Immunological Approaches to the Study of Plant Disease Resistance

Chapter

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Immunological Approaches to the Study of Plant Disease Resistance 1

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Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch023

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Arthur R. Ayers , Keith L. Wycoff , Patricia Loomis , Christine Sharetzsky , and Misty Bender 1

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Genetic Engineering Technology Program, Cedar Crest College, Allentown, PA 18104 Cellular and Developmental Biology Department, Harvard University, Cambridge, MA 02138 Study of complex biological phenomena i s often inhibited by lack of information on the identity of the molecules that mediate c r i t i c a l events. An example i s disease resistance, i n which plants recognize and respond to pathogens. Traditional immunochemical approaches using polyclonal antisera and, more recently, monoclonal antibodies, have been inadequate to elucidate pathogen antigens recognized by disease resistance gene products of the plant host. To overcome problems of tolerance and immunodominance, we have examined other more powerful immunological approaches including chemical immunosuppression (both general and specific), in vitro immunization and neonatal tolerization.

Disease resistance i n plants i s a complex phenomenon requiring control of hundreds of genes. The expression of genes involved in the accumulation of toxic secondary metabolites (phytoalexins), inhibitors of pathogen enzymes (protease and pectinase inhibitors), enzymes to degrade pathogen structural components (chitinase and endoglucanase) and plant wall components, is dependent on either direct recognition of pathogen constituents or action of these constituents on plant tissues (1). Thus, elaboration of plant defenses i s preceded by pathogen detection. Plants that succumb to pathogens are not deficient i n defenses, but rather are defective in their recognition a b i l i t y . The genes commonly identified by field tests as providing Current address: Plant Biology Laboratory, Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138 3

0097-6156/88/0379-0307$06.00/0 • 1988 American Chemical Society

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

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p r o t e c t i o n a g a i n s t a pathogen p e r m i t e x i s t i n g adequate defenses t o be m o b i l i z e d i n t h e p r e s e n c e o f t h e pathogen o f i n t e r e s t . These d i s e a s e r e s i s t a n c e genes a r e i d e n t i f i e d by t h e i r a b i l i t y t o i n t e r f a c e w i t h e x i s t i n g defenses and t h e r e b y c o n f e r pathogen-speci f i c res i stance.


Genetics o f Host-Pathogen I n t e r a c t i o n s The g e n e t i c r e l a t i o n s h i p between h o s t s and pathogens i s p r o v o c a t i v e i n b o t h i t s s i m p l i c i t y and i t s m o l e c u l a r ramifications. Each gene f o r d i s e a s e r e s i s t a n c e i n a p l a n t i s p a i r e d w i t h a complementary gene f o r a v i r u l e n c e i n t h e p a t h o g e n . O n l y a s i n g l e p a i r o f c o r r e s p o n d i n g genes among many i s r e q u i r e d to t r i g g e r the p l a n t ' s defenses. The g e n e r a l i t y o f t h e "Gene-for-Gene" r e l a t i o n s h i p i s thought t o e x t e n d from v i r u s e s and b a c t e r i a t o i n c l u d e f u n g a l pathogens, nematodes and i n s e c t p e s t s o f p l a n t s ( 4 ) . One m o l e c u l a r i n t e r p r e t a t i o n o f t h i s p r o f o u n d g e n e t i c r e l a t i o n s h i p i s t h a t p l a n t s produce r e c e p t o r s ( d i s e a s e r e s i s t a n c e gene p r o d u c t s ) t h a t b i n d pathogen m o l e c u l e s ( d i r e c t o r i n d i r e c t p r o d u c t s o f a v i r u l e n c e genes) and i n i t i a t e t h e s u i t e o f b i o c h e m i c a l r e s p o n s e s o b s e r v e d as r e s i s t a n c e ( 1 ) . F u n c t i o n o f R e s i s t a n c e Gene P r o d u c t s Knowledge o f t h e m o l e c u l a r a c t i o n o f d i s e a s e r e s i s t a n c e gene products w i l l determine the p o t e n t i a l a p p l i c a t i o n o f i s o l a t e d genes i n t r a n s g e n i c p l a n t s . The e x i s t e n c e o f d i s e a s e r e s i s t a n c e genes c a n now o n l y be d e t e c t e d b y t h e i n t e r a c t i o n o f p l a n t s w i t h a p p r o p r i a t e pathogens and t h u s l i m i t s i d e n t i f i c a t i o n o f needed r e s i s t a n c e genes t o h o s t p l a n t s . New genes f o r r e s i s t a n c e t o c o r n pathogens, f o r example, w i l l t h e r e f o r e , o n l y be found i n c o r n , b a s e d on t h e s e t y p e s o f i n f e c t i o n a s s a y s . T h i s approach i s o f l i m i t e d u t i l i t y , because b r e e d e r s a r e a l r e a d y v e r y s u c c e s s f u l a t e x p l o i t i n g e x i s t i n g s o u r c e s o f g e n e t i c v a r i a t i o n . What i s needed i s an u n d e r s t a n d i n g o f t h e f u n c t i o n o f t h e p r o d u c t s o f d i s e a s e r e s i s t a n c e genes, so t h a t t h e combined w o r l d p l a n t germ p l a s m c a n be e x p l o i t e d a s a s o u r c e f o r new r e s i s t a n c e genes. L i m i t a t i o n s o f S t u d i e s o f Gene S t r u c t u r e . I t i s l i k e l y that w i t h i n t h e n e x t y e a r o r two t h e f i r s t p l a n t d i s e a s e r e s i s t a n c e gene w i l l be c l o n e d and sequenced. T h i s m o l e c u l a r knowledge w i l l q u i c k l y l e a d t o t h e sequence o f t h e p r o t e i n gene p r o d u c t and i n f o r m a t i o n on t h e l o c a l i z a t i o n o f t h i s p r o t e i n , presumably on t h e c y t o p l a s m i c membrane. I t may a l s o be p o s s i b l e t o b e g i n t o p i e c e t o g e t h e r t h e i n t e r a c t i o n s w i t h o t h e r p l a n t components t h a t l e a d t o e l i c i t a t i o n o f p l a n t defenses. B u t a major p i e c e o f t h i s p u z z l e w i l l s t i l l be l a c k i n g ; what pathogen components i n t e r a c t w i t h t h i s p u t a t i v e membrane r e c e p t o r ? I t may be p o s s i b l e f o r us t o have v e r y d e t a i l e d i n f o r m a t i o n about a r e s i s t a n c e gene and i t s p r i m a r y gene p r o d u c t , b u t s t i l l n o t be a b l e t o use t h i s gene o u t s i d e o f t h e s p e c i e s from w h i c h i t was i s o l a t e d .


23. AYERSETAL.

Plant Disease Resistance

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A v i r u l e n c e genes have been i s o l a t e d a n d c h a r a c t e r i z e d ( 9 ) . Gene p r o d u c t s have been i d e n t i f i e d , b u t a r e n o t found o u t s i d e o f the pathogen. I t i s l i k e l y t h a t t h e gene p r o d u c t has e n z y m a t i c a c t i v i t y and y i e l d s a n i n d i r e c t p r o d u c t t h a t a c t u a l l y m e d i a t e s pathogen r e c o g n i t i o n b y i n t e r a c t i o n w i t h r e s i s t a n c e gene p r o d u c t s . But h e r e t o o , i n - d e p t h knowledge o f t h e gene a n d gene p r o d u c t does not l e a d d i r e c t l y t o a s t r a t e g y f o r implementing r e s i s t a n c e genes. What i s needed i s t h e i d e n t i t y o f t h e pathogen m o l e c u l e , p o t e n t i a l l y t h e i n d i r e c t p r o d u c t o f pathogen a v i r u l e n c e genes, t h a t mediates r e c o g n i t i o n by r e s i s t a n c e gene p r o d u c t s . C a r b o h y d r a t e s May M e d i a t e R e c o g n i t i o n . C a r b o h y d r a t e s have been p o s t u l a t e d t o mediate pathogen r e c o g n i t i o n and p l a n t s have been d e m o n s t r a t e d t o be e x q u i s i t e l y s e n s i t i v e t o a n d s p e c i f i c i n t h e i r b i n d i n g o f pathogen w a l l fragments, such a s t h e g l u c a n e l i c i t o r (1). The s t r u c t u r e o f t h e g l u c a n e l i c i t o r i s t o o s i m p l e t o a c c o u n t f o r t h e numerous a v i r u l e n c e genes p o s s e s s e d b y f u n g a l pathogens, s o t h e e l i c i t o r i s o b v i o u s l y n o t a s s o c i a t e d d i r e c t l y w i t h t h e a c t i o n s o f a v i r u l e n c e genes. O t h e r c a r b o h y d r a t e s , such as t h e complex o l i g o s a c c h a r i d e s t h a t d e c o r a t e t h e s u r f a c e s o f e x t r a c e l l u l a r fungal g l y c o p r o t e i n s , a r e b e t t e r candidates as the m e d i a t o r s o f r e s i s t a n c e . One p o s s i b i l i t y i s t h a t a v i r u l e n c e genes code f o r g l y c o s y l t r a n s f e r a s e s t h a t s p e c i f i c a l l y t r a n s f e r carbohydrate residues t o non-reducing terminal g l y c o s y l residues o f g l y c o p r o t e i n s . G l y c o p r o t e i n s would t h e n s e r v e a s c a r r i e r s r e f l e c t i n g the e x p r e s s i o n o f the complete a r r a y o f a v i r u l e n c e genes o f t h e pathogen ( 1 ) . F u n c t i o n May Lead t o B r o a d A p p l i c a t i o n i n T r a n s g e n i c C r o p P l a n t s . I f p l a n t r e s i s t a n c e genes s e r v e a s r e c e p t o r s f o r pathogen g l y c o s y l r e s i d u e s , knowledge o f t h i s f u n c t i o n i s v i t a l t o t h e deployment o f r e s i s t a n c e genes i n new t r a n s g e n i c c r o p v a r i e t i e s . S o u r c e s o f r e s i s t a n c e c o u l d be c h a r a c t e r i z e d b y r e c e p t o r s t h a t would b i n d t o pathogen g l y c o p r o t e i n s . Because i t i s l i k e l y t h a t t h e i n t e r f a c e between r e s i s t a n c e genes and defenses i s h i g h l y c o n s e r v e d , i t w i l l be p o s s i b l e t o use r e s i s t a n c e genes from a n y p l a n t a s l o n g a s t h e desired binding function i s present. Thus, b a s e d on f u n c t i o n , i t w i l l be p o s s i b l e t o u s e r e s i s t a n c e genes i d e n t i f i e d i n soybeans o r p o t a t o e s , f o r example, t o p r o v i d e needed r e s i s t a n c e t o c o r n pathogens. Immunological I d e n t i f i c a t i o n o f A n t i g e n s A s s o c i a t e d w i t h A v i r u l e n c e Genes A n t i b o d i e s p r o v i d e the p o t e n t i a l a n a l y t i c a l s e n s i t i v i t y and d i s c r i m i n a t i o n needed t o i d e n t i f y t h e s u b t l e b i o c h e m i c a l d i f f e r e n c e s between pathogen i s o l a t e s p o s s e s s i n g d i f f e r e n t a v i r u l e n c e genes, i . e . d i f f e r e n t pathogen r a c e s . Since avirulence genes a r e o b l i g a t o r i l y l i n k e d t o r e s i s t a n c e genes, i t f o l l o w s t h a t t h e s t r u c t u r e s a s s o c i a t e d w i t h p a r t i c u l a r a v i r u l e n c e genes a r e l i k e l y t o be t h o s e bound t o t h e p u t a t i v e r e s i s t a n c e gene r e c e p t o r .


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I f these assumptions are correct, i t should be possible to produce antibodies s p e c i f i c f o r each antigen associated with an avirulence gene and the s p e c i f i c i t y of these antibodies may mimic that of resistance gene products. Attempts to Produce Monospecific Polyclonal Antisera. Early characterization of antisera of mice and rabbits challenged with mycelial extracts and e x t r a c e l l u l a r glyoproteins of Phytophthora megasperma f.sp. glycinea (ftng), a fungal pathogen, showed that these components were very immunogenic, but antibodies raised to one race bound just as r e a d i l y to antigens from another (5). Attempts to create antisera s p e c i f i c f o r the immunizing race by absorption to antigens from another race l e d to a complete loss o f binding a c t i v i t y — a l l of the antibodies raised to one race bound to antigens from another. This was our f i r s t hint that a few antigens common to a l l races of the fungus were dominating the immunological response. Many other antigens were simply not y i e l d i n g antibodies i n the presence of the dominant antigens. Monoclonal Antibodies from Murine Hybridomas. Murine hybridomas are hybrid c e l l s formed by the fusion of a mouse cancer c e l l with lymphocytes committed to the secretion of antibodies. The lymphocytes are obtained from the spleens of mice immunized with antigens of i n t e r e s t , e.g. e x t r a c e l l u l a r glycoproteins from a fungal plant pathogen, and under normal circumstances would produce antibodies, but would be unable to divide. Fusion with cancer c e l l s y i e l d s hybrids that continue to divide, as well as secrete antibodies. Thus, from a population of lymphocytes from the spleen of an immunized mouse i t i s possible to generate numerous clones of hybridomas and each clone w i l l y i e l d a single antibody, i . e . monoclonal antibodies. A l i b r a r y of more that 50 monoclonal antibodies (mAbs) was created f o r the study of antigens p o t e n t i a l l y associated with the avirulence genes of Pmg (10). These antibodies were placed i n s i x groups based on binding patterns i n Western b l o t s of glycoproteins. In a l l but one case the antibodies bound to complex patterns of glycoproteins, suggesting an i n t e r a c t i o n with the oligosaccharides common to various groups o f glycoproteins. The carbohydrate nature of the dominant antigenic domains was further confirmed by the s e n s i t i v i t y of antibody binding to reagents and enzymes that modify the carbohydrate residues and not the protein component of glycoproteins. There was only a single example of a protein antigen for which mAbs were i s o l a t e d . These studies and further work evaluating the a b i l i t y of pairs o f mAbs to compete for the same antigenic determinants (K.L. Wycoff and A.R. Ayers, unpublished r e s u l t s ) , indicates that the e x t r a c e l l u l a r glycoproteins of t h i s fungus are decorated with only a small group of d i f f e r e n t oligosaccharides. Moreover, these oligosaccharides are the dominant antigenic determinants i n both e x t r a c e l l u l a r culture f i l t r a t e s and p u r i f i e d mycelial walls.


23. AYERSETAL.


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None of the mAbs i n our l i b r a r y showed q u a l i t a t i v e differences i n binding t o antigens from d i f f e r e n t races. The patterns of binding on Western b l o t s of glycoproteins from d i f f e r e n t races are d i f f e r e n t , but probably r e f l e c t random differences i n the proteins of various i s o l a t e s , rather than differences associated with p a r t i c u l a r avirulence genes. We think that i t i s l i k e l y that the antigenic determinants that make up the oligosaccharides of the e x t r a c e l l u l a r glycoproteins are i n antigenic competition. As a consequence of t h i s competition, antigenic determinants common to a l l races y i e l d antibodies, whereas the determinants associated with avirulence genes do not.

Deficiencies of Immunological

Approaches

Antibodies and mAbs i n p a r t i c u l a r are powerful tools f o r analysis of molecular structures, but complete exploitation of potential immunological approaches requires an assessment of the l i m i t a t i o n s , as well as the strengths o f the techniques. I t i s often assumed that any antigen injected into a mouse w i l l y i e l d antibodies. This i s not the case. Thus, an obvious strategy would be to s t a r t with polyclonal antisera, absorb out antibodies recognizing antigens common to two d i f f e r e n t races of pathogen, and y i e l d monospecific antisera. But, t h i s approach i s dependent on the production of antibodies f o r each of the antigens injected. Uniform production of antibodies to each determinant i n a mixture of i n j e c t antigens does not occur f o r several reasons. Immunodominance. Antigen presentation to the immune system i s a complex phenomenon, many steps and c e l l types are involved before antibody production begins. Antigens can i n essence compete f o r the l i m i t e d presentation system and some antigens can dominate the system to such an extent that other antigens f a i l to generate an immune response. In order to observe responses to a l l determinants i n a mixture, response to the dominant antigens must be controlled. Tolerance. Mice may also f a i l to respond to p a r t i c u l a r antigenic determinants, because those determinants are recognized as s e l f . It would not be surprising to find that many of the carbohydrate antigenic determinants of fungal glycoproteins are also present i n mice. We have already observed that some of our mAbs s p e c i f i c f o r pathogen carbohydrate residues bind to mouse immunoglobulins (K.L. Wycoff and A.R. Ayers unpublished r e s u l t s ) . I f antigenic determinants are recognized as s e l f , no antibodies are produced and the mice are said to be "tolerant"• We expect that some avirulence genes might be c r y p t i c because of tolerance, but we expect the majority of the antigenic determinants to be i d e n t i f i a b l e through antibody production.


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G e n e r a l Immunosuppression. A n t i b o d y p r o d u c t i o n b y B lymphocytes r e q u i r e s exposure t o a n t i g e n and subsequent c e l l d i v i s i o n . Reagents, such as a l k y l a t i n g drugs, t h a t i n t e r f e r e w i t h c e l l d i v i s i o n , e l i m i n a t e a n t i g e n - s t i m u l a t e d a n t i b o d y p r o d u c t i o n . These r e a g e n t s c a n f u n c t i o n a s g e n e r a l immunosuppressants. W i t h a g e n e r a l immunosuppressant, s u c h as c y c l o p h o s p h a m i d e , i t i s p o s s i b l e t o make a mouse t o l e r a n t t o an i n j e c t e d a n t i g e n , s o t h a t subsequent exposure t o t h e same a n t i g e n w i l l n o t r e s u l t i n antibody production (8). We have t e s t e d t h i s approach u s i n g p a i r s o f p r o t e i n s w i t h and w i t h o u t f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) a s an added a n t i g e n i c determinant. I n two s e p a r a t e s e r i e s o f e x p e r i m e n t s , we used e i t h e r b o v i n e serum a l b u m i n o r c a s e i n i n i n i t i a l i n j e c t i o n s f o l l o w e d by cyclophosphamide t r e a t m e n t . A f t e r two weeks, t h e m i c e r e c e i v e d immunizing i n j e c t i o n s o f e i t h e r t h e same p r o t e i n , o r t h e i n i t i a l p r o t e i n d e r i v i t i z e d w i t h F I T C . A second i d e n t i c a l immunizing i n j e c t i o n was g i v e n two weeks l a t e r and s e r a were e v a l u a t e d b y enzyme immunoassay a f t e r two more weeks. Control mice r e c e i v i n g no p r e t r e a t m e n t p r i o r t o t h e immunizing i n j e c t i o n s , p r o d u c e d a n t i b o d i e s t h a t bound t o p r o t e i n w i t h and w i t h o u t FITC d e r i v i t i z a t i o n . Response t o FITC on h e t e r o l o g o u s p r o t e i n s was detectable. A n t i b o d i e s from mice r e c e i v i n g s u p p r e s s i o n p r o t o c o l s showed g r e a t l y reduced b i n d i n g t o t h e p r o t e i n u s e d i n t h e s u p p r e s s i o n t r e a t m e n t , and enhanced b i n d i n g t o FITC p r o t e i n . S u b s t a n t i a l enhancement o f b i n d i n g t o FITC on h e t e r o l o g o u s p r o t e i n s was o b s e r v e d . These e x p e r i m e n t s i n d i c a t e t h a t g e n e r a l immunosuppression c a n be a v e r y p o w e r f u l t o o l f o r p r o d u c i n g t o l e r a n c e t o dominant a n t i g e n i c d e t e r m i n a n t s and e n h a n c i n g antibody p r o d u c t i o n t o minor a n t i g e n i c determinants.

We have a l s o a t t e m p t e d t o use g e n e r a l immunosuppression i n the search f o r a v i r u l e n c e gene-associated antigens ( 2 ) . In these e x p e r i m e n t s a n t i g e n s from one r a c e o f Pmg were u s e d f o r s u p p r e s s i o n w i t h c y c l o p h o s p h a m i d e , and a n t i g e n s from a second r a c e were u s e d f o r i m m u n i z a t i o n . A n t i s e r a showed a c l e a r enhancement f o r b i n d i n g t o t h e immunizing r a c e r e l a t i v e t o t h e s u p p r e s s i n g r a c e when compared b y E L I S A . Hybridomas d e r i v e d from t h e s e m i c e y i e l d e d a n t i b o d i e s i n t h e f i r s t s c r e e n i n g t h a t bound o n l y t o t h e immunizing a n t i g e n s . The r a c e - s p e c i f i c hybridomas a s a group showed s i m i l a r r e s p o n s e l e v e l s and were u n i f o r m l y r e c a l c i t r a n t t o c l o n i n g — t h e y c o u l d n o t be m a i n t a i n e d i n c e l l c u l t u r e . Subsequent s i m i l a r e x p e r i m e n t s have f a i l e d t o g i v e a s s t r i k i n g r e s u l t s i n terms o f enhancement o f b i n d i n g t o t h e immunizing r a c e a t t h e a n t i s e r u m l e v e l and a t t e m p t s t o g e n e r a t e r a c e - s p e c i f i c a n t i b o d i e s u s i n g t h i s methodology have been u n s u c c e s s f u l .



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S p e c i f i c Immunosuppression. T o x i c compounds c a n be l i n k e d t o an antigen to y i e l d conjugates t h a t can s e l e c t i v e l y e l i m i n a t e i m m u n o l o g i c a l response t o t h e a n t i g e n . We have u s e d c o p o l y m e r s o f D - g l u t a m i c a c i d and D - l y s i n e i n c o n j u g a t e s w i t h b i o t i n as a model t o t e s t t h i s approach o f s p e c i f i c immunosuppression. M i c e p r e t r e a t e d w i t h b i o t i n c o n j u g a t e d t o t h e copolymer were t o l e r a n t t o subsequent immunizing i n j e c t i o n s o f b i o t i n y l a t e d BSA — a n t i b o d i e s from t h e s e mice showed no b i n d i n g t o b i o t i n y l a t e d p l a t e surfaces i n ELISA. Mice without the s u p r e s s i n g pretreatment produced a n t i b o d i e s showing s u b s t a n t i a l b i o t i n b i n d i n g i n t h e same assay. S p e c i f i c immunosuppression shows g r e a t p o t e n t i a l f o r e l i m i n a t i n g t h e i n t e r f e r e n c e o f dominant a n t i g e n s i n i m m u n o l o g i c a l studies. F o r example, we b e l i e v e i t w i l l be p o s s i b l e t o use c o n j u g a t e s o f g l y c o p e p t i d e s w i t h t h e copolymer t o e l i m i n a t e t h e response t o t h e dominant a n t i g e n s common t o s e v e r a l d i f f e r e n t r a c e s o f Pmg, s o t h a t t h e mice c a n respond t o o t h e r a n t i g e n s , presumably i n c l u d i n g a v i r u l e n c e g e n e - a s s o c i a t e d a n t i g e n s . I n V i t r o I m m u n i z a t i o n . The c o n s t r a i n t s p l a c e d on i m m u n i z a t i o n b y t h e complex p r e s e n t a t i o n system o f t h e a n i m a l c a n be a v o i d e d b y exposure o f lymphocytes t o a n t i g e n s i n c u l t u r e ( 3 ) . We have performed p r e l i m i n a r y e x p e r i m e n t s ( K . L . Wycoff and A . R . A y e r s , u n p u b l i s h e d r e s u l t s ) u s i n g Pmg a n t i g e n s ( e x t r a c e l l u l a r g l y c o p r o t e i n s and p u r i f i e d m y c e l i a l w a l l s ) f o r i n v i t r o i m m u n i z a t i o n p r i o r t o hybridoma p r o d u c t i o n . A n t i b o d i e s from t h e hybridomas d e r i v e d from mice c h a l l e n g e d w i t h one r a c e o f Pmg were e v a l u a t e d f o r b i n d i n g t o a n t i g e n s from o t h e r r a c e s . None o f t h e a n t i b o d i e s was found t o be r a c e - s p e c i f i c . Examples o f s e v e r a l o f t h e mAbs from t h e s e e x p e r i m e n t s a r e b e i n g e v a l u a t e d f o r c o m p a r i s o n t o mAbs produced by i m m u n i z a t i o n i n v i v o . I n a d d i t i o n a l e x p e r i m e n t s , i n v i t r o i m m u n i z a t i o n was u s e d t o produce m o n o c l o n a l a n t i b o d i e s t o glucomannans p u r i f i e d from Pmg. Glucomannans have been s u g g e s t e d t o have r a c e - s p e c i f i c c h a r a c t e r i s t i c s ( 7 ) . As p a r t o f t h i s p r o j e c t , a s s a y s f o r m e a s u r i n g b i n d i n g o f a n t i b o d i e s t o glucomannans were d e v e l o p e d u s i n g e i t h e r i m m o b i l i z a t i o n o f glucommanans found a t t a c h e d t o p r o t e i n s i n s t a n d a r d enzyme immunoassays o r i m m o b i l i z a t i o n t h r o u g h r e d u c i n g t e r m i n i a v a i l a b l e on some glucommannans a f t e r o x i d a t i o n w i t h i o d i n e / p o t a s s i u m i o d i d e t r e a t m e n t under a l k a l i n e c o n d i t i o n s and l i n k a g e o f t h e r e s u l t i n g c a r b o x y 1 group t o Immulon C p l a t e s (Dynatech). A n o t h e r a s s a y approach t e s t e d was b i o t i n y l a t i o n o f glucomannans f o l l o w i n g p e r i o d a t e o x i d a t i o n . The o x i d i z e d glucomannans were b i o t i n y l a t e d w i t h b i o t i n h y d r a z i d e . B i n d i n g o f a n t i b o d i e s t o b i o t i n y l a t e d glucomannan was q u a n t i t a t e d b y b i n d i n g o f the t e s t a n t i b o d i e s t o immobilized goat anti-mouse I g , a d d i t i o n o f t h e b i o t i n y l a t e d glucomannan, and f i n a l a d d i t i o n o f s t r e p t a v i d i n - a l k a l i n e phosphatase c o n j u g a t e . These e x p e r i m e n t s showed d r a m a t i c d i f f e r e n c e s (10-20 f o l d ) i n t h e r e l a t i v e b i n d i n g o f some m o n o c l o n a l a n t i b o d i e s i n o u r Pmg


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l i b r a r y (10) to glucomannan from d i f f e r e n t races of Ftag. For example, while antibody KW2 showed an equivalent binding to glucomannans from Pmg races 1 and 7, MF7 showed a 20-fold preference f o r binding to glucomannan from race 7 r e l a t i v e to race 1. Antibodies generated by i n v i t r o immunization with Rug glucomannan on the other hand, showed no preferences of more than two f o l d . Similar r e s u l t s were obtained whether the assay used glucomannans immobilized through linked protein, through covalent linkage following oxidation or using b i o t i n y l a t e d glucomannans. Neonatal T o l e r i z a t i o n . Another potential strategy to eliminate response to dominant antigens and permit production of antibodies only to determinants that d i f f e r i n two antigen mixtures i s to expose mice to antigens before t h e i r immune systems reach maturity (6). Neonatal mice exposed to antigens w i l l be tolerant to the same antigens when challenged as adults. Exposure of neonatal mice to antigens from one race of Pmg should permit production of antibodies s p e c i f i c to a second race of Pmg when injected with antigens from that race at a l a t e r time. Another p r a c t i c a l application of t h i s strategy i s exposure of neonatal mice to antigens from organisms that commonly show crossreactions i n programs involved i n the development of antibodies diagnostic f o r p a r t i c u l a r pathogens. In preliminary experiments (S. Marshall and A.R. Ayers unpublished results) we have observed that the crossreaction of Phythium ultimum antigens with antibodies produced i n response to Pmg antigens may be g r e a t l y reduced by pretreatment of neonatal mice with P. ultimum. Conclusion We have focused on the use of antibodies f o r i d e n t i f i c a t i o n of the molecules that mediate pathogen recognition and lead to disease resistance i n plants. The immunological approaches described here should permit i d e n t i f i c a t i o n of avirulence gene-associated antigens whether those antigens are proteins or carbohydrates. Although the genetic evidence appears to favor carbohydrates as the pathogen components that mediate recognition by plant hosts, we continue to consider protein antigens as candidates. Several very powerful immunological approaches, including general and s p e c i f i c immunosuppression, i n v i t r o immunization, and neonatal t o l e r i z a t i o n have been i d e n t i f i e d as being of potential a p p l i c a b i l i t y . Our tests of these approaches have revealed that the techniques work well, but they also reveal the complexity of pathogens and the inadequacy of our knowledge. Examination of Pmg antigens by the production of a l i b r a r y of monoclonal antibodies revealed that less than a dozen antigens account f o r e s s e n t i a l l y the t o t a l immunological response to a mixture of Pmg e x t r a c e l l u l a r glycoproteins. This response can, however, be modified by immunological techniques that suppress antibody production to p a r t i c u l a r antigens. Even with these r e l a t i v e l y sophisticated tools, i t has proven d i f f i c u l t to screen


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for antibodies that can be used to identify the antigenic determinants associated with particular avirulence genes. Part of the difficulty may be technical. For example, i t i s d i f f i c u l t to identify antibodies that bind to antigens present as only a small percentage of a mixture of total antigens. Moreover, i t may not be possible to remove uninteresting antigens, because of linkage to the antigenic determinants of interest. Acknowledgment


Supported i n part by the United States Department of Agriculture (85-CRCR-1-1632). Literature Cited 1.

Ayers, A.R.; Goodell, J.J.; DeAngelis, P. In The Biochemical interactions of Plants with Other Organisms, Recent Advances in Phytochemistry, Volume 19; Conn, E . E . ; Cooper-Driver G; Swain, T., eds.; Plenum Press, 1985, pp. 1-20. 2. Ayers, A.R; Wycoff, K.L. In Molecular Strategies for Crop Protection; Arntzen, C.J.; Ryans C . , eds.; Alan R. Liss, Inc.: New York, 1987, pp. 71-81. 3. Boss, B.D. In Immunochemical Techniques, Part I, Hybridoma Technology and Monoclonal Antibodies, Methods i n Enzymology, Volume 121; Langone, J.J.; Van Vunakis, H eds.; Academic Press, Inc.: New York, 1986, pp. 27-33. 4. Day, P.R. Genetics of Host-Pathogen Interaction; W.H. Freeman and Company: San Francisco, 1974. 5. Goodell, J.J.; DeAngelis, P.; Ayers, A.R. In Cellular and Molecular Biology of Plant Stress, UCLA Symposia on Molecular and Cellular Biology, New Series, Volume 22; Key, J.L.; Kosuge, T., eds.; Alan R. Liss, Inc.: New York, 1984, pp. 447-457. 6. Hockfield S. Science 1987, 69:67-70. 7. Keen, N.T.; Yoshikawa, Y; Wang, M.C. Plant Physiol. 1983, 71,466-471. 8. Mathew, W.D.; Patterson, P.H. Cold Spring Harbor Symposium on Quantitative Biology. 1983, 48,625-531. 9. Swanson, J ; Kearney, B; Dahlbeck, D; Staskawicz, B. Molec. Pl.-Microbe Interact. 1988, 1,5-9. 10. Wycoff, K . L . ; Jellison, J.; Ayers, A.R. Plant Physiol. 1987, 85,508-515. RECEIVED May 31, 1988


Immunological Approaches to the Study of Plant Disease Resistance

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