d u c t i o n 1s o n l y one component o f the h y p e r s e n s i t i v e response. O t h e r s I n c l u d e the p r o d u c t i o n o f e n z y m a t l c a l l y a c t i v e p r o t e i n s such as c h l t l n a s e s , a n t i v i r a l p r o t e i n s and 3-1,3 g l u c a n a s e s as w e l l as p l a n t c e l l w a l l s t r u c t u r a l e l e m e n t s , I n c l u d i n g h y d r o x y p r o l i n e r i c h g l y c o p r o t e i n s , c a l l o s e and I1gn1n. The d e p o s i t i o n o f t h e s e s u b s t a n c e s I n t o p l a n t c e l l w a l l s s u r r o u n d i n g the pathogen I n f e c t i o n s i t e presumably c o n s t i t u t e s a b a r r i e r t o f u r t h e r pathogen I n g r e s s through the p l a n t . While none o f t h e s e mechanisms have been c o n v i n c i n g l y i m p l i c a t e d as p h y s i o l o g i c a l l y Important 1n d i s e a s e d e f e n s e , 1t 1s l i k e l y t h a t they c o n t r i b u t e toward r e s i s t a n c e t o a t l e a s t c e r t a i n types of pathogen. P h y t o a l e x i n s a r e low m o l e c u l a r w e i g h t , a n t i b i o t i c secondary m e t a b o l i t e s t h a t , as noted above, a r e produced i n d u d b l y by many p l a n t s p e c i e s and a r e I n v o l v e d w i t h d e f e n s e a g a i n s t pathogens [ f o r more d e t a i l e d r e c e n t r e v i e w s , see (1-10)]. P h y t o a l e x i n s a r e not p r e s e n t 1n v e g e t a t i v e p l a n t p a r t s 1n s i g n i f i c a n t q u a n t i t y but accumu l a t e r a p i d l y a t I n f e c t i o n s i t e s o f I n c o m p a t i b l e pathogens and 1n response t o t r e a t m e n t w i t h v a r i o u s s u b s t a n c e s o r s t i m u l i c a l l e d elldtors. P h y t o a l e x i n s a r e g e n e r a l t o x i c a n t s which a f f e c t the plasma membrane I n t e g r i t y o f m i c r o o r g a n i s m s , animal c e l l s a n d , I n d e e d , c e l l s o f the p l a n t s t h a t make them. Over 200 s p e c i e s o f p l a n t s have been shown t o produce p h y t o a l e x i n s and t h i s s u b s t a n t i a l number a priori I n d i c a t e s t h a t they are w i d e l y d i s t r i b u t e d , p h y s i o l o g i c a l l y Important components o f p l a n t disease defense. P h y t o a l e x i n s have r e c e n t l y been I s o l a t e d from s e v e r a l p l a n t t a x a 1n which they had not p r e v i o u s l y been found [ e . g . d t r u s (11); Coleostephus (12); sorghum (13); and c r u d f e r s (14,15)]. It was a l s o r e c e n t l y observed t h a t a l l o f 61 n a t u r a l l y collected a c c e s s i o n s r e p r e s e n t i n g 10 s p e c i e s o f Glycine produced one o r more members o f the f a m i l y o f c l o s e l y r e l a t e d p h y t o a l e x i n s t r i v i a l l y c a l l e d g l y c e o l l l n s (16). W h i l e the p r o d u c t i o n o f g l y c e o l U n s 1s r a r e o u t s i d e G 7 y c / / 7 e f i h e s e p h y t o a l e x i n s a r e h i g h l y c o n s e r v e d among members w i t h i n the genus. T h i s h i g h degree o f c o n s e r v a t i o n I m p l i e s a c r u c i a l r o l e f o r the p h y t o a l e x i n s 1n Glycine spp. Furthermore, w h i l e p h y t o a l e x i n s g e n e r a l l y e x h i b i t a n t i b i o t i c p r o p e r t i e s , many o f

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them have r e l a t i v e l y unusual chemical s t r u c t u r e s . In a d d i t i o n t o the l a r g e number o f novel s t r u c t u r e s a l r e a d y d e s c r i b e d , r e c e n t r e p o r t s have noted new and c h e m i c a l l y unique p h y t o a l e x i n s ( F i g u r e 1 ) . These I n c l u d e b r a s s l l e x l n , a s u l f u r c o n t a i n i n g i n d o l e d e r i v a t i v e ( 1 4 ) , t h e o r y z a l e x l n s from r i c e ( 1 7 ) , a b i p h e n y l p h y t o a l e x i n , m a g n o l o l , from CereIdlphyHum ( 1 5 ) , t h e n i t r o g e n c o n t a i n i n g compound d l a n t h a l e x i n (18) and t h e novel s p i r o k e t a l e n o l e t h e r p h y t o a l e x i n , m y c o s l n o l , from Coleostephus ( 1 2 ) . In a d d i t i o n , c e r t a i n secondary m e t a b o l i t e s p r e v i o u s l y c o n s i d e r e d as preformed m e t a b o l i t e s have been shown t o be produced as p h y t o a l e x i n s [ e . g . protoanemonln by Caltha palustrls (19) and a n t h o c y a n l d l n s 1n sorghum, shown t o behave as phytoalexins (13)]. Our u n d e r s t a n d i n g o f p h y t o a l e x i n s and t h e i r b i o l o g i c a l r o l e has been a i d e d by t h e r e c e n t f i n d i n g t h a t some o f them a r e p h o t o act Ivatab l e to molecules of greater b i o l o g i c a l a c t i v i t y ( 2 0 ) . For I n s t a n c e , i t was p r e v i o u s l y thought t h a t p h y t o a l e x i n s d i d not d i r e c t l y a f f e c t v i r u s e s 1n h y p e r s e n s l t l v e l y r e s p o n d i n g p l a n t t i s s u e . However, 2 , 7 - d 1 h y d r o x y c a d a l e n e , a p h o t o a c t i v a t a b l e c o t t o n p h y t o a l e x i n was r e c e n t l y shown t o I n a c t i v a t e c a u l i f l o w e r mosaic v i r u s In vitro ( 2 1 ) . This r a i s e s the p o s s i b i l i t y that c e r t a i n phytoalexins may cause v i r u s i n a c t i v a t l o n d u r i n g h y p e r s e n s i t i v e r e s i s t a n t p l a n t reactions. E v i d e n c e f o r t h e Involvement o f P h y t o a l e x i n s 1n R e s i s t a n c e A f t e r y e a r s o f f r e q u e n t l y c o n t r i v e d c o n t r o v e r s y on t h e r o l e o f p h y t o a l e x i n s 1n d i s e a s e r e s i s t a n c e , t h e y a r e now g e n e r a l l y regarded as bona f1de p h y s i o l o g i c a l l y Important a g e n t s . T h i s emerging consensus has r e s u l t e d from t h e c o n t i n u e d a c c u m u l a t i o n o f d a t a s u g g e s t i n g Importance i n r e s i s t a n c e and from g e n e t i c e x p e r i m e n t s showing t h a t t h e d e g r a d a t i o n o f h o s t p h y t o a l e x i n s by c e r t a i n p a t h o gens 1s r e q u i r e d f o r h i g h v i r u l e n c e ( 2 2 , 2 3 ) . An Important c o r r e l a r y o f t h e s e g e n e t i c e x p e r i m e n t s s t a t e s tuât 1f a pathogen e l i c i t s p h y t o a l e x i n s b u t cannot o t h e r w i s e t o l e r a t e o r d e t o x i f y them, then the p h y t o a l e x i n s c o n s t i t u t e an e f f e c t i v e d i s e a s e d e f e n s e mechanism. I t 1s not p o s s i b l e i n t h i s paper t o c o v e r t h e e x t e n s i v e l i t e r a t u r e linking phytoalexins with disease resistance, b ut the r e v i e w a r t i c l e s c i t e d e a r l i e r d i s c u s s much o f 1 t . The g e n e r a l l i n e s o f evidence Unking phytoalexins to resistance expression (with r e f e r e n c e s t h a t a r e I l l u s t r a t i v e bu t f a r from comprehensive) a r e : 9

( I ) G e n e t i c a l l y r e s i s t a n t p l a n t s I n v a r i a b l y produce h i g h e r l e v e l s o f p h y t o a l e x i n s than do s u s c e p t i b l e genotypes ( 2 4 , 2 5 ) . (II) pathogen produced decrease r e s i s t a n c e ( 2 6 ) .

suppressors

of

phytoalexin

production

( I I I ) chemical I n h i b i t o r s o f p r o t e i n s y n t h e s i s o r I n h i b i t o r s o f enzymes In p h y t o a l e x i n b l o s y n t h e t l c pathways d e c r e a s e p h y t o a l e x i n p r o d u c t i o n and negate r e s i s t a n c e ( 2 7 , 2 8 ) . (1v) race s p e c i f i c e l l d t o r s have been I s o l a t e d t h a t reproduce t h e s p e c i f i c i t y f o r p h y t o a l e x i n i n d u c t i o n o f t h e pathogen race from which t h e y were i s o l a t e d ( 2 9 , 3 0 ) .

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PhytoakxinsandTlmEUcUors

(ν) removal o f p h y t o a l e x i n s from a pathogen i n f e c t i o n s i t e resistance while exogenous a d d i t i o n of phytoalexins resistance (31).

117 decreases Increases

( v l ) l o c a l i z a t i o n s t u d i e s have shown t h a t p h y t o a l e x i n s a r e made a t the r i g h t p l a c e a t the r i g h t time and a t s u f f i c i e n t c o n c e n t r a t i o n t o a c c o u n t f o r I n h i b i t i o n o f t h e pathogen ( 3 2 , 3 3 ) . (v11) e l e c t r o n m i c r o s c o p e s t u d i e s have shown t h a t pathogens 1n r e s i s t a n t - r e a c t i n g p l a n t t i s s u e s e x h i b i t u l t r a s t r u c t u r a l abnormal i t i e s t h a t a r e e x t r e m e l y s i m i l a r t o t h o s e shown by t h e same organisms grown 1n c u l t u r e and exposed t o t h e pure p h y t o a l e x i n s ( 3 4 , 3 5 ) . E l i c i t o r s o f P h y t o a l e x i n s and the HR P l a n t pathogens e l a b o r a t e m e t a b o l i t e s I n t o t h e c u l t u r e medium o r c o n t a i n s u b s t a n c e s t h a t e l i c i t p l a n t t i s s u e s t o produce p h y t o a l e x i n s In t h e absence o f t h e pathogen I t s e l f . Generally, t h i s Involves s e v e r a l components ( e . g . 36) t h a t may appear 1n c u l t u r e f l u i d s , most o f w h i c h a r e not r a c e - s p e c T f l c — t h a t 1s they a r e produced by v a r i o u s I s o l a t e s o f t h e pathogen and cause s i m i l a r e f f e c t s on a range o f host c u l t i v a r s . The p h y s i o l o g i c Importance o f such e l i c i t o r s has g e n e r a l l y not been e s t a b l i s h e d , and DeVMt and Splkman (37) showed that n o n - s p e c i f i c glycoprotein e l l d t o r s present i n c u l t u r e f l u i d s o f Cladosporlum fulvum were not d e t e c t a b l e a t a l l In I n f e c t e d h o s t t i s s u e s , but a race s p e c i f i c e l l c i t o r was i s o l a t e d from I n f e c t e d l e a v e s , as w i l l be d i s c u s s e d l a t e r . D e s p i t e t h e r e s u l t i n g s u s p i c i o n that non-specific elldtors found 1n c u l t u r e fluids may not necessarily be o f physiological Importance in plant-pathogen I n t e r a c t i o n s , r o l e s have n e v e r t h e l e s s been c l a i m e d f o r some o f them. The major t y p e s o f n o n - s p e c i f i c e l i c i t o r s c h a r a c t e r i z e d have been g l u c a n s and c h l t l n d e r i v a t i v e s . The most s t u d i e d o f t h e s e a r e branched 3 - 1 , 3 - g l u c a n s e x t r a c t e d from t h e c e l l s w a l l s o f v a r i o u s f u n g i , p a r t i c u l a r l y Phytophthora s p p . A l b e r s h e l m ' s group e x t r a c t e d g l u c a n s from c e l l w a l l s o f P. megasperma f . s p . glydnea by a u t o c l a v l n g them 1n w a t e r o r w i t h m i l d a d d h y d r o l y s i s ( 3 8 ) . The e x t r a c t s were c a r e f u l l y f r a c t i o n a t e d I n t o t h e s m a l l e s t e l l c i t o r a c t l v e component, I d e n t i f i e d as a seven membered g l u c a n composed o f a 3-1,6-1 Inked p e n t a g l u c a n w i t h 3-1,3-1 Inked g l u c o s e r e s i d u e s on the second and f o u r t h r e s i d u e s (39) ( n - 1 , F i g u r e 2 ) . Yoshikawa e t a l . (40) r e c e n t l y I s o l a t e d homologues o f t h e h e p t a g l u c a n e l l d t o r from P. megasperma f . s p . glyclnea c e l l walls treated w i t h a p u r i f i e d 3 - 1 , 3 - e n d o g l u c a n a s e from soybean p l a n t s . These g l u c a n s shared t h e b a s i c h e p t a g l u c a n s t r u c t u r e o f a 3-1,6-1 i n k e d g l u c o s e backbone w i t h t h e p e n u l t i m a t e r e s i d u e s s u b s t i t u t e d w i t h P-l,3-1 inked glucose residues. However, the enzyme-released e l l d t o r s I n c l u d e d those w i t h l o n g e r 3 - 1 , 6 backbones than t h e h e p t a glucan (Figure 2 ) . S i g n i f i c a n t l y , t h e l a r g e r g l u c a n s w i t h n-2 o r g r e a t e r were much more p o t e n t e l l d t o r s than t h e h e p t a g l u c a n ( n = l ) . These more a c t i v e e l l d t o r s were not d e t e c t e d 1n t h e e a r l i e r work by A l b e r s h e l m ' s group ( 3 9 ) , p r o b a b l y because they exposed c e l l w a l l s t o the r e l a t i v e l y h a r s f i " t r e a t m e n t o f a d d e x t r a c t i o n . Unlike a d d , w h i c h a t t a c k s both 3 - 1 , 3 and 3 - 1 , 6 g l y c o s l d l c l i n k a g e s , t h e g l u c a n a s e used by Yoshikawa and co-workers (40) o n l y a t t a c k s I n t e r n a l 3 - 1 , 3 l i n k a g e s , thus p r e s e r v i n g the l o n g e r β-1,6-1 i n k e d e l l c i t o r back-

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CH3

α

H

BRASSILEXIN OH

ORYZALEXIN A

OH H C-C»C-C«C-C 3

OH CH

2

CH

2

MAGNOLOL

MYCOSINOL Ο

OH

DIANTHALEXINE F i g u r e 1.

Recently described phytoalexins.

G

'-G

L

η Figure 2.

Homologous p o l y g l u c a n e l l d t o r s I s o l a t e d from c e l l w a l l s o f Phytophthora megasperma f . s p . glydnea; η » 1 t o 5 o r g r e a t e r and a l l g l y c o s l d l c l i n k a g e s a r e 0 .

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6. KEEN

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bones. W h i l e t h e s e g l u c a n e l l d t o r s have c o n s i d e r a b l e a c t i v i t y 1n p h y t o a l e x i n e l l d t o r a s s a y s and a r e s u s p e c t e d t o p l a y a r o l e 1n d i s e a s e d e f e n s e , no e v i d e n c e has y e t d e f i n i t i v e l y l i n k e d them t o t h e o c c u r r e n c e o f d e f e n s e r e a c t i o n s 1n soybean p l a n t s I n f e c t e d w i t h an I n c o m p a t i b l e r a c e o f P. megasperma f . s p . glydnea. Indeed, a l l races o f t h e pathogen c o n t a i n t h e e l l d t o r s and a l l soybean c u l t i v a r s respond s i m i l a r l y t o g l u c a n p r e p a r a t i o n s . F i n a l l y , while glucans e f f i c i e n t l y e l i c i t p h y t o a l e x i n s 1n some p l a n t s such as s o y b e a n , they a r e r e l a t i v e l y poor e l l d t o r s 1n o t h e r p l a n t s ( e . g . 4 1 ) . Ch1t1n and c h l t o s a n a r e a l s o f u n g a l w a l l components t h a t e l l d t p h y t o a l e x i n s a n d / o r H g n l n a c c u m u l a t i o n ( 4 2 , 4 3 ) . As w i t h t h e g l u c a n e l l d t o r s , t h e p h y s i o l o g i c r o l e o f c h l t o s a n has not y e t been e s t a b l i s h e d and no r a c e s p e c i f i c p r o p e r t i e s have been n o t e d . Another e l l d t o r which has n o t y e t been shown t o p o s s e s s r a c e s p e c i f i c f e a t u r e s I s a g l y c o p r o t e i n from Pucdnla gramlnls f . s p . trltld cell w a l l s t h a t e l i c i t s t h e HR 1n wheat l e a v e s ( 4 4 ) . A f i n a l c l a s s o f non-specific elldtors worthy of note are ollgogalacturonlde e l l d t o r s I s o l a t e d from p l a n t c e l l w a l l s . B a i l e y (45) o b t a i n e d e v i d e n c e s u g g e s t i n g t h a t wounded p l a n t t i s s u e s produced""'endogenous elldtors which s u b s e q u e n t l y e l i c i t e d p h y t o a l e x i n responses i n surrounding healthy c e l l s . Bruce and West (46) and l a t e r s e v e r a l others showed t h a t certain microbial pecffc enzymes released e 1 1 d t o r - a c t 1 v e o l l g o g a l a c t u r o n l d e fragments from p l a n t c e l l w a l l s . A g a i n , t h e p h y s i o l o g i c r e l e v a n c e o f such e l l d t o r s has n o t been e s t a b l i s h e d , b u t 1t 1s a p p e a l i n g t o t h i n k t h a t t h e y may be o f Importance 1n t h e case o f p l a n t a t t a c k by pathogens t h a t produce r e l a t i v e l y low a c t i v i t i e s o f p e c t i n d e g r a d i n g enzymes. S i n c e t h e y cannot account f o r t h e s p e c i f i c i t i e s observed 1n g e n e - f o r - g e n e p l a n t - p a t h o g e n s y s t e m s , t h e p h y s i o l o g i c r o l e o f nons p e c i f i c e l l d t o r s , 1f a n y , must be 1n g e n e r a l r e s i s t a n c e . For I n s t a n c e , 1f p l a n t t i s s u e s respond r a p i d l y enough t o g l u c a n s , c h l t i n o r o t h e r η ο η - s p e d f l c e l l d t o r s from an I n v a d i n g p a t h o g e n , t h e r e s u l t i n g d e f e n s e r e a c t i o n may be s u f f i c i e n t t o Impede f u r t h e r pathogen development. S i m i l a r l y , p e c t l c enzymes produced by t h e pathogen may l i b e r a t e p l a n t o l l g o g a l a c t u r o n l d e e l l d t o r s t h a t l e a d to resistance. The s i t u a t i o n might a l s o be complex s i n c e p l a n t enzyme I n h i b i t o r s , u n f a v o r a b l e pH c o n d i t i o n s o r o t h e r f a c t o r s t h a t reduce t h e e f f i c i e n c y o f pathogen produced p e c t l c enzymes might Increase the p r o b a b i l i t y that e f f e c t i v e l e v e l s of o l l g o g a l a c t u r o n l d e e l l d t o r s c o u l d be formed a t t h e pathogen I n f e c t i o n s i t e ( 4 7 , 4 8 ) . A wide range o f ' a b i o t i c e l l d t o r s t h a t a r e n o t o b t a i n e d from pathogens f u n c t i o n a t v a r y i n g e f f i c i e n c i e s as p h y t o a l e x i n e l l d t o r s . These I n c l u d e heavy metal 1ons, p o l y a n l o n s , v a r i o u s a n t i b i o t i c s and d e t e r g e n t s and red-ox agents (see r e v i e w s noted e a r l i e r f o r d e t a i l s ) . The l a t t e r group I n c l u d e s g l u t a t h i o n e , r e p o r t e d t o be an e f f e c t i v e e l l d t o r 1n Phaseolus vulgaris by Wlngate e t a l . ( 4 9 ) . I t 1s p r o b a b l e t h a t most o f t h e a b i o t i c e l l d t o r s f u n c t i o n by c a u s i n g η ο η - s p e d f l c damage t o p l a n t t i s s u e which r e l e a s e s endogenous p l a n t e l l d t o r s , by b l o c k i n g p h y t o a l e x i n t u r n o v e r (50) o r by m1m1dng t h e p l a n t c e l l s i g n a l t r a n s d u c t i o n machinery ( s e e " B e l o w ) . 1

1

S u p p r e s s o r s and Enhancers M e t a b o l i t e s have been I s o l a t e d from pathogens t h a t s u p p r e s s p h y t o a l e x i n p r o d u c t i o n by a h o s t p l a n t 1n t h e p r e s e n c e o f o t h e r w i s e f u n c t i o n a l e l l d t o r s ( e . g . 2 6 ) . The e l a b o r a t i o n o f such s u p p r e s s o r s

120


1s a l o g i c a l p a t h o g e n i c s t r a t e g y f o r overcoming g e n e r a l r e s i s t a n c e and they may a c c o r d i n g l y be more w i d e s p r e a d among pathogens than i s currently appreciated. Doke, Kuc and a s s o c i a t e s have p r e s e n t e d e v i d e n c e I n d i c a t i n g t h a t s u p p r e s s o r s may a l s o f u n c t i o n 1n a g e n e - f o r - g e n e system I n v o l v i n g s i n g l e disease r e s i s t a n c e genes. The potàto-Phytophthora Infestans system I n v o l v e s t h e I n t e r p l a y o f s e v e r a l s i n g l e d i s e a s e r e s i s t a n c e genes and many pathogen r a c e s . The s u p p r e s s o r model as a p p l i e d t o t h i s system h o l d s t h a t a l l fungus r a c e s e l a b o r a t e non-specific e l i c i t o r s when I n f e c t i n g p o t a t o [ p o s s i b l y a r a c h l d o n l c and r e l a t e d f a t t y a d d s ( 5 1 ) ] . Races t h a t a r e not r e s t r i c t e d 1n a c e r t a i n p l a n t r e s i s t a n c e genotype have been proposed t o produce s p e c i f i c s u p p r e s s o r s u b s t a n c e s t h a t r e p r e s s t h e p h y t o a l e x i n r e s p o n s e . Doke e t a l . (52) p r e s e n t e d e v i d e n c e s u g g e s t i n g t h a t t h e race s p e c i f i c s u p p r e s s o r s might be m y c o l a m l n a r a n s , s m a l l o l l g o g l u c a n s p r e s e n t i n t h e c y t o p l a s m o f v a r i o u s Phytophthora s p p . The s u p p r e s s o r work has been c r i t i c i z e d because 1t i s a t odds w i t h g e n e t i c d a t a on g e n e - f o r - g e n e systems and t h e s u p p r e s s o r s have n e i t h e r been I s o l a t e d and c h a r a c t e r i z e d nor proven t o a c c o u n t f o r s p e c i f i c i t y . To c o m p l i c a t e t h e p o t a t o - P . Infestans system f u r t h e r , Man1ara e t al. (53) and Pre1s1g and Kuc (54) r e p o r t e d t h e p r e s e n c e 1n f u n g a l homogenates o f s u b s t a n c e s , a g a i n I d e n t i f i e d as o l l g o g l u c a n s , t h a t behaved as ' e n h a n c e r s ' o f t h e d e f e n s e r e a c t i o n e l i c i t e d by a r a c h l d o n l c a d d in potato. A d d i t i o n a l work w i l l be r e q u i r e d t o d e t e r m i n e whether t h e v a r i o u s s u p p r e s s o r s and enhancers a r e p h y s i o l o g i c a l l y Important 1n r e s i s t a n c e e x p r e s s i o n . Race S p e c i f i c

Elicitors

As noted e a r l i e r , s p e c i f i c r e s i s t a n c e o c c u r s 1n o n l y c e r t a i n p l a n t c u l t i v a r s a g a i n s t c e r t a i n pathogen b i o t y p e s . In c l a s s i c work, F l o r (55) and o t h e r s showed t h a t I n v o c a t i o n o f t h e HR 1s d e t e r m i n e d by the a c t i v i t y o f s i n g l e genes 1n t h e h o s t ( c a l l e d d i s e a s e r e s i s t a n c e genes) and 1n t h e pathogen ( c a l l e d a v l r u l e n c e g e n e s ) . Because r e s i s t a n c e genes a r e w i d e l y used In a g r i c u l t u r e f o r t h e c o n t r o l o f p l a n t d i s e a s e s , t h e r e 1s c o n s i d e r a b l e I n t e r e s t In u n d e r s t a n d i n g t h e biochemical basis of t h i s s p e c i f i c i t y . The e l i c i t o r - r e c e p t o r model has f r e q u e n t l y been proposed t o e x p l a i n s p e c i f i c i t y In such genef o r - g e n e systems and 1s s u p p o r t e d by c o n s i d e r a b l e g e n e t i c and biochemical evidence (56-58). The model p r e d i c t s t h a t a pathogen race c a r r y i n g a s p e c i f i c a v l r u l e n c e gene produces a unique m e t a b o l i t e , c a l l e d a race s p e c i f i c e l l d t o r , w h i l e r a c e s l a c k i n g t h e f u n c t i o n a l a v l r u l e n c e gene do not make t h i s e l l c i t o r . P l a n t s c a r r y i n g the complementary d i s e a s e r e s i s t a n c e gene produce a unique r e c e p t o r f o r the e l l d t o r m o l e c u l e . F o l l o w i n g e l 1c1 t o r - r e c e p t o r b i n d i n g , unknown Intermediate events r e s u l t i n s p e c i f i c derepression o f c e r t a i n p l a n t g e n e s , c a l l e d d i s e a s e response g e n e s . T h e i r e x p r e s s i o n l e a d s t o t h e v a r i o u s consequences o f t h e HR, I n c l u d i n g p h y t o a l e x i n a c c u m u l a t i o n (58). Our own r e s e a r c h group r e p o r t e d t h e f i r s t e v i d e n c e s u g g e s t i n g o c c u r r e n c e o f r a c e s p e c i f i c e l l d t o r s i n Phytophthora megasperma f . s p . glydnea (59) and Pseudomonas syrlngae p v . glydnea (60). P r e p a r a t i o n s from v a r i o u s r a c e s o f t h e s e pathogens e x h i b i t e d g r e a t e r e l l d t o r a c t i v i t y on I n c o m p a t i b l e soybean c u l t i v a r s than on c o m p a t i b l e cultivars. D e s p i t e t h e p r o m i s i n g r e s u l t s , however, 1t was not p o s s i b l e t o I s o l a t e and c h a r a c t e r i z e t h e e l l d t o r s , a problem t h a t has been noted w i t h o t h e r p l a n t - p a t h o g e n systems as w e l l . Such

6. KEEN

Phytoalexins andTheirElicitors

121

d i f f i c u l t i e s may be caused by l a b i l i t y o f t h e r e l e v a n t e l l d t o r s o r t h e i r p r o d u c t i o n 1n q u a n t i t y o n l y when t h e pathogens have been I n o c u l a t e d I n t o host t i s s u e . More r e c e n t r e s e a r c h i n s e v e r a l l a b o r a t o r i e s has I l l u m i n a t e d t h e s e p o s s i b i l i t i e s . C r u c e f l x e t a l . (61) searched I n t e n s i v e l y f o r race s p e c i f i c e l l d t o r s 1n t h e Bremla-lettuce host-pathogen system. Infection s t r u c t u r e s o f t h e o b l l g a t e l y p a t h o g e n i c fungus were a s s a y e d f o r e f f e c t s i n l e t t u c e c o t y l e d o n s and p r o t o p l a s t s o f known r e s i s t a n c e genotypes. While t h e presence o f race s p e c i f i c e l i c i t o r s o f t h e HR was I n d i c a t e d from c e r t a i n I s o l a t e s o f t h e f u n g u s , b i o a s s a y i n c o n s i s t e n c y and t h e d i f f i c u l t y 1n I s o l a t i n g s i g n i f i c a n t q u a n t i t i e s o f t h e p u t a t i v e e l l d t o r s posed s e r i o u s p r o b l e m s . The a u t h o r s a l s o s p e c u l a t e d t h a t t h e s p e c i f i c e l l d t o r s 1n t h i s system might be l a b i l e m e t a b o l i t e s t h a t were o n l y s e c r e t e d a t c e r t a i n s t a g e s o f t h e I n f e c t i o n p r o c e s s 1n t h e p l a n t h o s t . The p r o d u c t i o n o f race s p e c i f i c HR e l l d t o r s o n l y d u r i n g h o s t I n f e c t i o n was a l s o suggested by t h e e a r l i e r work o f Jones and D e v e r a l l (62) w i t h t h e vfheàt-Pucdnla recondlta system and has been d r a m a t i c a l l y shown t o o c c u r i n t h e I n t e r a c t i o n o f Cladosporlum fulvum and tomato by De W i t and c o workers. Several I n v e s t i g a t o r s had searched u n s u c c e s s f u l l y f o r race s p e c i f i c e l l d t o r s 1n C. fulvum by f r a c t i o n a t i n g f u n g a l c u l t u r e s grown on common l a b o r a t o r y m e d i a . De Wit and c o - w o r k e r s (37,63) I n o c u l a t e d t h e fungus I n t o a c o m p a t i b l e tomato c u l t i v a r and subs e q u e n t l y e x t r a c t e d I n t e r c e l l u l a r f l u i d s from t h e I n o c u l a t e d l e a v e s . These l e a f e x t r a c t s c o n t a i n e d m e t a b o l i t e s o f presumed f u n g a l o r i g i n that elicited the h y p e r s e n s i t i v e reaction only 1n subsequent b i o a s s a y s on tomato c u l t i v a r s c a r r y i n g complementary r e s i s t a n c e genes t o a v l r u l e n c e genes 1n t h e o r i g i n a l l y i n o c u l a t e d f u n g a l r a c e . Thus, a s p e c i f i c e l l d t o r was produced o n l y by f u n g a l isolates c a r r y i n g a unique a v l r u l e n c e gene and o n l y when t h e fungus grew 1n the h o s t . One o f t h e p u t a t i v e race s p e c i f i c e l l d t o r s , a s s o c i a t e d w i t h pathogen a v l r u l e n c e gene avrCf9, has r e c e n t l y been I s o l a t e d and i d e n t i f i e d as a 27 amino a d d , c y s t e 1 n e - r 1 c h p e p t i d e (29) ( F i g u r e 3 ) . The p e p t i d e may be a p r o c e s s e d p r o d u c t o f t h e pathogen A9 a v l r u l e n c e gene, but t h i s has not y e t been t e s t e d by c r i t i c a l g e n e t i c e x p e r i ments. Another characterized race specific elldtor 1s v l c t o r l n , produced by Helm1nthospor1um vlctorlae. This metabolite s p e c i f i c a l l y e l i c i t s t h e HR a t p l c o m o l a r c o n c e n t r a t i o n s o n l y on o a t c u l t i v a r s c a r r y i n g t h e gene Pc-2, t h a t c o n f e r s r e s i s t a n c e t o t h e r u s t p a t h o g e n , Pucclnla coronata ( 6 4 ) . The s t r u c t u r e o f v i c t o r l n has been d e t e r mined by Wolpert e t a l . (65) as a b a s i c p e p t i d e c o n t a i n i n g unusual amino a d d r e s i d u e s . Mayama e t a l . (64) showed t h a t v i c t o r l n e l i c i t e d t h e h y p e r s e n s i t i v e response and p h y t o a l e x i n p r o d u c t i o n o n l y 1n Pc-2 o a t U n e s . I t t h e r e f o r e appears t h a t v i c t o r l n 1s a race s p e c i f i c e l l d t o r o f t h e o a t HR and t h a t P. coronata r a c e s c a r r y i n g Tyr- Cys-Asn-Ser-Ser-Cys-Thr-Arg-Ala-Phe-AspCys-Leu-Gly-Gln-Cys-Gly-Arg-CysAsp-Phe-His-Lys-Leu-Gln-Cys-Val Figure 3.

Race s p e c i f i c e l l d t o r from Cladosporlum fulvum. All r e l e v a n t amino a d d s a r e assumed t o be (L) and t h e f i r s t amino a d d 1s t h e N-term1nus.

122


the a v l r u l e n c e gene complementing Pc-2 must c o n t a i n an analogous e l l c i t o r . W o l p e r t and Macko (66) r e c e n t l y I d e n t i f i e d a p r o t e i n t h a t may f u n c t i o n as a s p e c i f i c r e c e p t o r f o r v i c t o r l n 1n t h e Pc-2 o a t g e n o t y p e . However, 1t remains t o be e s t a b l i s h e d whether t h i s p r o t e i n I s t h e p r i m a r y p r o d u c t o f t h e Pc-2 r e s i s t a n c e g e n e . Recent r e s u l t s w i t h t o b a c c o mosaic v i r u s (TMY) have I l l u m i n a t e d t h e b a s i s o f h o s t range s p e c i f i c i t y w i t h t h i s v i r u s . K n o r r and Dawson (67) I d e n t i f i e d m u t a t i o n s 1n t h e c o a t p r o t e i n gene o f TMY t h a t r e s u l t e d 1n h y p e r s e n s i t i v e r e s i s t a n t r e a c t i o n s on t o b a c c o p l a n t s c a r r y i n g the N r e s i s t a n c e gene r a t h e r than the normal s u s c e p t i b l e response. More r e c e n t l y , t h e same group has p r o v i d e d e v i d e n c e I n d i c a t i n g t h a t t h e c o a t p r o t e i n I t s e l f and not v i r a l RNA o r o t h e r v i r a l gene p r o d u c t s I s a s p e c i f i c e l l d t o r o f t h e HR. The most e l e g a n t o f t h e s e e x p e r i m e n t s I n v o l v e d I n t r o d u c t i o n o f the mutant c o a t p r o t e i n gene I n t o N t o b a c c o p l a n t s . The r e g e n e r a t e d , t r a n s g e n i c p l a n t s e x h i b i t e d numerous n e c r o t i c f o l i a r l e s i o n s , s i m i l a r t o p l a n t s I n o c u l a t e d w i t h the v i r u s ( C u l v e r , J . and Dawson, W., Univ. of C a l i f o r n i a , R i v e r s i d e , personal communication, 1989). W h i l e 1t cannot be e n t i r e l y r u l e d out t h a t t h e TMV c o a t p r o t e i n may have an unknown e n z y m a t i c f u n c t i o n which l e a d s t o an a c t i v e e l l d t o r , t h e r e s u l t s I n d i c a t e t h a t t h e c o a t p r o t e i n I t s e l f f u n c t i o n s as an e l l d t o r o f the HR. 1

1

A v l r u l e n c e Genes and S p e c i f i c

Elldtors

Our u n d e r s t a n d i n g o f g e n e - f o r - g e n e c o m p l e m e n t a r i t y and t h e r o l e o f r a c e s p e c i f i c e l i c i t o r s has been c o n s i d e r a b l y f u r t h e r e d by t h e c l o n i n g o f a v l r u l e n c e genes from s e v e r a l b a c t e r i a l pathogens ( f o r r e v i e w see 5 7 ) . A v l r u l e n c e (avr) genes have been c l o n e d from s e v e r a l members o f the Pseudomonas syrlngae and Xanthomonas campestrls groups and some o f them have been sequenced and t h e i r gene p r o d u c t s characterized. Thus f a r , a l l sequenced avr genes encode s i n g l e protein products. S u r p r i s i n g l y , a l l of the p r o t e i n s lack s i g n a l p e p t i d e sequences, I n d i c a t i n g t h a t t h e y o c c u r 1n t h e b a c t e r i a l c y t o p l a s m and a r e not s e c r e t e d t o the medium o r t o t h e c e l l s u r f a c e . Coupled w i t h the absence o f d e t e c t a b l e e l l d t o r a c t i v i t y from c e r t a i n avr p r o t e i n s ( e . g . 6 8 , 6 9 ) , t h i s I n d i c a t e s t h a t the gene p r o d u c t s themselves may not d i r e c t l y e l l d t the p l a n t HR. Research w i t h the avrO gene c l o n e d from P. syrlngae p v . tomato has o f f e r e d f u r t h e r I n s i g h t I n t o t h i s a s p e c t o f avr gene f u n c t i o n . Four d i f f e r e n t a v l r u l e n c e genes have been c l o n e d from P. syrlngae p v . tomato ( 7 0 , Shen and Keen, u n p u b l i s h e d ) t h a t , when I n t r o d u c e d I n t o t h e r e l a t e d b a c t e r i u m P. syrlngae pv. glydnea, caused them t o e l l d t h y p e r s e n s i t i v e r e a c t i o n s on some but not a l l soybean c u l t i v a r s . These b a c t e r i a l genes t h e r e f o r e behaved as race s p e c i f i c avr genes 1n P.s. glydnea. S u r p r i s i n g l y , one o f them a l s o t u r n e d out t o be I d e n t i c a l t o e w A , p r e v i o u s l y c l o n e d from r a c e 6 o f P.s. glydnea (69,61). Thus, the avr genes from P . s . tomato not o n l y f u n c t i o n 1n P . s . glydnea, but one o f them 1s I d e n t i c a l t o an avr gene p r e s e n t 1n a P . s . glydnea race. On the o t h e r hand, a f u n c t i o n a l copy o f one o f the o t h e r P.s. tomato a v l r u l e n c e genes (avru) has not been I d e n t i f i e d 1n any I s o l a t e o f P . s . glydnea. K o b a y a s M and Keen ( m a n u s c r i p t 1n p r e p a r a t i o n ) sequenced the avrO gene from P . s . tomato and found t h a t 1t encoded a s i n g l e p r o t e i n p r o d u c t o f 34 kD. I t was a l s o observed t h a t Escherichia coll cells

6. KEEN

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123

e x p r e s s i n g avru e l i c i t e d HR 1n p r e c i s e l y t h e same c u l t i v a r s as d i d P.s. glydnea race 4 c e l l s c a r r y i n g t h e gene. N e i t h e r t h e p a r t i a l l y p u r i f i e d avrl) p r o t e i n n o r l y s e d E. coll c e l l s e x p r e s s i n g avrO e l i c i t e d t h e HR 1n t h e a p p r o p r i a t e soybean c u l t i v a r s . However, s u p e r n a t a n t f l u i d s o f t h e s e E. coll c u l t u r e s c o n t a i n e d a low m o l e c u l a r w e i g h t f a c t o r t h a t s p e c i f i c a l l y e l i c i t e d t h e HR o n l y 1n Incompatible soybean cultivars (Keen, N. T., Tamakl, S., T h o r d a l - C h r i s t e n s e n , H . , G e r h o l t , D., and K o b a y a s h l , D., u n p u b l i s h e d data). The avrt) gene p r o d u c t may t h e r e f o r e p o s s e s s an e n z y m a t i c f u n c t i o n t h a t c o n v e r t s a normal E. coll m e t a b o l i t e t o an e l l d t o r a c t l v e compound which 1s s e c r e t e d from t h e c e l l s . F u r t h e r work has a l s o shown t h a t w i l d t y p e P.s. tomato but not P.s. glydnea produces the e l l d t o r . However, I n t r o d u c t i o n o f t h e c l o n e d avrQ gene I n t o P.s. glydnea l e a d s t o p r o d u c t i o n o f t h e e l l d t o r and t h e s e c e l l s e l l d t t h e HR 1n a p p r o p r i a t e soybean c u l t i v a r s . The avrO e l l d t o r has been p u r i f i e d by t h e use o f C18 and phenyl HPLC columns (Tamakl, S . , S t a y t o n , Μ., G e r h o l t , D., and Keen, N. T . , u n p u b l i s h e d d a t a ) , b u t I t s s t r u c t u r e 1s not y e t known. Low l e v e l s o f t h e e l l d t o r a r e produced by P.s. tomato c e l l s , but an avru mutant s t r a i n 1s d e f i c i e n t . These and o t h e r g e n e t i c e x p e r i m e n t s prove t h a t t h e e l l d t o r s p e c i f i e d by avrO m e d i a t e s a v l r u l e n c e gene f u n c t i o n and 1s t h e agent which I n i t i a t e s p l a n t d e f e n s e 1n soybean c u l t i v a r s c a r r y i n g t h e complementary r e s i s t a n c e g e n e . I t 1s t h e r e f o r e t h e f i r s t race s p e c i f i c e l l d t o r t h a t has been I s o l a t e d and proven t o account f o r g e n e - f o r - g e n e s p e c i f i c i t y . The r e s u l t s w i t h avru a r e a l s o c o n s i s t e n t w i t h t h e e l 1 c 1 t o r - r e c e p t o r model d i s c u s s e d earlier. I t w i l l be o f f u r t h e r I n t e r e s t t o see 1 f , as p r e d i c t e d , a s p e c i f i c r e c e p t o r f o r t h e avru e l l d t o r o c c u r s 1n soybean c u l t i v a r s c a r r y i n g t h e complementary r e s i s t a n c e gene. S i n c e avrV l e a d s t o HR on some but not a l l soybean c u l t i v a r s , the g e n e - f o r - g e n e r e l a t i o n s h i p p r e d i c t s t h e p r e s e n c e o f a c l a s s i c a l s i n g l e r e s i s t a n c e gene 1n those c u l t i v a r s t h a t a r e r e s i s t a n t t o b a c t e r i a c a r r y i n g avru. T h i s p o s s i b i l i t y has been t e s t e d 1n more r e c e n t work I n v o l v i n g c r o s s e s o f c o m p a t i b l e and I n c o m p a t i b l e soybean cultivars. F2 progeny o f c r o s s e s I n v o l v i n g soybean c u l t i v a r s Acme χ N o r c h l e f and Flambeau χ M e r i t s e g r e g a t e d 1n a 3:1 manner f o r both r e s i s t a n c e t o P.s. glydnea c a r r y i n g avrt and f o r s e n s i t i v i t y t o the avrt e l l d t o r (Keen, Ν. T . , and B u z z e l l , R. I . , u n p u b l i s h e d data). S e n s i t i v i t y t o t h e e l l d t o r and r e s i s t a n c e t o Psg c a r r y i n g the gene were dominant and c o - I n h e r i t e d 1n a l l o f c a . 600 s e g r e g a t i n g F2 and F 3 progeny t e s t e d . The t i g h t l i n k a g e o f e l l d t o r s e n s i t i v i t y and r e s i s t a n c e I m p l i e s t h a t t h e e l l d t o r 1s t h e b a c t e r i a l agent which p h y s i o l o g i c a l l y I n i t i a t e s t h e HR 1n r e s i s t a n t p l a n t s . The presence 1n soybean o f a r e s i s t a n c e gene complementary t o avrV 1s s u r p r i s i n g 1n view o f t h e f a c t t h a t a f u n c t i o n a l avrt) gene has not been I d e n t i f i e d 1n any race o f P.s. glydnea. What t h e r e f o r e 1s t h e f u n c t i o n o f t h i s soybean r e s i s t a n c e gene? Does 1t c o n f e r r e s i s t a n c e t o o t h e r b a c t e r i a c a r r y i n g avri), such as P.s. tomato? How E l l d t o r s

I n i t i a t e Phytoalexin

Production

Recent p r o g r e s s has o c c u r r e d 1n u n d e r s t a n d i n g t h e b i o c h e m i s t r y t h a t o c c u r s 1n p l a n t t i s s u e s t r e a t e d w i t h p h y t o a l e x i n e l l d t o r s . This work has been f a c i l i t a t e d by t h e use o f c l o n e d p l a n t genes encoding enzymes 1n p h y t o a l e x i n b l o s y n t h e t l c pathways. Thus, In legumes, 1t

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has been p o s s i b l e t o use c l o n e d genes o f p h e n y l a l a n i n e ammonia l y a s e and c h a l c o n e s y n t h a s e as probes t o m o n i t o r e x p r e s s i o n o f t h e s e d e f e n s e response genes f o l l o w i n g pathogen I n f e c t i o n o r e l l d t o r t r e a t m e n t ( s e e 5 8 ) . These e x p e r i m e n t s have demonstrated I n c r e a s e d e x p r e s s i o n o f £he d e f e n s e response genes r a p i d l y a f t e r e l l d t o r t r e a t m e n t ( 7 2 ) , t h e r e f o r e s u g g e s t i n g t h e o c c u r r e n c e o f few I n t e r m e d i a t e s t e p s between e l l d t o r r e c o g n i t i o n and p l a n t gene a c t i v a t i o n . W h i l e p l a n t d i s e a s e r e s i s t a n c e genes I n v o l v e d w i t h g e n e - f o r - g e n e r e s i s t a n c e a s w e l l a s r e s i s t a n c e genes c o n f e r r i n g g e n e r a l r e s i s t a n c e have been p o s t u l a t e d t o encode c e l l s u r f a c e r e c e p t o r s , v e r y l i t t l e d i r e c t e v i d e n c e 1s a t hand. Yoshikawa e t a l . (73) produced t h e f i r s t e v i d e n c e d i r e c t l y I n d i c a t i n g t h a t soybean ceTTs p o s s e s s r e c e p t o r s f o r t h e f u n g a l g l u c a n e l l d t o r , myco l a m i n a r a n . S i n c e mycolamlnaran 1s n o t r a c e s p e c i f i c , t h e p u t a t i v e r e c e p t o r must be a s s o c i a t e d w i t h g e n e r a l and n o t s p e c i f i c r e s i s t a n c e . Schmidt and Ebel (74) and C o s l o e t a l . (75) c o n f i r m e d o c c u r r e n c e o f t h e g l u c a n r e c e p t o r on t h e soybean plasma membrane, s e t t i n g t h e s t a g e f o r I t s I s o l a t i o n . It s h o u l d a l s o be noted t h a t r e c e p t o r s f o r t h e Cladosporlum peptide, f o r t h e o a t Pc-2 r e s i s t a n c e gene a f f e c t e d by v i c t o r l n , and t h e p u t a t i v e soybean r e c e p t o r f o r t h e e l l d t o r s p e c i f i e d by avrO c o u l d a l s o be I s o l a t e d by u s i n g a f f i n i t y t e c h n i q u e s w i t h t h e v a r i o u s e l l d t o r s as p r o b e s . I t 1s n o t y e t c l e a r whether r e c e p t o r / e l 1 c 1 t o r complexes d i r e c t l y I n t e r a c t w i t h DNA t o d e r e p r e s s p h y t o a l e x i n b l o s y n t h e t l c genes o r whether o t h e r second messengers e x i s t . F o r I n s t a n c e , work by Hadwlger (76) s u g g e s t s t h a t e l l d t o r s o r e l 1 d t o r - r e c e p t o r complexes may d i r e c t l y I n t e r a c t w i t h p l a n t DNA, thus changing I t s t r a n s c r i p t i o n . Recent e x p e r i m e n t s 1n C h r i s Lamb's l a b o r a t o r y have a l s o I l l u m i n a t e d the b a s i s f o r gene d e r e p r e s s i o n . Phaseolus p h e n y l a l a n i n e ammonia l y a s e and c h a l c o n e s y n t h a s e genes were found t o c o n t a i n DNA elements upstream o f t h e c o d i n g r e g i o n s t h a t f u n c t i o n e d a s s i l e n c e r and a c t i v a t o r r e g i o n s ( s e e 5 8 ) . These r e g i o n s a r e assumed t o I n t e r a c t w i t h as y e t u n i d e n t i f i e d p r o t e i n s t o a c t i v a t e gene e x p r e s s i o n 1n response t o e l l d t o r s o r I n o c u l a t i o n w i t h I n c o m p a t i b l e pathogen r a c e s . S e v e r a l groups have o b t a i n e d d a t a I n d i c a t i n g t h a t changes 1n c a l c i u m f l u x from e l 1 c 1 t o r - t r e a t e d p l a n t c e l l s may s e r v e a s a second messenger 1n p h y t o a l e x i n I n i t i a t i o n (77-80). The a l t e r e d c a l c i u m f l u x may In t u r n be caused by r a p i d d e p o l a r i z a t i o n / p o l a r i z a t i o n and redox p e r t u r b a t i o n o f t h e p l a n t c e l l membrane (56; 81-85). For I n s t a n c e , p r o d u c t i o n o f s u p e r o x i d e a n i o n , hydrogen p e r o x i d e and h y d r o x y l r a d i c a l s have been observed In p l a n t t i s s u e s undergoing h y p e r s e n s i t i v e r e a c t i o n s (86-89). Uses o f E l l d t o r s E l l d t o r s have p r e v i o u s l y been u s e f u l 1n s t u d i e s on t h e mechanisms o f p l a n t d e f e n s e and promise g r e a t e r u t i l i t y 1n t h e f u t u r e . This s h o u l d be e s p e c i a l l y t r u e a s a d d i t i o n a l r a c e s p e c i f i c e l l d t o r s a r e I s o l a t e d and t h e i r e f f e c t s t e s t e d on I n c o m p a t i b l e p l a n t g e n o t y p e s . In a d d i t i o n , e l l d t o r s may prove u s e f u l 1n o t h e r a s p e c t s o f p l a n t biology. Some o f t h e e s t a b l i s h e d and p o t e n t i a l uses o f e l l d t o r s a r e shown I n T a b l e I .

6. KEEN

Table I. 1. 2. 3. 4. 5. 6.

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As agents f o r the s p e c i f i c I n d u c t i o n o f p l a n t e n z y m a t i c pathways l e a d i n g to p h y t o a l e x i n s ( 3 , 4 ) . For the p r o d u c t i o n o f e x o t i c secondary m e t a b o l i t e s by c u l t u r e d p l a n t c e l l s ( 1 9 , 9 0 ) . S c r e e n i n g p l a n t c u l t i v a r s and b r e e d i n g U n e s f o r pathogen r e s i s t a n c e ( 9 1 , 9 2 ) . For s t u d i e s on r e c e p t o r b i o l o g y 1n p l a n t c e l l s ( 6 6 , 7 3 ) . In s t u d i e s on p l a n t - p a t h o g e n s p e c i f i c i t y and c e l l - c e l l s i g n a l l i n g (57,58). F o r use as s p e c i f i c h e r b i c i d e s ( 9 3 ) .

E l l d t o r s have been w i d e l y used 1n s t u d i e s o f d e f e n s e gene a c t i v a t i o n ( f o r r e v i e w s see 3 , 5 8 ) . S i n c e I t 1s s i m p l e r t o t r e a t p l a n t c e l l s w i t h e l l d t o r s , as opposed t o I n t a c t p a t h o g e n s , r e s u l t s a r e not compromised by o t h e r components o f the p a t h o g e n . There has been the t e n d e n c y , however, t o assume t h a t 1f a s u b s t a n c e causes I n d u c t i o n o f e a r l y d e f e n s e genes such as p h e n y l a l a n i n e ammonia l y a s e , then 1t Is n e c e s s a r i l y a p h y t o a l e x i n e l l d t o r . Ebel e t a l . (94) showed, however, t h a t some s u b s t a n c e s which a c t i v a t e e a r l y d e f e n s e genes do not l e a d t o p h y t o a l e x i n a c c u m u l a t i o n . Therefore, e l l d t o r s should be c a r e f u l l y checked t o ensure t h a t they i n f a c t a c t i v a t e p h y t o a l e x i n pathways. There has r e c e n t l y been c o n s i d e r a b l e I n t e r e s t 1n the use o f e l l d t o r s t o a c t i v a t e b l o s y n t h e t l c pathways l e a d i n g t o r e l a t i v e l y e x o t i c p l a n t secondary m e t a b o l i t e s ( 1 9 , 9 5 - 9 8 ) . I n d e e d , 1t may be p o s s i b l e t o e f f i c i e n t l y I s o l a t e r a r e m e t a b o l i t e s from c u l t u r e d p l a n t c e l l s f o l l o w i n g t h e i r exposure t o a p p r o p r i a t e e l l d t o r s . For example, T y l e r e t a l . (90) used f u n g a l e l l d t o r s t o produce s a n g u l n a r l n e from Papaver c e l l s u s p e n s i o n c u l t u r e s and reused the same c e l l s w i t h a new round o f medium and e l l d t o r . Such semi c o n t i n u o u s p r o d u c t i o n schemes may p e r m i t the economic I s o l a t i o n o f r a r e secondary metabo l i t e s t h a t behave as p h y t o a l e x i n s . W h i l e η ο η - s p e d f l c e l l d t o r s may have u t i l i t y 1n s e l e c t i n g d i s e a s e and I n s e c t r e s i s t a n t b r e e d i n g l i n e s ( 9 1 , 9 2 ) , race s p e c i f i c e l i c i t o r s would seem t o have the g r e a t e s t p o t e n t i a l use i n t h i s area. Thus, b r e e d i n g U n e s s e g r e g a t i n g f o r a c e r t a i n d i s e a s e r e s i s t a n c e gene c o u l d be screened r e l a t i v e l y e a s i l y 1f a s p e c i f i c e l l c i t o r complementing the r e s i s t a n c e gene was a v a i l a b l e . As noted e a r l i e r , l i m i t e d e x p e r i m e n t a l r e s u l t s I n d i c a t e t h a t p l a n t r e c e p t o r s may p e r c e i v e e l l c i t o r s t i m u l i . Race specific e l l d t o r s a r e a l s o b e i n g used as a f f i n i t y probes f o r i s o l a t i o n o f the c o r r e s p o n d i n g p l a n t r e c e p t o r s . These 1n t u r n a r e p o s t u l a t e d t o be the p r i m a r y p r o d u c t s o f p l a n t d i s e a s e r e s i s t a n c e g e n e s , and t h e i r I s o l a t i o n would p r e s e n t a r a t i o n a l e f o r c l o n i n g the r e s i s t a n c e genes. Plant-pathogen s p e c i f i c i t i e s Involving e i t h e r s p e c i f i c resistance or general r e s i s t a n c e c l e a r l y Involve d i f f e r e n t i a l r e c o g n i t i o n of pathogens. In some cases t h i s may be more c o m p l i c a t e d than pathogen d i s p l a y o f a c o n s t i t u t i v e m e t a b o l i t e t h a t 1s r e c o g n i z e d by the resistant plant. For I n s t a n c e , c e r t a i n b a c t e r i a l a v l r u l e n c e genes

126


are I n d u d b l y expressed f o l l o w i n g I n f e c t i o n o f the p l a n t . This I m p l i e s t h a t e i t h e r t h e s p e c i f i c p l a n t environment o r c e r t a i n p l a n t s i g n a l m o l e c u l e s may be r e q u i r e d t o a c t i v a t e t h e e x p r e s s i o n o f a v l r u l e n c e genes. The s p e c i f i c e l l d t o r , v i c t o r l n , was c o n s i d e r e d f o r many y e a r s t o be a h o s t s e l e c t i v e t o x i n , u n t i l 1 t was shown t o I n t e r a c t w i t h the Pc-2 d i s e a s e r e s i s t a n c e gene by e l i c i t i n g t h e h y p e r s e n s i t i v e response 1n o a t p l a n t s . S i m i l a r l y , the peptide e l l d t o r associated w i t h t h e ACf9 a v l r u l e n c e gene o f Cladosporlum fulvum and t h e avrt e l l d t o r c o u l d be c o n s i d e r e d a s h o s t s p e c i f i c t o x i n s 1 f I n f o r m a t i o n was n o t a v a i l a b l e U n k i n g them t o r e s i s t a n c e e x p r e s s i o n . Since p u r i f i e d p r e p a r a t i o n s o f a l l t h e s e e l l d t o r s cause e x t e n s i v e n e c r o s i s on s e n s i t i v e p l a n t c u l t i v a r s , 1 t may be p o s s i b l e t o use t h e e l l d t o r s themselves o r s u i t a b l e d e r i v a t i v e s as s p e c i f i c h e r b i c i d e s ( 9 3 ) . For example, I n j e c t i o n o f t h e avru e l l d t o r I n t o a s i n g l e p r i m a r y soybean l e a f o f a s e n s i t i v e c u l t l v a r r e s u l t 1n n e c r o s i s n o t o n l y o f t h e I n f i l t r a t e d l e a f b u t a l s o t h e o t h e r p r i m a r y l e a f on t h e p l a n t (Tamakl, S . , and S t a y t o n , Μ., U n i v . o f Wyoming, u n p u b l i s h e d d a t a , 1989). T h i s I n d i c a t e s a degree o f s y s t e m i c ! t y f o r t h e e l l d t o r w h i c h would be u s e f u l 1n a p o t e n t i a l h e r b i c i d e . Acknowledgments The a u t h o r acknowledges t h e c o n t r i b u t i o n s o f S . T a m a k l , D. K o b a y a s h l , H. T h o r d a l - C h M s t e n s e n , S . G o l d , H. Shen, J . L o r a n g , D. G e r h o l t , B. S t a s k a w l c z and H. S t a y t o n and t h e N a t i o n a l S c i e n c e F o u n d a t i o n t o our work w i t h avr genes and t h e avrV e l l d t o r .

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Phytoalexins and Their Elicitors

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