Chapter 2
Tentoxin: A Cyclic Tetrapeptide Having Potential Herbicidal Usage Alan R. Lax and Hurley S. Shepherd Microbial/Plant Technology Research Unit, Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, LA 70179
Tentoxin, a chlorosis-inducing cyclic tetrapeptide from Alternaria alternata, shows promise for use as a biorational herbicide and to identify novel target enzyme systems for the design of other selective herbicides. Specific effects on thylakoid and chloroplast envelope ATPases and on protein transport into the chloroplast have been identified as potential herbicidal target sites. A major impediment to development of tentoxin as a commerical herbicide is the limited production of the toxin by A. alternata. Identification of fungal viruses associated with tentoxin producing strains of the fungus provides some promise of increasing biosynthetic capabilities through traditional fermentation technology, semisynthetic production, or through bioengineering of organisms to provide economical levels of the toxin.
The w i d e s p r e a d u s e o f h e r b i c i d e s i n t h e a g r i c u l t u r e o f i n d u s t r i a l i zed n a t i o n s i s w e l l e s t a b l i s h e d . Continued a p p l i c a t i o n o f such compounds h a s l e d t o t h e s e l e c t i o n o f weeds h a v i n g r e s i s t a n c e t o e s t a b l i s h e d h e r b i c i d e s a n d t o weeds p h y s i o l o g i c a l l y more c l o s e l y r e s e m b l i n g t h e crop species they a f f e c t . Reduced t i l l a g e a g r i c u l t u r a l p r a c t i c e s have c r e a t e d a n i n c r e a s e d r e l i a n c e on h e r b i c i d e s w h i l e e n v i r o n m e n t a l c o n c e r n s d i c t a t e a more j u d i c i o u s u s e o f p o t e n t i a l l y d e t r i m e n t a l c o m p o u n d s . The c o s t o f h e r b i c i d e development v i a t r a d i t i o n a l approaches has r i s e n because o f t h e e x t r e m e n u m b e r s o f c o m p o u n d s w h i c h m u s t be s c r e e n e d t o y i e l d a successful product. Increasingly industry i s turning t o natural p r o d u c t s t o p r o v i d e new compounds o r c h e m i s t r i e s s a t i s f y i n g b o t h e f f i c a c y and safety c r i t e r i a f o r commercial development. One n e w s t r a t e g y i s t h e u s e o f p a t h o g e n i c m i c r o o r g a n i s m s i n a b i o l o g i c a l c o n t r o l program (1). Two s u c h h e r b i c i d e s , C o l l e g o a n d
This chapter not subject to U.S. copyright Published 1988 American Chemical Society
2. LAX AND SHEPHERD
Tentoxin: Potential Herbicide
25
D e v i n e , have been c o m m e r c i a l l y e x p l o i t e d but have e x p e r i e n c e d l i m i t e d use because o f t h e l i m i t e d g e o g r a p h i c a l d i s t r i b u t i o n and e c o n o m i c i m p o r t a n c e o f t h e i r weed h o s t s ( 1 , 2 ) . Other c o n s t r a i n t s t o d i r e c t m i c r o b i a l use i n v o l v e the l o n g e v i t y o f the propagules p r i o r t o a p p l i c a t i o n , p o s s i b l e e n v i r o n m e n t a l l i m i t a t i o n s , and storage s p a c e l i m i t a t i o n s ( 3 ) . The p o t e n t i a l f o r e x p l o i t a t i o n o f t h e s e b i o l o g i c a l h e r b i c i d e s h a s b e e n t h o r o u g h l y r e v i e w e d a n d w i l l n o t be considered f u r t h e r here (1-3). The p o t e n t i a l f o r t h e u s e o f m i c r o b i a l l y d e r i v e d t o x i n s a s p e s t i c i d e s has a l s o been thoroughly reviewed r e c e n t l y ( 2 , 3 ) . In the p r e s e n t r e v i e w , we i d e n t i f y one s u c h t o x i n f o r w h i c h c o m m e r c i a l development has been suggested f o r e x a m i n a t i o n i n g r e a t e r d e t a i l . T h i s r e v i e w i s m e a n t n o t t o be e n c y c l o p e d i c o r e x h a u s t i v e , b u t r a t h e r t o s u g g e s t a n d d e f i n e a r e a s o f r e s e a r c h w h i c h may p e r m i t f u r t h e r u t i l i z a t i o n of n a t u r a l products i n an i n t e g r a t e d pest c o n t r o l program. T e n t o x i n has been s e l e c t e d f o r c o n s i d e r a t i o n b e c a u s e o f i t s b r o a d a c t i v i t y on a number o f m a j o r weed s p e c i e s i n c o r n and soybean w i t h o u t any d i s c e r n a b l e e f f e c t on t h o s e c r o p s ( 2 ) . T o x i n s p r o d u c e d by p l a n t
pathogens
Many p a t h o g e n i c o r g a n i s m s p r o d u c e t o x i n s h a v i n g g r e a t e r o r l e s s e r degrees o f host s p e c i f i c i t y which cause o r c o n t r i b u t e t o the pathogenicity of the producing organism (3). I t i s not a broad l o g i c a l jump t o c o n s i d e r t h e u s e o f s u c h t o x i n s r a t h e r t h a n t h e producing organism i n a b i o r a t i o n a l pest c o n t r o l program. A number o f p h y t o t o x i n s p r o d u c e d by m i c r o o r g a n i s m s a r e known w h i c h i n d u c e symptoms on o n l y t h o s e s p e c i e s o r e v e n v a r i e t i e s w i t h i n a s p e c i e s which a r e s u s c e p t i b l e t o the pathogen, thus h o s t - s p e c i f i c t o x i n s . E x a m p l e s o f t h e s e h o s t s p e c i f i c t o x i n s a r e AM a n d AK t o x i n s p r o d u c e d by A l t e r n a r i a c a u s i n g d i s e a s e symptoms o n a p p l e a n d p e a r , r e s p e c t i v e l y , a n d HC a n d HMT t o x i n s p r o d u c e d b y H e l m i n t h o s p o r i u m s p p . c a u s i n g symptoms o n s u s c e p t i b l e m a i z e v a r i e t i e s ( 4 , 5 ) . These t o x i n s among many o t h e r s a r e known p r i m a r i l y b e c a u s e o f t h e i r association with disease-causing organisms of commercially v a l u a b l e crop s p e c i e s , w i t h few exceptions ( 6 ) . L i t t l e i s known o f h o s t s p e c i f i c t o x i n s p r o d u c e d by p a t h o g e n s o f w e e d s , i n p a r t b e c a u s e o f t h e p a u c i t y o f p h y s i o l o g i c a l r e s e a r c h on s u c h h o s t / p a t h o g e n relationships. T a r g e t i n g weeds h a v i n g m a j o r e c o n o m i c i m p a c t t o d i s c o v e r s u c h h o s t - s p e c i f i c t o x i n s s h o u l d p r o v e t o be a f r u i t f u l area of research (6). P o t e n t i a l l i m i t a t i o n s t o the h e r b i c i d a l use of h o s t - s p e c i f i c t o x i n s l i e i n t h e i r extreme s p e c i f i c i t y w i t h the p r o b a b i l i t y o f c o n t r o l l i n g o n l y a s i n g l e weed s p e c i e s , and t h e p o s s i b i l i t y o f r e s i s t a n c e w i t h i n weed b i o t y p e s b e c a u s e o f e x t e n s i v e c o - e v o l u t i o n o f such host-pathogen combinations. The d i s c o v e r y o f such t o x i n s c o u l d however, provide the b a s i s f o r c h e m i c a l manipulation to increased t o x i c i t y or decreased s p e c i f i c i t y r e s u l t i n g i n a n i n c r e a s e d r a n g e o f w e e d s t h a t c o u l d be c o n t r o l l e d (3). S t r u c t u r e / a c t i v i t y i n v e s t i g a t i o n and c h a r a c t e r i z a t i o n o f t h e m o l e c u l a r m e c h a n i s m s u n d e r l y i n g t h e s p e c i f i c i t i e s o f s u c h compounds c o u l d r e s u l t i n t h e d e s i g n o f s i m p l e r , more c h e a p l y p r o d u c e d s y n t h e t i c a n a l o g s as h e r b i c i d e s ( 3 ) . Such s t u d i e s a l s o c o u l d provide novel target s i t e s f o r the design of p e s t i c i d e s having the requisite s p e c i f i c i t i e s f o r c l o s e l y a l l i e d crop/weed a s s o c i a t i o n s s i n c e most n a t u r a l p r o d u c t t o x i n s f o r w h i c h a m o l e c u l a r t a r g e t s i t e
26 a r e known d i f f e r herbicides (3). Non-host-specific
BIOLOGICALLY ACTIVE NATURAL PRODUCTS
from s i t e s a f f e c t e d by
present
commercial
toxins
P o t e n t i a l l y more p r o m i s i n g r e s e a r c h l i e s i n non-host s e l e c t i v e t o x i n s w h i c h a f f e c t s p e c i e s o t h e r than those i n f e c t e d by the p r o d u c i n g organism. Several f a c t o r s c o n t r i b u t e to t h i s p r o b a b l i t y : 1) c o n s i d e r a b l y more i s u n d e r s t o o d c h e m i c a l l y and p h y s i o l o g i c a l l y about the non-host s p e c i f i c t o x i n s and 2) the p o t e n t i a l l y b r o a d e r range of weeds w h i c h may be c o n t r o l l e d t h r o u g h a s i n g l e a p p l i c a t i o n . The remainder of t h i s d i s c u s s i o n w i l l f o c u s p r i m a r i l y upon the d i s c o v e r y and c h a r a c t e r i z a t i o n of one such t o x i n , t e n t o x i n , and assessment of i t s promise as a h e r b i c i d e . Tentoxin i s a c y c l i c t e t r a p e p t i d e produced by the fungus A l t e r n a r i a a l t e r n a t a ( 4 ) . I n d u s t r y has e x p r e s s e d i n t e r e s t i n commercial development of t e n t o x i n , among o t h e r p h y t o t o x i n s , and a t h o r o u g h d i s c u s s i o n of s u c c e s s e s i n u n d e r s t a n d i n g t h i s t o x i n demonstrates p o t e n t i a l f o r d i s c o v e r y and development of h i t h e r t o unknown compounds. As w e l l , p o t e n t i a l s h o r t c o m i n g s demonstrate the problems w h i c h must be surmounted i n the development of t h i s u n d e r - r e p r e s e n t e d c l a s s of pesticides. W h i l e s e v e r a l i m p o r t a n t h e r b i c i d e s have been s y n t h e s i z e d as a n a l o g u e s of n a t u r a l p r o d u c t s ( 3 ) , h e r e i n we w i l l c o n s i d e r o n l y the a c t i v i t y and b i o s y n t h e s i s of the n a t u r a l t o x i n . A f u l l c o n s i d e r a t i o n of the s y n t h e s i s of the t o x i n and i t s analogues as w e l l as f u n c t i o n a l d e t e r m i n a n t s of i t s t o x i c i t y i s p r o v i d e d i n a n o t h e r c h a p t e r of t h i s volume. Discovery As w i t h the d i s c o v e r y of most p r e s e n t l y known p h y t o t o x i n s the d i s c o v e r y of t e n t o x i n r e s u l t e d from i t s t o x i c e f f e c t s on an important crop species. Cotton's s u s c e p t i b i l i t y to c h l o r o s i s i n d u c e d by t e n t o x i n r e s u l t e d i n c h l o r o t i c s e e d l i n g s and diminished stands of c o t t o n (4,7). The c a u s a l o r g a n i s m a s s o c i a t e d w i t h the s e e d l i n g d i s e a s e was A l t e r n a r i a t e n u i s (-A. a l t e r n a t a ) ( 4 , 7 ) . I t was found t h a t c e l l - f r e e c u l t u r e f i l t r a t e s of the fungus when a p p l i e d as a s o i l d r e n c h r e s u l t e d i n i d e n t i c a l symptoms, thus e s t a b l i s h i n g an e x t r a c e l l u l a r m e t a b o l i t e as the c a u s a l agent of the c h l o r o s i s (4,7). The d i s c o v e r y of many o t h e r t o x i n s w h i c h are p r i m a r y d e t e r m i n a n t s of p l a n t d i s e a s e have r e s u l t e d from t h e i r e f f e c t on c o m m e r c i a l l y i m p o r t a n t c r o p s p e c i e s . Examples of such d i s c o v e r i e s i n c l u d e the h o s t - s e l e c t i v e t o x i n s AM, AK, HMT, and HC toxins (3). To d a t e , however, t h e r e have been o n l y few of the many known f u n g a l pathogen t o x i n s d e s c r i b e d as a r e s u l t of t h e i r e f f e c t s on n o n - c r o p s p e c i e s . T h i s l a c k of d i s c o v e r y p r o b a b l y r e f l e c t s the l i m i t e d r e s e a r c h i n t h i s a r e a r a t h e r than a p a u c i t y of t o x i n s a f f e c t i n g weed s p e c i e s . I t i s i n t h i s a r e a t h a t major s t u d i e s may be made, e s p e c i a l l y t h r o u g h t a r g e t i n g of one or s e v e r a l p a r t i c u l a r l y troublesome weeds and t h o r o u g h e x a m i n a t i o n of pathogens w h i c h i n f e c t t h e s e weeds. I n d u s t r y i s i n f a c t d e d i c a t i n g some e f f o r t i n the a r e a of weed/pathogen r e s e a r c h w i t h the p o t e n t i a l f o r d i s c o v e r y of new c l a s s i c a l b i o l o g i c a l c o n t r o l organisms and/or t o x i n s d e r i v e d from them ( 3 ) .
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LAX AND SHEPHERD
Tentoxin: Potential Herbicide
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W h i l e many o f t h e c u r r e n t l y known t o x i n s show e x t r e m e s p e c i f i c i t y w i t h i n the genera or even w i t h i n s p e c i e s w h i c h t h e i r p r o d u c i n g m i c r o o r g a n i s n s a f f e c t , we k n o w o f no r e p o r t s o f t h e s y s t e m a t i c e x a m i n a t i o n of the p o s s i b i l i t y t h a t these t o x i n s or t h e i r a n a l o g u e s may a f f e c t d i s t a n t l y r e l a t e d w e e d s p e c i e s . Mode o f a c t i o n a n d
identification
of p o t e n t i a l t a r g e t
sites.
A f t e r i s o l a t i o n and the d i s c o v e r y o f the t o x i n s t r u c t u r e , f o l l o w s c h a r a c t e r i z a t i o n of the m o l e c u l a r s i t e s of t o x i c i t y . The n a t u r e o f t h e s i t e o f a c t i o n i s known f o r s e v e r a l o f t h e e a r l i e r m e n t i o n e d p h y t o t o x i n s w h i l e f o r others the m o l e c u l a r t a r g e t remains unelucidated (2,3). E a r l y r e s u l t s c o n c e r n i n g t e n t o x i n mode o f a c t i o n f o c u s e d on i t s i n h i b i t i o n o f t h e A T P a s e a c t i v i t y o f c h l o r o p l a s t c o u p l i n g f a c t o r 1 (CFn.) ( 8 ) . T e n t o x i n a t some c o n c e n t r a t i o n s was shown t o i n h i b i t A T P a s e a c t i v i t y o f t h e CF i s o l a t e d from t o x i n - s e n s i t i v e but not i n s e n s i t i v e s p e c i e s ( 8 ) . M o r e o v e r , i n i s o l a t e d c h l o r o p l a s t s t e n t o x i n was shown t o i n h i b i t coupled photosynthetic e l e c t r o n t r a n s p o r t t h u s a c t i n g as an e n e r g y transfer i n h i b i t o r (9). The r e s u l t i n g l o s s o f m e t a b o l i c e n e r g y was thought t h e r e f o r e to r e s u l t i n the l a c k of c h l o r o p l a s t development and c h l o r o s i s c h a r a c t e r i s t i c o f t e n t o x i n e f f e c t s . Electron microscopic e x a m i n a t i o n , however, r e v e a l e d t h a t t e n t o x i n d i s r u p t e d d e v e l o p m e n t i n a p i c a l p l a s t i d s and e t i o p l a s t s , b o t h t i s s u e s i n w h i c h e f f e c t s on C F x - d e p e n d e n t p h o s p h o r y l a t i o n s h o u l d h a v e no e f f e c t s ( 1 0 12). Moreover, the t e n t o x i n t r e a t e d p l a s t i d s m o r p h o l o g i c a l l y resembled p l a s t i d s of a mutant of Hosta w h i c h l a c k e d p o l y p h e n o l o x i d a s e (PPO) (13). I n a s e r i e s o f e x p e r i m e n t s , V a u g h n a n d Duke d e m o n s t r a t e d t h a t PPO a c t i v i t y was a b s e n t f r o m t e n t o x i n t r e a t e d p l a s t i d s , e t i o p l a s t s and a p i c a l p l a s t i d s and t h a t t h e n u c l e a r l y e n c o d e d , i n a c t i v e enzyme a c c u m u l a t e d a t t h e c h l o r o p l a s t e n v e l o p e (10-12). These r e s u l t s suggested t h a t a p r o c e s s i n v o l v e d i n the t r a n s p o r t o f PPO i n t o t h e c h l o r o p l a s t may be i n v o l v e d i n t h e t o x i n syndrome. T h a t t h i s t r a n s p o r t i n h i b i t i o n i s s p e c i f i c and n o t a r e s u l t o f o v e r a l l e n e r g y (ATP) d i s r u p t i o n i s d e m o n s t r a t e d by t h e presence i n t o x i n - t r e a t e d p l a s t i d s of o t h e r n u c l e a r l y encoded p r o t e i n s , n o t a b l y f e r r e d o x i n - N A D P " * - r e d u c t a s e (FNR) (14), l i g h t h a r v e s t i n g c h l o r o p h y l l c o m p l e x (LHC) ( L a x u n p u b l i s h e d ) and small s u b u n i t of r i b u l o s e b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e (Lax unpublished), a l l p l a s t i d i c enzymes w h i c h h a v e b e e n d e m o n s t r a t e d t o be t r a n s p o r t e d i n t o t h e c h l o r o p l a s t i n a n e n e r g y - d e p e n d e n t m a n n e r . F i g u r e 1 shows t h a t o n l y m i n o r d i f f e r e n c e s e x i s t i n t h e polypeptide p r o f i l e o f e t i o p l a s t s f r o m c o n t r o l and t e n t o x i n - t r e a t e d p l a n t s , a g a i n i n d i c a t i v e o f a s e l e c t i v e d i s r u p t i o n o f i m p o r t o f o n l y one or a few n u c l e a r l y - e n c o d e d c h l o r o p l a s t p r o t e i n s . That only minor d i f f e r e n c e s are noted e a r l y i n ontogeny p r i o r to p l e i o t r o p i c e f f e c t s due t o l a c k o f c h l o r o p l a s t d e v e l o p m e n t s t r e n g t h e n t h e a r g u m e n t t h a t o n l y one o r a f e w s p e c i f i c p r o t e i n s a r e a f f e c t e d . X
W h i l e some o f t h e a b o v e e f f e c t s o f t e n t o x i n c o u l d be m e d i a t e d t h r o u g h i n h i b i t i o n o f e n e r g y t r a n s d u c t i o n by t e n t o x i n * s k n o w n e f f e c t s on C F , s e v e r a l f u r t h e r l i n e s of evidence i n d i c a t e t h a t the p r i m a r y l e s i o n c a u s e d by t e n t o x i n t r e a t m e n t l i e s i n o t h e r c e l l u l a r processes. F i r s t , o n l y one o f t h r e e C F isozymes i s o l a t e d through e l e c t r o p h o r e s i s show t e n t o x i n s e n s i t i v i t y , a n d t h e s e n s i t i v i t y o f X
X
28
Figure
BIOLOGICALLY ACTIVE NATURAL PRODUCTS
1.
P o l y p e p t i d e p r o f i l e o f c o n t r o l (C) and t e n t o x i n treated (T) e t i o p l a s t s i s o l a t e d f r o m dark-grown l e t t u c e cotyledons. P r o t e i n s were s o l u b i l i z e d i n l i t h i u m d o d e c y l s u l f a t e and e l e c t r o p h o r e s e d on polyacryamide s l a b g e l s . P r o t e i n s were s i l v e r stained. Note the minor d i f f e r e n c e s i n p r o t e i n profiles. S m a l l arrows i n d i c a t e absence and l a r g e arrows denote p r e s e n c e o f p r o t e i n s i n the t e n t o x i n p r o f i l e compared t o the c o n t r o l p r o f i l e s .
2.
LAX AND SHEPHERD
29
Tentoxin: Potential Herbicide
t h i s isozyme i s seen o n l y a t low t o x i n c o n c e n t r a t i o n s , while c h l o r o s i s m e d i a t e d by t e n t o x i n t r e a t m e n t shows no a l l e v i a t i o n a t higher toxin concentrations (15). S e c o n d l y , Zea m a y s , w h i c h i s i n s e n s i t i v e i n v i v o t o c h l o r o s i s c a u s e d by t e n t o x i n , h a s a CF isozyme w h i c h i s t o x i n s e n s i t i v e . F i n a l l y , the most c o m p e l l i n g a r g u m e n t a g a i n s t C F - m e d i a t e d c h l o r o s i s i s p r o v i d e d by t h e s e n s i t i v i t y t o t e n t o x i n o f a CFo. d e f e c t i v e m u t a n t o f O e n o t h e r a hookeri (15). While tentoxin-mediated c h l o r o s i s does not appear t o be s o l e l y o r p r i m a r i l y d e p e n d e n t u p o n t e n t o x i n ' s e f f e c t s on CF t h e r e i s no a r g u m e n t t h a t t e n t o x i n a n d some o f i t s a n a l o g u e s do p o s s e s s a s p e c i e s s p e c i f i c e f f e c t on CF w h i c h may p r o v e v a l u a b l e i n t h e d e s i g n o f CF e f f e c t o r s w h i c h c o u l d be e f f e c t i v e a s s e l e c t i v e herbicides. More r e c e n t e v i d e n c e f r o m our l a b i n d i c a t e s t h a t a n o t h e r m o l e c u l a r t a r g e t s i t e f o r t e n t o x i n m e d i a t e d c h l o r o s i s may e x i s t a t t h e c h l o r o p l a s t e n v e l o p e , t h e known s i t e o f a c c u m u l a t i o n o f i n a c t i v e PPO i n t e n t o x i n - t r e a t e d t i s s u e s . F i g u r e 2 shows t h e s e n s i t i v i t y o f one o f two c h l o r o p l a s t e n v e l o p e a s s o c i a t e d A T P a s e s w h i c h c a n be s e p a r a t e d e l e c t r o p h o r e t i c a l l y . W h i l e i t r e m a i n s t o be d e m o n s t r a t e d that these envelope-associated ATPases are a s s o c i a t e d w i t h p r o c e s s e s i n v o l v e d i n p r o t e i n i m p o r t i n t o the c h l o r o p l a s t , i t has been d e m o n s t r a t e d f o r a number o f n u c l e a r - c o d e d p r o t e i n s t h a t s u c h i m p o r t i s an ATP-dependent p r o c e s s ( 1 6 , 1 7 ) . T h a t t h e r e a r e a t l e a s t two envelope a s s o c i a t e d ATPases h a v i n g d i f f e r e n t s e n s i t i v i t i e s to t e n t o x i n c o u l d be i n v o k e d t o e x p l a i n t h e d i f f e r e n t s e n s i t i v i t i e s o f t h e k n o w n i m p o r t e d p r o t e i n s ( e . g . , PPO, FNR, SS) t o i n h i b i t i o n by tentoxin. I t i s a l s o p o s s i b l e t h a t the above mentioned e n v e l o p e a s s o c i a t e d ATPases are u n r e l a t e d to the import processes. D i f f e r e n t i a l s e n s i t i v i t i e s of these envelope ATPases to t e n t o x i n suggest yet another molecular s i t e f o r b i o r a t i o n a l n a t u r a l product (or analogue) h e r b i c i d e s ( 2 ) . Regardless of whether these envelope-bound ATPases are i n v o l v e d i n t h e s p e c i f i c t r a n s p o r t p r o c e s s e s k n o w n t o a f f e c t PPO, t h e t i g h t c o r r e l a t i o n b e t w e e n PPO t r a n s p o r t i n h i b i t i o n a n d c h l o r o s i s i d e n t i f i e s t h i s p r o c e s s i t s e l f as a p o t e n t i a l h e r b i c i d a l t a r g e t site. An i m p o r t a n t p o i n t h e r e i s t h a t w h e t h e r o r n o t the n a t u r a l p r o d u c t i t s e l f may be c o m m e r c i a l l y e x p l o i t e d , r e s e a r c h i n t o i t s t a r g e t s i t e may p r o v i d e c l u e s t o one o r m o r e t a r g e t e n z y m e s y s t e m s w h i c h may be e x p l o i t e d a s h e r b i c i d a l t a r g e t s . A f t e r the i d e n t i f i c a t i o n of p o t e n t i a l h e r b i c i d a l m i c r o b i a l p r o d u c t s , f i e l d e f f i c a c y a n d p r o d u c t i o n e c o n o m i c s m u s t be a s s e s s e d . In the case of t e n t o x i n these parameters are t i g h t l y coupled because of the g e n e r a l l y low y i e l d s of the t o x i n i n s t a n d a r d fermentation and t h e r e s u l t a n t e x p e n s e o f l a r g e s c a l e f i e l d t e s t s . In f a c t , to our k n o w l e d g e t e n t o x i n has n e v e r been t e s t e d i n the f i e l d , a l t h o u g h i n c o r p o r a t i o n o f c r u d e c u l t u r e f i l t r a t e s i n t o s o i l i n g r o w t h chamber s t u d i e s has d e m o n s t r a t e d t h e p o t e n t i a l e f f i c a c y o f t e n t o x i n ' s use as a h e r b i c i d e i n c o r n and s o y b e a n ( 2 ) . W h i l e the b i o s y n t h e t i c pathway t o t e n t o x i n i s unknown, r a t h e r more a t t e n t i o n has been p a i d t o i t s b i o s y n t h e s i s t h a n t o f i e l d efficacy. S e v e r a l methods have been u t i l i z e d t o q u a n t i t a t e t o x i n p r o d u c t i o n i n c l u d i n g b i o l o g i c a l a s s a y , HPLC, and t h i n l a y e r chromatography (18), Using a semiquantitative s e e d l i n g c h l o r o s i s bioassay, A l t e r n a r i a i s o l a t e s e l e c t i o n to maximize t o x i n production X
x
X
X
X
30
BIOLOGICALLY ACTIVE NATURAL PRODUCTS
c a n be p e r f o r m e d . M e d i a c o m p o s i t i o n a n d c u l t u r e c o n d i t i o n s t o be d i s c u s s e d b e l o w h a v e b e e n d e m o n s t r a t e d t o h a v e t r e m e d o u s e f f e c t on t o x i n p r o d u c t i o n , b u t t o d a t e t h e maximum y i e l d o f t e n t o x i n h a s b e e n 90 mg/1 (19,20). Moreover these y i e l d s are r e a l i z e d only a f t e r prolonged s t i l l c u l t u r e (18). By t h e a d d i t i o n o f p r e v i o u s l y m e t a b o l i z e d c u l t u r e f i l t r a t e s to A l t e r n a r i a c u l t u r e s , the t o x i n y i e l d may be i n c r e a s e d t e m p o r a l l y , h o w e v e r , t h e f i n a l y i e l d i s l i t t l e a l t e r e d (18), suggesting the presence of a p r e c u r s o r ( s ) i n the m e t a b o l i z e d c u l t u r e b r o t h s . I t i s c u r r e n t l y unknown w h e t h e r t e n t o x i n i s s y n t h e s i z e d on r i b o s o m e s as a h i g h e r m o l e c u l a r w e i g h t p r e c u r s o r w h i c h i s s u b s e q u e n t l y c l e a v e d , m o d i f i e d and c y c l i z e d o r w h e t h e r i t s s y n t h e s i s p r o c e d e s v i a e n z y m a t i c ( s u c h as a n c o v e n i n i n S t r e p t o m y c e s s p . a n d s u b t i l i s i n A i n B a c i l l u s s u b t i l i s ) (21), or non-ribosomal s y n t h e s i s a s i s k n o w n f o r g r a m i c i d i n and t y r o c i d i n e ( i n b a c t e r i a ) o r C y l - 1 and HC t o x i n s i n f u n g i ( 2 1 ) . The a d d i t i o n o f p r o t e i n s y n t h e s i s i n h i b i t o r s s u c h a s cycloheximide a n d e m e t i n e h a d no e f f e c t on t o x i n b i o s y n t h e s i s , l e a d i n g to the c o n c l u s i o n t h a t t e n t o x i n b i o s y n t h e s i s proceedes v i a a n o n - r i b o s o m a l pathway (18). However, i n t h e s e e x p e r i m e n t s the i n h i b i t o r s w e r e added i n l a t e g r o w t h phase and t h e p o s s i b i l i t y o f higher molecular weight, ribosomally synthesized precursor a c c u m u l a t i o n p r i o r t o i n h i b i t o r a d d i t i o n s c a n n o t be r u l e d o u t . Dot b l o t s of c u l t u r e f i l t r a t e s of t o x i n producing s t r a i n s of A l t e r n a r i a using a n t i s e r a to t e n t o x i n ( r a i s e d i n r a b b i t against p u r i f i e d t e n t o x i n ) i n d i c a t e d g r e a t e r amounts of i m m u n o l o g i c a l l y reactive m a t e r i a l t h a n c o u l d be a s c r i b e d t o t h e t o x i n i t s e l f (Lax unpublished). Western b l o t t i n g , f o l l o w i n g e l e c t r o p h o r e s i s of p r o t e i n s i s o l a t e d from v a r i o u s A l t e r n a r i a s t r a i n s , i n d i c a t e d the p r e s e n c e o f h i g h m o l e c u l a r w e i g h t p r o t e i n s r e c o g n i z e d by a n t i s e r a t o t e n t o x i n i n t o x i n p r o d u c i n g , but not n o n - p r o d u c i n g i s o l a t e s . These d a t a suggested the p o s s i b l e a c c u m u l a t i o n of a h i g h m o l e c u l a r w e i g h t p r e c u r s o r t o t e n t o x i n w h i c h c o u l d be s u b s e q u e n t l y p r o c e s s e d t o y i e l d the f i n a l p r o d u c t . I n o u r a t t e m p t s t o i s o l a t e mRNA f r o m A l t e r n a r i a t o p e r f o r m i n v i t r o t r a n s l a t i o n s , we c o n s i s t e n t l y f o u n d u n i q u e RNAs i n s t r a i n s which produced t e n t o x i n . T h e s e RNA s p e c i e s w e r e s e n s i t i v e t o RNAse d i g e s t i o n i n low s a l t but not h i g h s a l t , i n d i c a t i v e of d o u b l e s t r a n d e d RNA c h a r a c t e r i s t i c o f p r e v i o u s l y d e s c r i b e d f u n g a l v i r u s e s (22). C e s i u m c h l o r i d e and s u c r o s e d e n s i t y g r a d i e n t u l t r a centrifugation yielded p u r i f i e d isodiametric virus p a r t i c l e s having a d i a m e t e r o f -30 nm ( 2 3 ) a l s o c h a r a c t e r i s t i c o f p r e v i o u s l y described fungal v i r u s e s (22). E l e c t r o p h o r e t i c s e p a r a t i o n of the v i r a l p r o t e i n o f two s u c h v i r u s e s h a s d e m o n s t r a t e d a t l e a s t two p r o t e i n s ( F i g u r e 3A) i n e a c h , a t l e a s t one o f w h i c h i s r e c o g n i z e d by a n t i s e r a t o t e n t o x i n upon w e s t e r n b l o t t i n g ( F i g u r e 3B). As i n d i c a t e d p r e v i o u s l y , t e n t o x i n b i o s y n t h e s i s i s dependent upon c u l t u r e c o n d i t i o n s s u c h as t e m p e r a t u r e and a e r a t i o n . Tentoxin b i o s y n t h e s i s i s m a x i m a l a t ~28°C ( 1 9 ) a n d i s g r e a t l y r e d u c e d a t t e m p e r a t u r e s up t o 35°C ( 1 9 ) w h i l e t h e g r o w t h r a t e i s u n a f f e c t e d . T o x i n p r o d u c t i o n i s p r e v e n t e d i n shake c u l t u r e under c o n d i t i o n s o t h e w i s e i d e n t i c a l w i t h s t i l l c u l t u r e s (Lax u n p u b l i s h e d ) . Elevated t e m p e r a t u r e s and s h a k i n g a l s o r e d u c e l e v e l s o f v i r u s a s s o c i a t e d w i t h t o x i n p r o d u c i n g s t r a i n s ( F i g u r e s 4A, 4 B ) . The a d d i t i o n o f
LAX AND SHEPHERD
Figure
2.
Tentoxin: Potential Herbicide
D i f f e r e n t i a l s e n s i t i v i t y of c h l o r o p l a s t envelope ATPases of pea. E n v e l o p e membranes w e r e i s o l a t e d f r o m i n t a c t pea c h l o r o p l a s t s and s o l u b i l i z e d i n CHAPS. F o l l o w i n g e l e c t r o p h o r e t i c s e p a r a t i o n ATPase a c t i v i t y was d e t e c t e d i n t h e a b s e n c e ( C ) o r p r e s e n c e ( T ) o f 10 tentoxin. Deposition of a white Ca (P0*)2 p r e c i p i t a t e i n d i c a t e s a c t i v i t y . Note a b s e n c e o f a c t i v i t y o f t h e more s l o w l y migrating species i n the presence of t e n t o x i n .
JJLM
3
Figure
3.
A n a l y s i s of proteins from v i r u s p a r t i c l e s of s t r a i n s 1 and 2 o f A^ a l t e r n a t a p u r i f i e d by C s C l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . A) P r o t e i n s o f l i t h i u m dodecyl sulfate-disrupted v i r u s p a r t i c l e s separated by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Two m a j o r p r o t e i n s are v i s i b l e f o r each s t r a i n f o l l o w i n g s i l v e r s t a i n i n g . B) W e s t e r n b l o t o f A ) , p r o b e d w i t h a n t i s e r a to tentoxin. Note i m m u n o r e a c t i v i t y of a t l e a s t one b a n d i n e a c h c a s e .
32
BIOLOGICALLY ACTIVE NATURAL PRODUCTS
F i g u r e 4.
D o u b l e - s t r a n d e d ( v i r a l ) RNAs i s o l a t e d f r o m c o n t r o l (C) and t r e a t e d (T) A. a l t e r n a t a c u l t u r e s . A l l t r e a t m e n t s were c a r r i e d out two o r more times on each o f the s t r a i n s . R e p r e s e n t a t i v e r e s u l t s from s t r a i n 2 a r e shown. T e n t o x i n was d e t e c t a b l e o n l y i n c o n t r o l s , e x c e p t as n o t e d , a) D o u b l e - s t r a n d e d RNA from 28°C (C) and 34°C (T) c u l t u r e s , b ) Doubles t r a n d e d RNA from S t i l l (C) and Shaking (T) cultures, c ) D o u b l e - s t r a n d e d RNA from c u l t u r e s grown i n the absence (C) and p r e s e n c e (T) o f c y c l o h e x i m i d e , (0.2 mM on l e f t , 0.4 mM on r i g h t . A low l e v e l of t e n t o x i n was d e t e c t e d i n the 0.2 mM cycloheximide treatment.
2. LAX AND SHEPHERD
Tentoxin: Potential Herbicide
33
cycloheximide in early growth phase has been reported to reduce fungal virus (24); such addition to toxin-producing strains concomitantly reduces toxin biosynthesis and virus concentration (Figure 4C). All of these experiments have been carried out several times with at least two tentoxin-producing strains. A firmer understanding of the biosynthetic processes will allow better assessment of the potential for increasing production to a commercially acceptable level. Whether this increase can be realized through traditional microbiological methods, the design of a semisynthetic pathway, or through more advanced molecular techniques remains to be seen. This appears to be the major hurdle to be overcome in the exploitation of tentoxin as a broad spectrum herbicide in the future. From the foregoing it should be realized that deployment of tentoxin as a herbicide will at best be several years away. As with other chemically complex natural products a major limitation is production (2,3). Another major concern which is beyond the scope of this chapter is toxicological testing and licensing which are dependent upon regulatory agencies. Information regarding tentoxin*s mode of action would indicate a low mammalian toxicity with all evidence pointing to an exclusively chloroplast localized activity. Tentoxin like other fungal natural products should be relatively non-persistent in the environment and therefore environmentally safer than other more persistent compounds. Although only a few microbially derived herbicides are on the market today, there is great hope that natural products can f i l l the need for highly selective and safe herbicides. Tentoxin is but one of thousands of compounds that offers such potential. Certainly further research is needed and warranted to fulfill this vast untapped potential. Literature Cited 1.
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Luke, H.H.; Gracen, V.E. Jr.; In Microbial Toxins; Kadis, S; Ciegler, A., Ajl, S.J., Eds.; Academic: New York, 1972, Chapter 6, pp 139-168.
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Steele, J.A.; Uchytil, T.F.; Durbin, R.D.; Bhatnagar, P.; Rich, D.H. Proc. Nat. Acad. Sci. U.S.A. 1976. 73, 2245-8.
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Vaughn, K. C.; Duke, S.O., Physiol. Plant. 1984, 60, 257-61.
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Lax, A.R.; Vaughn, K.C.; Sisson, V.A.; Templeton, G.E., Photosynth. Res., 1985, 6, 113-20.
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Lax, A.R.; Vaughn, K.C., Physiol. Plant. 1986, 66, 384-391.
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Schmidt, G.W.; Bartlett, S.G.; Grossman, A.R., Cashmore, A.R.; Chua, N.-H., J. Cell. Biol. 1981, 91, 468-78.
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Pain, D.; Blobel, G., Proc. Natl. Acad. Sci. USA. 1987, 84, 3288-92.
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Sheu, J.-T.; Talburt, D.E., Appl. Env. Microbiol. 1986, 51, 368-72.
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Liebermann, B.; Oertel, B., Z. Allg. Mikrobiol., 1983, 23, 50311.
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Kleinkauf, H.; vonDöhren, H., Ann. Rev. Microbiol. 1987, 41, 259-89.
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Buck, K.W.; Ackermann, H.-W.; Bozarth, R.F.; Bruenn, J.A.; Koltin, Y.; Rawlinson, C.J., Ushiyama, R.; Wood, H.A. Intervirology, 1984, 22, 17-23.
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RECEIVED May 26, 1988
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