Plant Cell Wall Polymers - American Chemical Society

Chapter 28

Virulence-Inducing Phenolic Compounds Detected by Agrobacterium tumefaciens Paul A. Spencer and G. H. N. Towers

Downloaded by UNIV QUEENSLAND on April 20, 2013 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch028

Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 2B1, Canada

Construction of vir::lacZ fusion reporter genes and subsequent analysis of their expression in strains of Agrobacterium tumefaciens has permitted the discovery of a class of phytochemicals that this pathogen detects and which induce virulence. Preliminary screening of a variety of commercially available phenolics revealed that some were active as vir inducers. Two acetophenones were isolated from transformed tobacco root cultures. The results of a recent study by us indicate that there exists a range of virulence-inducing plant phenolics which are not limited to acetophenones but include chalcones as well as cinnamic acid derivatives. Among the latter are acids, alcohols and esters known to be associated with plant cell walls or implicated in lignin biosynthesis, a discovery which suggests that this wide host range pathogen likely responds to chemicals common to all susceptible hosts. We are currently studying signal compounds and natural inhibitors in relation to the host range of Agrobacterium strains. Activity was detected in extracts from a grapevine tissue culture, grapevine bark, and flavan-containing fractions obtained from grapes. In addition, as yet unidentified compounds inhibitory to vir -induction have been discovered. It is n o w k n o w n t h a t the i n i t i a l i n t e r a c t i o n between p l a n t s a n d b a c t e r i a of the R h i z o b i a c e a e is a c h e m i c a l d e t e c t i o n b y the m i c r o b e o f a s u s c e p t i ble host, i . e . , t h e host produces c o m p o u n d s w h i c h act as signals for the m i c r o b i a l p a t h o g e n o r s y m b i o n t . T h e m i c r o b e responds t o these signals b y expression o f genes necessary i n subsequent stages o f the i n t e r a c t i o n . F o r a few o f the R h i z o b i a c e a e some s i g n a l c o m p o u n d s i n v o l v e d have been identified (1-7). 0097-6156/89/0399-0383$06.00A) © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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T h e s i g n a l c o m p o u n d s o f p l a n t - Agrobacterium tumefaciens i n t e r a c t i o n s have received m u c h a t t e n t i o n . T h i s g r a m negative s o i l b a c t e r i u m , w h i c h causes c r o w n g a l l disease o f a w i d e variety o f dicotyledonous p l a n t s (8), is responsible for a neoplastic g r o w t h of the p l a n t tissue b y passing T - D N A , a p a r t o f its t u m o r i n d u c i n g p l a s m i d ( p T i ) , into the host p l a n t genome (9-14). T h i s T - D N A includes genes w h i c h encode enzymes of a u x i n (15,16) a n d c y t o k i n i n (17,18) biosynthesis, w h i c h are expressed i n the t r a n s f o r m e d p l a n t cell (19,20). T h e m i c r o b e is a useful vector for genetic engineering i n p l a n t s because c e r t a i n of the n o r m a l T - D N A genes m a y be replaced w i t h new genes o f interest. P l a n t cells infected w i t h the b a c t e r i u m c o n t a i n i n g the m o d i f i e d T i - p l a s m i d are used to generate transgenic p l a n t s . D e t e c t i o n of susceptible host cells a n d e a r l y stages of tumorogenesis are m a i n l y c o n t r o l l e d by a set of p T i genes k n o w n as the v i r u l e n c e (vir) genes (21,22). T h e s e genes are expressed u p o n c o c u l t i v a t i o n of the b a c t e r i a w i t h host p l a n t cells (23,24) . Because of their role i n the early stages of tumorogenesis, a n d therefore their c e n t r a l i m p o r t a n c e i n t r a n s f o r m a t i o n of p l a n t genomes, research has been directed t o u n d e r s t a n d i n g the m e c h a n i s m i n v o l v e d i n vir gene expression a n d i d e n t i f y i n g the v i r gene p r o d u c t s . T w o o f the vir genes ( A a n d G ) are r e g u l a t o r y i n n a t u r e (25,26). vir A is also a host range d e t e r m i n a n t a n d is thought to be the e n v i r o n m e n t a l sensor of the p l a n t - d e r i v e d inducer molecules (27). A t least one more vir locus (virC) is connected w i t h host range (28-30), a n d another (the virO o p e r o n ) is now k n o w n to encode a n endonuclease w h i c h recognizes a n d cleaves the left a n d right b o r d e r sequences of T - D N A (31). T h e virB region encodes p o l y p e p tides s i m i l a r t o those i n v o l v e d i n b a c t e r i a l c o n j u g a t i o n (32). R e c e n t l y i t was d e t e r m i n e d t h a t virE encodes a single s t r a n d e d D N A - b i n d i n g p r o t e i n (33). A c t i v a t i o n of v i r gene expression is k n o w n to result i n the p r o d u c t i o n o f m u l t i p l e s i n g l e - s t r a n d e d T - D N A molecules w i t h i n the b a c t e r i u m (34). B o l t o n et al. (35) f o u n d t h a t a m i x t u r e of s i m p l e , low m o l e c u l a r weight p h e n o l i c c o m p o u n d s c o u l d be used to i n d u c e expression of most of the vir genes. S t a c h e l et al. (7) i d e n t i f i e d two active s i g n a l c o m p o u n d s , acetosyringone ( A S ) a n d α-hydroxyacetosyringone ( H O - A S ) , f r o m tobacco tissues. In t h a t r e p o r t a few other related c o m p o u n d s were assayed at one or more c o n c e n t r a t i o n s for t h e i r v i r - i n d u c i n g a c t i v i t y . T h i s c o m p r i s e d a very b r i e f s t r u c t u r e - a c t i v i t y s t u d y w h i c h presented some i n f o r m a t i o n about the s t r u c t u r a l features r e q u i r e d to confer a c t i v i t y . A t the concentrations tested, none of these c o m p o u n d s d i s p l a y e d the level of a c t i v i t y observed w i t h acetosyr i n g o n e . It was suggested t h a t Agrobacterium is a t t r a c t e d to susceptible p l a n t tissues b y f o l l o w i n g a c o n c e n t r a t i o n gradient o f these virulence i n d u c i n g substances, a n d some results w h i c h s u p p o r t t h i s i d e a were o b t a i n e d b y A s h b y et al. (36,37). T h e c o m p o u n d s A S a n d H O - A S have come to be regarded as the u n i q u e c h e m i c a l s w h i c h Agrobacterium detects i n n a t u r e a n d w h i c h trigger the i n i t i a l events w i t h i n the b a c t e r i u m , r e s u l t i n g i n t u m o r f o r m a t i o n . However, it has yet t o be s h o w n t h a t these acetophenones, w h i c h i n fact have never pre v i o u s l y been r e p o r t e d as n a t u r a l l y o c c u r r i n g p h y t o c h e m i c a l s , are the s i g n a l c o m p o u n d s p r o d u c e d by any other susceptible hosts. A S has been used to


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boost the t r a n s f o r m a t i o n efficiency (38). H o w e v e r , t r a n s f o r m a t i o n of soyb e a n cells was p r o m o t e d b y a d d i n g either A S or s y r i n g a l d e h y d e [ l b ] to the i n o c u l u m (39). I n a d d i t i o n , v i r u l e n c e i n d u c i n g w o u n d exudates o b t a i n e d f r o m a host p l a n t e x t e n d e d the n o r m a l host range of Agrobacierium to i n clude a m o n o c o t crop p l a n t (40). W e propose t h a t o t h e r p h y t o c h e m i c a l s are i n v o l v e d i n the i n d u c t i o n o f v i r u l e n c e i n Agrobacterium. W e recently r e p o r t e d the v i r - i n d u c i n g a c t i v i t y over a range of concent r a t i o n s of a v a r i e t y of p l a n t - d e r i v e d p h e n o l i c c o m p o u n d s w i t h s t r u c t u r e s related to t h a t of acetosyringone a n d discussed the s t r u c t u r a l features necessary for the a c t i v a t i o n o f v i r genes (41). T h e a c t i v i t i e s of some c i n n a m i c a c i d d e r i v a t i v e s , chalcones, a n d of the l i g n i n precursors s i n a p y l a l c o h o l a n d c o n i f e r y l a l c o h o l were e x a m i n e d . A n u m b e r of these c o m p o u n d s are of w i d e s p r e a d occurrence, a n d others such as the m o n o l i g n o l s are u b i q u i t o u s i n angiosperms a n d g y m n o s p e r m s . In t h i s r e p o r t we review the results of o u r s t r u c t u r e - a c t i v i t y a n a l y s i s of v i r - i n d u c t i o n a n d discuss some p r e l i m i n a r y results o f o u r search for s i g n a l c o m p o u n d s for a g r a p e v i n e i s o l a t e o f A. tumefaciens. R e s e a r c h has revealed t h a t the v i r A gene p r o d u c t is l o c a t e d at the b a c t e r i a l cell surface where i t l i k e l y acts as the e n v i r o n m e n t a l sensor of p l a n t d e r i v e d s i g n a l c o m p o u n d s (27). T h e v i r A l o c i of l i m i t e d host range ( L H R ) a n d w i d e host range ( W H R ) s t r a i n s of Agrobacterium were sequenced a n d the p r e d i c t e d gene p r o d u c t s c o m p a r e d . T h e gene p r o d u c t s were f o u n d to have diverged most s t r o n g l y i n t h e i r p u t a t i v e p e r i p l a s m i c d o m a i n . T h e r e fore we considered t h a t differences i n these gene p r o d u c t s m i g h t c o r r e s p o n d to differences i n t h e i r specificity for s i g n a l c o m p o u n d s , a n d t h i s i n p a r t m a y e x p l a i n the differences observed i n the host range of these two types of Agrobacterium. T o prove t h i s h y p o t h e s i s , i n d u c t i o n of other vir l o c i i n the presence of L H R host p l a n t cells, or b y w o u n d exudates thereof, h a d t o be d e m o n s t r a t e d . T h e s i g n a l c o m p o u n d s t h e n h a d t o be i s o l a t e d a n d i d e n t i f i e d . I n i t i a l l y , however, o n l y a v i r B : : / a c Z gene-fusion c o n t a i n i n g L H R s t r a i n o f Agrobacterium ( A 8 5 6 / p S M 2 4 3 c d ) was a v a i l a b l e . I n o u r p r e l i m i n a r y e x p e r i m e n t s w i t h t h i s s t r a i n , v i r expression was n o t g r e a t l y i n d u c e d b y c o c u l t i v a t i o n w i t h host p l a n t cells or b y p u r i f i e d w o u n d - i n d u c e d p h e n o l i c c o m p o u n d s ; therefore we w i s h e d to e x a m i n e v i r - i n d u c t i o n i n a different c o n s t r u c t , n a m e l y , the L H R s t r a i n c a r r y i n g a lacL f u s i o n to a different vir gene. W e have p r e p a r e d a v i r E : : / a c Z gene f u s i o n - c o n t a i n i n g L H R s t r a i n , A 8 5 6 / p S M 3 5 8 c d , b y t r i p a r e n t a l m a t i n g a n d have used t h i s s t r a i n , a l o n g w i t h the s t r a i n A 8 5 6 / p S M 2 4 3 c d , to e x a m i n e v i r gene expression i n the l i m i t e d host range (grapevine) s t r a i n A 8 5 6 . P h e n o l i c w o u n d exudates f r o m the leaves a n d stems of Vitis lubrusca, as w e l l as exudates p r o d u c e d by t w o g r a p e v i n e c a l l u s cultures ( Vitis sp. cv. S e y v a l a n d V. lubruscana cv. S t e u b e n ) a n d e x t r a c t s o b t a i n e d f r o m two varieties o f grapes (red a n d green seedless) a n d g r a p e v i n e b a r k (cv. C o n c o r d ) were e x a m i n e d for v i r - i n d u c i n g c o m p o u n d s . W e have established t h a t the specificity of L H R s i g n a l c o m p o u n d s is u n l i k e t h a t p r e v i o u s l y described for W H R A. tumefaciens (41) i n t h a t vir gene expression is not as g r e a t l y i n d u c e d b y acetosyringone.



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T h i s i n d i c a t e s t h a t , u n l i k e the W H R s t r a i n s of Agrobacterium, the L H R s t r a i n s are less sensitive to p h e n y l p r o p a n o i d m e t a b o l i t e s . P r e l i m i n a r y results i n d i c a t e t h a t the v i r u l e n c e of grapevine isolates of Agrobactenum may be influenced b y the presence o f c e r t a i n higher m o l e c u l a r weight p h e n o l i c esters of grape flavans i n a d d i t i o n to less p o l a r , c h l o r o f o r m soluble phenolics present i n aqueous g r a p e v i n e - s t e m w o u n d exudates. A n u n d e r s t a n d i n g of the p h y t o c h e m i s t r y of vir gene expression i n b o t h W H R (7,35,41) a n d i n L H R Agrobacterium s h o u l d p r o v i d e an i n t e r e s t i n g a n d p o t e n t i a l l y useful m o d e l s y s t e m of host range c o n t r o l i n p l a n t - b a c t e r i a l i n t e r a c t i o n s . N e w insights i n t o r e g u l a t i o n of host range are of i m p o r t a n c e i n p l a n t b i o c h e m i s t r y , biotechnology a n d p a t h o l o g y i n t h a t c h e m i c a l clues are p r o v i d e d w h i c h c o u l d allow for extension of t h i s pathogen's host range t o i n c l u d e species w h i c h are refractory to t r a n s f o r m a t i o n . Results and Discussion In o u r a n a l y s i s o f the c h e m i c a l s t r u c t u r e s w h i c h are a c t i v e vir-inducers (41) i t was f o u n d t h a t the c o m p o u n d s fell i n t o four groups: (1) acetophenones a n d related s t r u c t u r e s , (2) m o n o l i g n o l s , (3) h y d r o x y c i n n a m i c acids a n d t h e i r esters, a n d (4) chalcone derivatives ( F i g . 1). E a c h c o m p o u n d h a d either a g u a i a c y l or a s y r i n g y l nucleus, a n d w i t h the exception of the m o n o l i g n o l s , possessed a c a r b o n y l g r o u p . M o s t were of c o m m o n occurrence i n vascular p l a n t s . T h e a c t i v i t y curves w i t h increasing c o n c e n t r a t i o n of a n u m b e r of viri n d u c i n g p h e n o l i c c o m p o u n d s are s h o w n i n F i g u r e s 2 a ( m o n o l i g n o l s , c h a l cones a n d acetophenones) a n d 2b (phenolic acids a n d their m e t h y l esters). R e g a r d i n g the m o n o l i g n o l s , we e m p h a s i z e d the b a c t e r i u m ' s a b i l i t y to res p o n d t o the presence of these l i g n i n precursors. T h i s result established t h a t Agrobactenum m a y be capable of d e t e c t i n g cells w h i c h are u n d e r g o i n g l i g n i n synthesis or cell w a l l repair a n d thereby target those cells for t r a n s f o r m a t i o n . Agrobacterium responded e q u a l l y w e l l to the presence of l i g n i n d e g r a d a t i o n p r o d u c t s (7,35,41) a n d therefore the virulence of the m i c r o b e c a n be considered as sensitive to l i g n i n metabolites i n general. T h e u n i q u e a c t i v i t y curves of the chalcones [ 4 a & 4 b ] represent an i n t e r e s t i n g a d d i t i o n to the list of effective phenolics. T h e m e t h y l esters of ferulic [3d], s y r i n g i c [ I f ] , a n d s i n a p i c acids [3f] e x h i b i t e d s i g n i f i c a n t l y greater a c t i v i t y t h a n the c o r r e s p o n d i n g free acids ( F i g . 2b). Some effects of esterification are discussed below. T h e e t h y l esters tested were less a c t i v e a g a i n ( d a t a not s h o w n ) , p e r h a p s due to altered h y d r o p h i l i c i t y of the c o m p o u n d or some steric h i n d r a n c e at the b a c t e r i a l receptor site not evident w i t h the m e t h y l esters. In a p l a t e assay for viri n d u c t i o n we discovered t h a t the glucose ester of ferulic a c i d was a n effective i n d u c e r of vir gene expression. It is likely t h a t such glucose esters, released u p o n w o u n d i n g of the host p l a n t , act as s i g n a l c o m p o u n d s . P h e n o l i c g l y cosides, such as glucoferulaldehyde, were i n a c t i v e . W e assayed a n u m b e r o f other phenolic c o m p o u n d s , i n c l u d i n g aurones, flavones, flavanones, flavanols, lignans a n d 5 - h y d r o x y c i n n a m i c a c i d d e r i v a tives, b u t they a l l d i s p l a y e d l i t t l e or no a c t i v i t y . T h e lack of a c t i v i t y of


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587

R2 H

H

la

OMe

b

H

c

CH

3

d

CH

3

H OMe

e

OH

OMe

f

OMe

OMe

2a b

R H OMe

3a b c d e f

Ri CH OH OH OMe OMe OMe

CH OH


2

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3

Ri Η Η OMe Η OH OMe

OMe .OH HO.

4a b

R Η OMe

OH F i g u r e 1. T h e s t r u c t u r e s of the w r - i n d u c i n g p h e n o l i c c o m p o u n d s e m p l o y e d i n o u r s t r u c t u r e - a c t i v i t y a n a l y s i s (41).



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CONCENTRATION (μΜ) F i g u r e 2. T h e virulence i n d u c i n g a c t i v i t y o f (a) m o n o l i g n o l s , chalcones, a n d acetophenones, a n d (b) phenolic acids a n d their m e t h y l esters. F o l l o w i n g i n c u b a t i o n w i t h a c o m p o u n d i n aqueous s o l u t i o n , /?-galactosidase a c t i v i t y i n a s t r a i n o f Agrobacterium c a r r y i n g a virE.lacZ fusion p l a s m i d ( A 3 4 8 / p S M 3 5 8 ) was assayed as a n i n d i c a t o r o f v i r gene i n d u c t i o n . Abréviations used: A S = acetosyringone; C O N . A L C O H O L = coniferyl alcohol; S I N . A L C O H O L = sinapyl alcohol; C H A L C O N E A = 2',4',4-trihydroxy3 - m e t h o x y chalcone; C H A L C O N E Β = 2', 4', 4 - t r i h y d r o x y - 3 , 5 d i m e t h o x y chalcone.


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5 - h y d r o x y f e r u l i c a c i d was of interest because i t was recently i d e n t i f i e d as one of the cell w a l l b o u n d acids i n m o n o c o t s (42). A t t h a t t i m e we h a d not d e m o n s t r a t e d i n h i b i t i o n of v i r - i n d u c t i o n b y a p h e n o l i c c o m p o u n d , a l t h o u g h we s p e c u l a t e d a b o u t the occurrence of p h e n o l i c w r - i n h i b i t o r s i n m o n o c o t s . Indeed, we have been i n f o r m e d of u n i d e n t i f i e d w r - i n h i b i t o r s recently iso l a t e d f r o m Zea mays ( E . W . N e s t e r , p e r s o n a l c o m m u n i c a t i o n ) , a n d have i n i t i a t e d w o r k o n the i d e n t i f i c a t i o n of w r - i n h i b i t o r s f r o m Vitis species. I n a d d i t i o n to the i n a c t i v e c o m p o u n d s listed above, each of the c o m p o u n d s used b y B o l t o n et ai (35) was assayed i n d i v i d u a l l y , a n d of these o n l y v a n i l l i n [ l a ] p r o d u c e d a n y significant v i r - i n d u c t i o n . Interestingly, at the c o n c e n t r a t i o n s e x a m i n e d by us the r e m a i n i n g c o m p o u n d s (gallic, βr e s o r c y l i c , p y r o g a l l i c , p - h y d r o x y b e n z o i c , a n d p r o t o c a t e c h u i c acids, a n d c a t echol) were essentially i n a c t i v e . N o n e of these i n a c t i v e c o m p o u n d s has a g u a i a c y l or s y r i n g y l nucleus. W e have observed low level i n d u c t i o n b y g a l l i c a c i d at higher c o n c e n t r a t i o n s (e.g., I m M , d a t a not s h o w n ) . T h e possible significance of t h i s result w i t h respect to v i r - i n d u c t i o n b y grape flavans is discussed below. T h e results i n d i c a t e d t h a t two basic s t r u c t u r a l features were r e q u i r e d t o confer a c t i v i t y u p o n a c o m p o u n d : (1) g u a i a c y l or ( u s u a l l y c o n f e r r i n g enhanced a c t i v i t y ) s y r i n g y l s u b s t i t u t i o n on a benzene r i n g , a n d (2) a carb o n y l g r o u p o n a s u b s t i t u e n t p a r a to the h y d r o x y s u b s t i t u e n t o n the r i n g . M o n o l i g n o l s , however, are a c t i v e even t h o u g h there is no c a r b o n y l f u n c t i o n i n the side c h a i n i n the p a r a p o s i t i o n . W h e n present, the c a r b o n y l c a r b o n m a y be one or three c a r b o n a t o m s removed f r o m the r i n g . H o w e v e r , to confer m a x i m a l a c t i v i t y , i n the l a t t e r case there m u s t be a double b o n d between the c a r b o n y l c a r b o n a n d the r i n g , as is present i n the chalcones a n d c i n n a m i c a c i d derivatives. F u r t h e r m o r e , the c a r b o n y l g r o u p of a free a c i d is less effective t h a n t h a t of the c o r r e s p o n d i n g ester. E s t e r i f i c a t i o n a l ters the s o l u b i l i t y of the c o m p o u n d . In a d d i t i o n , esterification prevents one o x y g e n of the c a r b o x y l g r o u p f r o m f o r m i n g a p a r t i a l d o u b l e b o n d , thereby r e n d e r i n g the c a r b o n y l group more reactive. In these cases, a n d the case of the aldehydes a n d chalcones, t h i s c a r b o n y l g r o u p forms the t e r m i n u s of a c o n j u g a t e d double b o n d s y s t e m r u n n i n g f r o m the h y d r o x y l g r o u p a n d t h r o u g h the r i n g . T h e presence of a C r i n g i n the flavonoids tested v i r t u a l l y a b o l i s h e d a c t i v i t y , i n d i c a t i n g t h a t the more t y p i c a l flavonoids are not a c t i v e i n t h i s cell-cell s i g n a l l i n g . T h e s e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s are different f r o m those r e p o r t e d for the a c t i v a t i o n of nod genes i n Rhizobium species (1-6). H y d r o x y l a t e d flavones, isoflavones or flavanones i n n M to μ Μ c o n c e n t r a t i o n s i n d u c e ex p r e s s i o n of nod genes. E a c h Rhizobium species is not o n l y h i g h l y specific for its host p l a n t species b u t also d i s p l a y s a h i g h degree of specificity towards its s i g n a l c o m p o u n d . I n c o n t r a s t , the o r i g i n a l s t r a i n of A. tumefaciens, from w h i c h the s t r a i n used i n our s t u d y was d e r i v e d , e x h i b i t e d a w i d e host range ( W H R ) a n d , as we have seen, a c o m p a r a t i v e l y lower degree of s i g n a l c o m p o u n d specificity. F u r t h e r m o r e , some of the very c o m p o u n d s w h i c h induce vir genes i n Agrobacterium s t r o n g l y i n h i b i t nod gene a c t i v a t i o n by these flavonoids (1). A t higher concentrations most of the w r - i n d u c i n g phenolics


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were b a c t e r i o s t a t i c even against Agrobacterium ( d a t a not s h o w n ) , a n d pre s u m a b l y they act i n t h i s way against Rhizobium species, or they m a y act m o r e d i r e c t l y b y c o m p e t i t i v e i n h i b i t i o n of n o d - i n d u c t i o n . A n u m b e r o f these a c t i v e c o m p o u n d s are of widespread occurence d i cotyledons. T h e l i g n i n precursors are u b i q u i t o u s i n susceptible hosts. It is t e m p t i n g t o conclude t h a t the presence o f a n y one of these c o m p o u n d s alone w o u l d determine whether a given p l a n t is susceptible to i n f e c t i o n b y Agrobacterium. However, m o n o c o t s also p r o d u c e these c o m p o u n d s , even e x u d i n g t h e m i n t o the rhizosphere f r o m i n t a c t roots (43) a n d yet, w i t h few exceptions (44-47), they lie outside of the n a t u r a l host range of a n y s t r a i n of Agrobacterium. T h i s l i m i t a t i o n of host range r e m a i n s a significant p r o b l e m i n the use o f t h i s o r g a n i s m as a vector for genetic engineering i n m o n o c o t s . T h e a t t a c h m e n t of Agrobacterium to m o n o c o t cells has been r e p o r t e d (48, a n d references t h e r e i n ) . T h e r e f o r e the u n d e r l y i n g m e c h a n i s m of host range d e t e r m i n a t i o n appears t o d e p e n d , at least i n p a r t , o n the p h y t o c h e m i s t r y o f the i n t e r a c t i o n . T h e recently established presence of w r - i n h i b i t o r s i n a m o n o c o t a n d , as w i l l be discussed, L H R host exudates s u p p o r t s the concept o f a p h e n o l i c m i l i e u i n w h i c h v i r - i n d u c e r s c o m p e t e w i t h v i r - i n h i b i t o r s . It m a y be t h a t a s o p h i s t i c a t e d a p p l i c a t i o n of inducer c o m p o u n d s w i l l p e r m i t the T i - p l a s m i d - m e d i a t e d t r a n s f o r m a t i o n o f p l a n t species n o r m a l l y resistant to infection. O u r i n i t i a l e x p e r i m e n t s , u s i n g /?-galactosidase a c t i v i t y i n A 8 5 6 / p S M 2 4 3 c d as a n i n d i c a t o r o f v i r - i n d u c t i o n , suggested t h a t w o u n d - i n d u c e d phenolics f r o m the leaves of V. lubrusca d i d not i n c l u d e any L H R s i g n a l c o m p o u n d s . W e considered the f o l l o w i n g p o s s i b i l i t i e s : (1) t h a t the source of the n a t u r a l L H R s i g n a l c o m p o u n d m i g h t be tissue specific (e.g., l i m i t e d t o the roots or c r o w n of the g r a p e v i n e ) , (2) t h a t u n l i k e the W H R s i g n a l c o m p o u n d s , these u n k n o w n chemicals m a y not be low m o l e c u l a r weight phenolics (e.g., p h e n y l p r o p a n o i d s or acetophenones) e x t r a c t able w i t h the solvents used, a n d (3) t h a t the s i g n a l c o m p o u n d c o u l d be a p h y t o a l e x i n p r o d u c e d o n l y after i n f e c t i o n o f the grapevine tissue. In order to c o n f i r m our results w i t h A 8 5 6 / p S M 2 4 3 c d we prepared the s t r a i n A 8 5 6 / p S M 3 5 8 c d as described. T h e p l a s m i d p S M 3 5 8 c d c o n t a i n s a virE::lacZ gene f u s i o n , a n d i t was f o u n d t h a t the higher levels of βgalactosidase a c t i v i t y w h i c h are i n d u c i b l e f r o m t h i s construct (35) p e r m i t ted d e t e c t i o n o f even v a n i s h i n g l y s m a l l a m o u n t s of i n d u c i n g c o m p o u n d . T h e results w i t h t h i s new s t r a i n confirmed our i n i t i a l results; neither acetosy ringone i t s e l f nor a n y of the isolated w o u n d - i n d u c e d p h e n o l i c c o m p o u n d s f r o m grape leaves g r e a t l y i n d u c e d the v i r u l e n c e genes of the L H R A. tume faciens. O b v i o u s l y either the techniques used to isolate c o m p o u n d s f r o m the host m a t e r i a l were not a p p r o p r i a t e for the i s o l a t i o n of L H R inducers, or the L H R s t r a i n cannot efficiently detect the W H R s i g n a l c o m p o u n d s due to i t s different vir A gene p r o d u c t . A s s u m i n g the former to be the case, we considered t h a t the L H R i n d u c ers m i g h t be m o r e p o l a r i n n a t u r e a n d were therefore e x c l u d e d b y s e p a r a t o r y techniques for c o m p o u n d s such as those k n o w n to i n d u c e W H R A. tumefa ciens. P r o a n t h o c y a n i d i n m o n o m e r s (i.e., catechins, flavan-3-ols), oligomers,


28.

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a n d esters s h o u l d be prevalent i n the w o o d y tissue o f the g r a p e v i n e . S u c h c o m p o u n d s have been s t u d i e d i n grapes w i t h respect t o w i n e q u a l i t y (49) a n d recently r e v i e w e d w i t h respect to t h e i r possible p h y s i o l o g i c a l role i n c o n n e c t i o n w i t h l i g n i n (50). I n v i e w of the c h e m i c a l s p e c i f i c i t y of v i r u l e n c e i n d u c t i o n i n W H R Agrobacterium (41), a feature w h i c h i n i t s s e n s i t i v i t y was n o t s h a r e d b y the L H R s t r a i n used i n t h i s s t u d y , i t was n o t e w o r t h y t h a t these flavans have been envisaged as f u n c t i o n a l l y connected w i t h l i g n i n . I n terestingly, i n a review o n p r o a n t h o c y a n i d i n s a n d l i g n i n c h e m i s t r y , Stafford (50) m e n t i o n e d the p h e n o m e n o n of p l a n t p h e n o l i c c o m p o u n d s as m o l e c u l a r signals for Rhizobium a n d Agrobacterium, a n d recognized the p o t e n t i a l " i n f o r m a t i o n a l " f u n c t i o n of b o t h types of c o m p o u n d s . W e a n a l y z e d the flavan-containing e x t r a c t s o b t a i n e d f r o m t w o l o c a l l y available varieties of grapes a n d detected a c t i v i t y i n the f r a c t i o n s c o n t a i n i n g , a m o n g other p h e n o l i c s , flavan m o n o m e r s , d i m e r s a n d esters. T h e s e c o m p o n e n t s were separated b y c h r o m a t o g r a p h y o n S e p h a d e x L H 20. F r a c t i o n s w i t h s i m i l a r t h i n layer c h r o m a t o g r a p h i c ( T L C ) profiles were p o o l e d a n d at least 3 o u t of 12 s u c h p o o l e d samples c o n t a i n e d substances w h i c h res u l t e d i n w r - i n d u c t i o n i n the L H R s t r a i n . S i m i l a r l y , g r a p e v i n e b a r k flavans were e x a m i n e d , b u t o n l y c o m p o u n d s i n d u c i n g very low levels of vir gene e x p r e s i o n were f o u n d . I n fact, i n a d d i t i o n to vz'r-inducers, a v i r - i n h i b i t o r y e x t r a c t was o b t a i n e d f r o m g r a p e v i n e b a r k . In p l a t e assays, t h i s L H R hostd e r i v e d i n h i b i t o r y substance c o m p l e t e l y prevented W H R w r - i n d u c t i o n by acetosyringone. R e p e a t e d T L C of a c t i v e , p o o l e d fractions f r o m R e d F l a m e grapes revealed m a j o r spots w i t h R / ' s c o r r e s p o n d i n g to those of c a t e c h i n a n d e p i c a t e c h i n , i d e n t i c a l color r e a c t i o n w i t h p-toluenesolfonic a c i d s p r a y reagent, a n d c o e l u t i o n o f t r i m e t h y l s i l a n e ( T M S ) d e r i v a t i v e s b y G C w i t h reference s a m p l e s of these flavans. H o w e v e r c a t e c h i n a n d e p i c a t e c h i n were assayed w i t h the b a c t e r i a l s t r a i n s described a n d no a c t i v i t y was d e t e c t e d . T M S d e r i v a t i z e d samples of a c t i v e grape flavans were e x a m i n e d b y G C - M S , b u t a search for the m o l e c u l a r ions of a n u m b e r of k n o w n v i r - i n d u c i n g phenolics y i e l d e d negative results. Isolated c o m p o u n d s were collected after s e p a r a t i o n by H P L C a n d assayed for w r - i n d u c t i o n i n the s t r a i n s d e s c r i b e d . It b e c a m e a p p a r e n t t h a t the l i m i t e d host range s t r a i n responded to a l l the substances t h a t i n d u c e d vir expression i n the w i d e host range s t r a i n , b u t the L H R s t r a i n was less sensitive to the same substances a n d as a result was considered a less sens i t i v e bioassay o r g a n i s m . Therefore work o n i s o l a t i o n of g r a p e v i n e - d e r i v e d s i g n a l c o m p o u n d s c o n t i n u e d u s i n g the more sensitive W H R s t r a i n a n d p l a t e assay s y s t e m d e s c r i b e d . U s i n g A 3 4 8 / p S M 3 5 8 as o u r bioassay o r g a n i s m w i t h w h i c h to detect the w r - i n d u c i n g substances, we i s o l a t e d b y H P L C the a c t i v e c o m p o u n d present i n a m i x t u r e o b t a i n e d f r o m gel filtration o n S e p h a d e x L H - 2 0 . B e t ter r e s o l u t i o n o f the c o m p o n e n t s of the grape flavan m i x t u r e was achieved b y gel f i l t r a t i o n w i t h S e p h a d e x G 25. I n t h i s w a y f r a c t i o n s e n r i c h e d for the i n d u c i n g c o m p o u n d s m a y be o b t a i n e d . T h i s m a y p r o v i d e samples f r o m w h i c h we c a n efficiently isolate new s i g n a l c o m p o u n d s i n sufficient q u a n -



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t i t y to p e r m i t s t r u c t u r e e l u c i d a t i o n . However, w i t h the d a t a at h a n d , we can m a k e the f o l l o w i n g educated guess as to the n a t u r e of the active s t r u c ture i n the active grape flavan m i x t u r e . C o n s i d e r i n g the facts t h a t g a l l i c a c i d was r e p o r t e d to induce v i r u l e n c e (35), t h a t epicatechin-gallate ( F i g . 3) was p r e v i o u s l y reported f r o m grapes (49), a n d t h a t esters of p h e n o l i c acids e x h i b i t e d enhanced a c t i v i t y , we suggest t h a t a flavan ester such as epicatechin-gallate is the active c o m p o n e n t i n our grape flavan f r a c t i o n s . Esters i n c l u d i n g phenolic acids w i t h g u a i a c y l or s y r i n g y l nuclei s h o u l d exh i b i t enhanced a c t i v i t y . T w o other sources of s i g n a l c o m p o u n d s f r o m L H R host tissues were f o u n d . O n e source was the S e y v a l callus c u l t u r e (see E x p e r i m e n t a l ) a n d the other was the aqueous exudate p r o d u c e d i n a b u n d a n c e u p o n c u t t i n g new grapevine stems i n the s p r i n g , when the sap flow was great. A n other grapevine callus c u l t u r e (obtained f r o m V. lubrascana cv. S t e u b e n , a n a t u r a l host of b o t h L H R a n d W H R Agrobacterium), its exudates, a n d fractions p a r t i t i o n e d t h e r e f r o m , as w e l l as exudates f r o m m a t u r e Nicotiana glauca p l a n t s , were i n c a p a b l e of i n d u c i n g vir gene expression i n any of the s t r a i n s used. A p p a r e n t l y not a l l callus cultures were e q u a l l y capable of p r o d u c i n g w r - i n d u c i n g m i x t u r e s of substances. P e r h a p s the two cultures differed i n the a m o u n t s of w r - i n h i b i t o r y substances p r o d u c e d . F i n a l l y , i n c o n s i d e r a t i o n of the n a t u r a l s e t t i n g i n w h i c h the L H R strains infect their host, we felt it w o r t h w h i l e to e x a m i n e the copious aqueous exudates of cut g r a p e v i n e stems. T h e c h l o r o f o r m soluble f r a c t i o n of such an e x u d a t e f r o m V. lubrusca was s t r o n g l y active i n a plate assay a n d is b e i n g further characterized. Conclusion In c o n c l u s i o n , b o t h w r - i n d u c i n g a n d w r - i n h i b i t o r y substances were p r o duced f r o m hosts a n d nonhosts of strains of A. tumefaciens. The comp o u n d s i n v o l v e d covered a range of p o l a r i t y a n d m o l e c u l a r weight, a n d t h i s likely reflects ester or other linkages between k n o w n lower m o l e c u l a r weight, w r - i n d u c i n g , c i n n a m i c a c i d derivatives a n d other organic c o m p o u n d s such as sugars a n d p r o a n t h o c y a n i d i n monomers (flavan-3-ols) or oligomers. It has been d e m o n s t r a t e d (7,35,41) t h a t A. tumefaciens is sensitive to the m o n o m e r s i n v o l v e d i n l i g n i n biosynthesis. T h e d a t a presented here suggest Agrobacterium m a y also be sensitive to c o m p o u n d s such as c a t e c h i n - or e p i c a t e c h i n - g a l l a t e , w h i c h l i n k s w r - i n d u c t i o n w i t h the m o n o m e r s of p r o cyanidin polymers. W e are c o n t i n u i n g our efforts to identify b o t h i n d u c i n g a n d i n h i b i t o r y c o m p o u n d s f r o m g r a p e v i n e c u l t i v a r s a n d other hosts of Agrobactenum. W i t h a more complete u n d e r s t a n d i n g of the c h e m i c a l signals i n v o l v e d , it s h o u l d be possible to induce T i - m e d i a t e d t r a n s f o r m a t i o n i n v i r t u a l l y a n y p l a n t species. C l e a r l y such a d i s t a n t goal w i l l also require a synthesis of d a t a f r o m a n u m b e r of fields of research.




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Experimental Bacterial Strains. In order to m o n i t o r w r - i n d u c t i o n i n the L H R s t r a i n A 8 5 6 a virEr.lacZ gene f u s i o n - c o n t a i n i n g p l a s m i d ( p S M 3 5 8 c d ) was i n t r o d u c e d b y t r i p a r e n t a l m a t i n g . T h i s was done so t h a t /?-galactosidase a c t i v i t y i n A 8 5 6 / p S M 3 5 8 c d c o u l d be assayed as an i n d i c a t o r of w r - i n d u c t i o n . T h i s p l a s m i d c o n t a i n e d the virE region of a W H R p T i i n the absence of other vir l o c i , so o n l y the L H R vir A gene p r o d u c t acted as the e n v i r o n m e n t a l sensor of s i g n a l c o m p o u n d s . T h e donor s t r a i n of E. coli ( J C 2 9 2 6 / p S M 3 5 8 c d ) was m a i n t a i n e d o n L B m e d i u m (51) c o n t a i n i n g 100 / i g / m L k a n a m y c i n . T h e s t r a i n o f E. coli w h i c h contained the helper p l a s m i d ( J C 2 9 2 6 / p R K 2 0 1 3 ) was m a i n t a i n e d o n L B m e d i u m c o n t a i n i n g 30 ^ g / m L s p e c t i n o m y c i n . A 8 5 6 was resistant to c h l o r a m p h e n i c o l , r i f a m p i c i n , a n d n a l i d i x i c a c i d (40 μ g / m L , 10 μ g / m L , a n d 20 μ g / m L , r e s p e c t i v e l y ) . Resistance to these a n t i b i o t i c s were used i n a d d i t i o n to k a n a m y c i n resistance to select for A 8 5 6 / p S M 3 5 8 c d . T h i s s t r a i n was m a i n t a i n e d o n A B m e d i u m (51) c o n t a i n i n g 100 μ g / m L k a n a m y c i n . I n a d d i t i o n , the L H R s t r a i n A 8 5 6 / p S M 2 4 3 c d ( p r o v i d e d by D r . E u g e n e N e s t e r , U n i v e r s i t y of W a s h i n g t o n ) was used to m o n i t o r vir gene i n d u c t i o n a n d was also m a i n t a i n e d on A B m e d i u m c o n t a i n i n g 100 μ g / m L kanamycin. Plant Materials. T h e callus cultures used i n t h i s e x p e r i m e n t ( Vitis sp. cv. S e y v a l a n d V. lubrascana cv. Steuben) were also p r o v i d e d by D r . Nester. H e a l t h y , m a t u r e leaves of a n u m b e r of Vitis c u l t i v a r s were o b t a i n e d f r o m the U n i v e r s i t y of B r i t i s h C o l u m b i a B o t a n i c a l G a r d e n s a n d also f r o m l o c a l p r i v a t e l y o w n e d vines. R e d seedless ( F l a m e red) a n d G r e e n seedless grapes, i m p o r t e d f r o m C h i l e , were o b t a i n e d f r o m a l o c a l grocery store. N. glauca seedlings were o b t a i n e d f r o m the A g r i c u l t u r e C a n a d a Research S t a t i o n at U . B . C . , a n d raised i n our greenhouse. Isolation of LHR ν'ιτ-Inducers. C o n d i t i o n e d m e d i u m was o b t a i n e d f r o m 5 varieties of Vitis b y c u t t i n g about 20 fresh leaves a n d stems i n t o 1 c m pieces a n d p l a c i n g t h e m i m m e d i a t e l y into 1.5 L of sterile p H 5.7 M u r a s h i g e a n d S k o o g ( M S ) m e d i u m (52). C o n d i t i o n e d M S m e d i u m f r o m callus cultures of the Vitis c u l t i v a r S t e u b e n was o b t a i n e d by b r e a k i n g up h e a l t h y c a l l i f r o m 6-8 p e t r i plates i n 500 m L of sterile p H 5.7 M S m e d i u m . A f t e r 8-12 hours at r o o m t e m p e r a t u r e , the p l a n t m a t e r i a l was removed a n d the c o n d i t i o n e d m e d i u m was filtered, then processed i m m e d i a t e l y . W o u n d i n d u c e d p h e n o l i c aglycones were p a r t i t i o n e d f r o m the c o n d i t i o n e d m e d i u m u s i n g three v o l umes each of e t h y l acetate or d i e t h y l ether, a n d phenolic glycosides were p a r t i t i o n e d f r o m the c o n d i t i o n e d m e d i u m u s i n g three volumes of n - b u t a n o l . T h e solvents were removed b y r o t a r y e v a p o r a t i o n a n d each e x t r a c t was resuspended i n a s m a l l v o l u m e of 100% m e t h a n o l . M e t h a n o l i c e x t r a c t s were m a d e d i r e c t l y f r o m healthy, m a t u r e c a l l i so t h a t a c o m p a r i s o n between the w o u n d i n d u c e d a n d n a t u r a l l y present c o m p o u n d s c o u l d be m a d e . T h e c o m p o u n d s present i n these m i x t u r e s were separated either by c o l u m n c h r o m a t o g r a p h y on p o l y a m i d e ( S C 6 - A C ) or by b a n d i n g on p r e p a r a tive p o l y a m i d e ( A C 6) T L C plates. E a c h b a n d was collected, the c o m p o u n d



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e l u t e d f r o m the p o l y a m i d e a n d t h e n tested for v i r - i n d u c t i o n i n the Agrobac tenum s t r a i n s A 8 5 6 / p S M 3 5 8 c d a n d A 8 5 6 / p S M 2 4 3 c d . G r a p e flavans were i s o l a t e d f r o m the t w o varieties of c o m m e r c i a l l y available grapes a n d f r o m the b a r k of Vitis g r a p e v i n e c u l t i v a r C o n c o r d ac c o r d i n g t o the m e t h o d s of C z o c h a n s k a et ai (46). CHCI3, E t O A c , a n d aqueous f r a c t i o n s o f b o t h grape varieties were assayed for v i r - i n d u c i n g ac t i v i t y i n b o t h A 8 5 4 / p S M 2 4 3 c d a n d A 8 5 6 / p S M 3 5 8 c d . T h e E t O A c frac t i o n s f r o m the flavan e x t r a c t i o n s o f b o t h varieties of grapes a n d b a r k were r e s u s p e n d e d either i n 1 0 0 % e t h a n o l or m e t h a n o l for c h r o m a t o g r a p h y on S e p h a d e x L H 20. T h e f r a c t i o n s were e x a m i n e d b y T L C o n cellulose ( M e r c k , 0 . 1 m m ) developed w i t h s e c - B u O H : A c O H : H 2 0 (14:1:5), a n d s i m i l a r frac t i o n s were p o o l e d a n d t h e n assayed for a c t i v i t y or a l t e r n a t i v e l y they were screened for a c t i v i t y a n d groups of active f r a c t i o n s were p o o l e d . G r a p e flavan f r a c t i o n s were also separated o n S e p h a d e x G 25 u s i n g 10 m M N a C l as the d e v e l o p i n g solvent. T L C o n cellulose ( s e c - B u O H : A c O H : H 0 ; 14:1:5) of a c t i v e , p o o l e d f r a c t i o n s revealed at least 5 or m o r e m a j o r c o m p o u n d s . G C showed t h a t there were c o n s i d e r a b l y m o r e c o m p o u n d s also present. H P L C of the active fractions was p e r f o r m e d o n a V a r i a n m o d e l 50200000 e q u i p p e d w i t h a n a n a l y t i c a l W a t e r s C - 1 8 c o l u m n (0.5 x 3 0 c m ) a n d u s i n g solvent A : 5 % acetic a c i d i n H 2 O , a n d B : a c e t o n i t r i l e , u n d e r the f o l l o w i n g c o n d i t i o n s 9 5 % A : 5 % B , 10 m i n . , c h a n g i n g to 2 5 % Β i n 10 m i n . , t h e n 3 0 % Β i n 10 m i n . a n d m a i n t a i n e d for 10 m i n . , a n d finally to 4 0 % i n 10 m i n . U V absorbance was m o n i t o r e d at 254 or 275 n m . T h e i s o l a t e d c o m p o u n d s were t e s t e d for v i r - i n d u c t i o n as described below. 2

v i r - I n d u c t i o n Assay. F o r the s t r u c t u r e - a c t i v i t y s t u d y , /?-galactosidase ac t i v i t y was assayed as a measure of vir-gene i n d u c t i o n i n a w i d e host range s t r a i n w h i c h c a r r i e d a virE.JacZ gene f u s i o n . T h e c o m p o u n d s tested were dissolved i n D M S O a n d d i l u t e d i n c i t r a t e - p h o s p h a t e buffered p H 5.70 M S m e d i u m (52) t o a final c o n c e n t r a t i o n of 0 . 1 % D M S O . 100 μΐ, of b a c t e r i a l cells f r o m a n o v e r n i g h t c u l t u r e of A 3 4 8 / p S M 3 5 8 (23) were i n o c u l a t e d i n t o each 25 x 1 5 0 m m c u l t u r e t u b e a n d s u b j e c t e d to c o n t i n u o u s s h a k i n g at 200 R P M a n d at 2 8 ° C for 8 hours to allow for i n d u c t i o n of virEv.lacl ex p r e s s i o n . C e l l d e n s i t y was d e t e r m i n e d b y m e a s u r i n g absorbance at 600 n m a n d 1 m l a l i q u o t s were r e m o v e d for /?-galactosidase assay essentially as d e s c r i b e d b y M i l l e r (53). E a c h p o i n t o n the a c t i v i t y curve of a test c o m p o u n d represented the av erage o f the results of each c o n c e n t r a t i o n tested i n t r i p l i c a t e . w r - I n d u c t i o n was s t r o n g l y p H dependent (24, o u r r e s u l t s , d a t a not s h o w n ) , so the buffer s y s t e m was used to m i n i m i z e v a r i a t i o n i n p H . S t a n d a r d d e v i a t i o n s r a r e l y reached 1 0 % , the average b e i n g 4 . 7 % (n = 92) for results of 100 M i l l e r u n i t s a n d above. I n the search for L H R w r - i n d u c e r s , each p h e n o l i c aglycone or other f r a c t i o n to be tested was dissolved i n D M S O a n d d i l u t e d i n p H 5.50 or 5.70 M S m e d i u m t o a final c o n c e n t r a t i o n of 0 . 1 % D M S O ( p h e n o l i c glycosides a n d the grape flavan f r a c t i o n s were dissolved d i r e c t l y i n ρ Η 5.50 or 5.70 M S m e d i u m ) . S u b s e q u e n t e x p e r i m e n t s revealed a more a c i d i c p H o p t i m u m for



396


w r - i n d u c t i o n i n t h e L H R s t r a i n , so some e x p e r i m e n t s were c o n d u c t e d at p H 5.0. S t a n d a r d s o f acetosyringone, c a t e c h i n , e p i c a t e c h i n , a n d a p r o a n t h o c y a n i d i n p o l y m e r were assayed for L H R w r - i n d u c t i o n . 100 μ L o f cells f r o m a n overnight c u l t u r e o f A 8 5 6 / p S M 3 5 8 c d or A 8 5 6 / p S M 2 4 3 c d was i n o c u l a t e d i n t o each 25 x 1 5 0 m m test t u b e c o n t a i n i n g 10 m L o f M S or 0 . 1 % D M S O - M S s o l u t i o n w i t h various concentrations o f t h e test substances, a n d a l l tubes were s u b j e c t e d t o 200 r p m for 10-24 h a n d at 28° C t o allow for i n d u c t i o n of vir expression. /?-Galactosidase a c t i v i t y was t h e n assayed as described b y M i l l e r (53). A l t e r n a t i v e l y , t h e f o l l o w i n g screening assay was used t o identify v i r - i n d u c i n g f r a c t i o n s . H P L C fractions were collected, reduced t o d r y ness under v a c u u m , resuspended i n a s m a l l a m o u n t o f M e O H a n d a few μΐ, o f each was a p p l i e d t o a filter p a p e r disc. T h e discs were p l a c e d o n a M 9 (51) agar p l a t e c o n t a i n i n g 0 . 1 % 5-bromo-4-chloro-3indolyl-/?-D-galactopyranoside (Xgal) with a lawn of A 8 5 6 / p S M 3 5 8 c d or A 8 5 6 / p S M 2 4 3 c d a n d the plates were i n c u b a t e d at 28° C for 2 4 h or u n t i l blue zones ( i n d i c a t i n g /?-galactosidase a c t i v i t y ) developed s u r r o u n d i n g a n y disc. Gas Chromatography. In our G C analyses, N , 0 - b i s - ( T r i m e t h y l s i l y l ) T r i f l u o r o a c e t a m i d e ( B S T F A ) - d e r i v a t i z e d s t a n d a r d s o f k n o w n w r - i n d u c i n g pheno lics f a i l e d t o correspond i n r e t e n t i o n t i m e t o a n y o f the d e r i v a t i z e d samples of t h e most active grape flavan fractions. C a t e c h i n a n d e p i c a t e c h i n were t e n t a t i v e l y identified by G C . Acknowledgments W e w o u l d like t o give s p e c i a l t h a n k s t o D r . Eugene W . Nester w h o p r o v i d e d the Agrobactenum strains A 3 4 8 / p S M 3 5 8 a n d A 8 5 6 / p S M 2 4 3 c d , a n d the b a c t e r i a l s t r a i n s f r o m w h i c h we prepared A 8 5 6 / p S M 3 5 8 c d . W e also t h a n k D o n C h a m p a g n e for useful discussions a n d Felipe B a l z a for c o n d u c t i n g mass spectroscopy. W e are grateful to L a c e y Samuels for p e r m i s s i o n t o use her S E M figures i n o u r s y m p o s i u m p r e s e n t a t i o n . P . A . S . was s u p p o r t e d b y a U n i v e r s i t y G r a d u a t e F e l l o w s h i p at the U n i v e r s i t y o f B r i t i s h C o l u m b i a . T h e research was f u n d e d b y the N a t u r a l Sciences a n d E n g i n e e r i n g Research Council of Canada. Literature Cited

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Plant Cell Wall Polymers - American Chemical Society

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