Chapter 29
Properties of a Cutinase-Defective Mutant of Fusarium solani Anne H . Dantzig Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285
The fungal plant pathogen Fusarium solani produces an extracellular enzyme, cutinase, which catalyzes the degradation of the bipolymer, cutin, in the plant cuticle. The enzyme was repressed when the microorganism was grown on a medium containing glucose and induced to high levels by cutin or its hydrolysis products, the true inducers. In the present study, culture filtrates contained basal levels of cutinase when Fusarium was grown on 0.5% acetate as the sole carbon source and high levels of cutinase when grown on cutin. After mutagenesis, a cutinase-defective mutant of Fusarium was identified by screening acetate-grown colonies for a loss of enzyme activity. The mutant exhibited an 80-90% reduction in cutinase activity under several growth conditions due to a quantitative reduction in a qualitatively normal enzyme. The mutant also exhibited a reduction in virulence in the pea stem bioassay. Taken together, these data indicated that a growth condition exists where the cutinase enzyme was neither induced nor repressed and was present in basal levels. This condition may pose the pathogen for rapid enzyme induction when in the proximity of the plant cuticle. The cutinase-defective mutant was either a regulatory mutant with an altered expression of cutinase, or a mutant modified in its ability to excrete the enzyme. T h e b i o p o l y m e r c u t i n is a m a j o r constituent o f the p l a n t cuticle t h a t p r o vides a protective covering for p l a n t s ( 1 , 2 ) . A t the t i m e o f i n f e c t i o n , a n u m b e r o f f u n g a l pathogens secrete a n e x t r a c e l l u l a r h y d r o l y t i c e n z y m e , cutinase, w h i c h facilitates the d e g r a d a t i o n o f c u t i n i n t o i t s constituent C\e~ to C i s - l e n g t h h y d r o x y f a t t y acids ( 3 , 4 ) . Since the e n z y m e is believed t o 0097-6156/89/0399-0399$06.00/0 © 1989 American Chemical Society
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p l a y a n i m p o r t a n t role i n v i r u l e n c e (5), m a n y studies have been c o n d u c t e d to d e t e r m i n e its b i o c h e m i c a l properties a n d p h y s i o l o g i c a l role d u r i n g p a t h o genesis. T o date the most comprehensive studies have been c a r r i e d o u t b y K o l a t t u k u d y a n d coworkers w i t h a f u n g a l p l a n t p a t h o g e n of peas, Fusarium solani f. sp. pisi. W h e n g r o w n o n c u t i n , the fungus p r o d u c e d two isozymes of cutinase of m o l e c u l a r weights 2 2 , 4 0 0 a n d 2 1 , 2 0 0 , w i t h the former b e i n g the precursor of the l a t t e r , m a t u r e f o r m (6-8). T h e c D N A for the c u t i nase gene was i s o l a t e d , sequenced, a n d the p r i m a r y a m i n o a c i d sequence deduced ( 9 , 1 0 ) . D N A h y b r i d i z a t i o n studies i n d i c a t e d t h a t more t h a n one gene was present i n Fusarium solani (9). A l t h o u g h details of the r e g u l a t i o n of the expression of cutinase at the D N A level were not established, p h y s i o l o g i c a l studies i n d i c a t e d t h a t the r e g u l a t i o n of the enzyme was likely to be c o m p l e x . T h e e n z y m e u n d e r w e n t c a t a b o l i t e repression a n d was i n d u c e d to h i g h levels w h e n c u t i n , or its h y d r o l y z e d p r o d u c t s , were p r o v i d e d as the sole c a r b o n source (11). Since c u t i n has a large m o l e c u l a r weight, it is not likely to penetrate the f u n g a l cell w a l l ; the d e g r a d a t i o n p r o d u c t s are therefore believed to be the true inducers of the enzyme ( 1 1 , 1 2 ) . T h u s , the presence of the cutinase e n z y m e seemed to be required for its o w n i n d u c t i o n i n order to generate the s m a l l m o l e c u l a r weight inducers. C o n s e q u e n t l y , i t seemed p l a u s i b l e t h a t g r o w t h c o n d i t i o n s m i g h t exist i n w h i c h cutinase were present i n b a s a l q u a n t i t i e s , thereby p o s i n g the p a t h o g e n for r a p i d e n z y m e i n d u c t i o n w h e n presented w i t h the c u t i n b i o p o l y m e r . T h e present s t u d y was u n d e r t a k e n t o e x a m i n e the r e g u l a t i o n of cutinase b y a n alternate c a r b o n source t h a t m i g h t p e r m i t b a s a l levels of the e n z y m e t o be synthesized, a n d t h e n to subsequently use t h i s g r o w t h c o n d i t i o n for the i s o l a t i o n o f a cutinase-defective m u t a n t . T h e details of t h i s work have been p r e v i o u s l y p u b l i s h e d i n Ref. 13. Results A c e t a t e is k n o w n to be a g o o d c a r b o n source for f u n g i a n d w o u l d be expected to be the u l t i m a t e d e g r a d a t i o n p r o d u c t of c u t i n (14). T h e effect of acetate o n the p r o d u c t i o n of cutinase b y the T - 8 s t r a i n of F. solani was exa m i n e d a n d c o m p a r e d w i t h t h a t of glucose. Since previous studies showed t h a t h y d r o l y s i s of the a r t i f i c i a l substrate p - n i t r o p h e n y l b u t y r a t e , P N B , was specifically h y d r o l y z e d by cutinase i n the T - 8 s t r a i n , t h i s a c t i v i t y was used to measure cutinase levels (8). F i g u r e 1 i l l u s t r a t e s t h a t b a s a l levels of c u t i nase a c t i v i t y were detected i n the g r o w t h m e d i u m w h e n T - 8 was g r o w n o n 0.5 a n d 1.5% acetate, b u t not 2 . 0 % glucose, as the sole c a r b o n source. A s s h o w n i n F i g u r e 2 A , T - 8 p r o d u c e d h i g h levels of cutinase i n the g r o w t h m e d i u m when g r o w n on apple c u t i n as the sole c a r b o n source, a n d was repressed by i n c r e a s i n g concentrations of glucose; b y c o n t r a s t , F i g u r e 2 B showed t h a t a d d i t i o n of increasing concentrations of acetate was less repressive t h a n glucose. These d a t a suggest t h a t the e n z y m e was present at low concentrations when the o r g a n i s m was g r o w n o n acetate. Since g r o w t h of F. solani o n acetate m e d i u m p e r m i t t e d p r o d u c t i o n o f cutinase, t h i s g r o w t h c o n d i t i o n was used for the i s o l a t i o n of a m u t a n t
29.
DANTZIG
Cutinase-Defective Mutant of Fusarium solani
401
F i g u r e 1. Specific a c t i v i t y of cutinase as a f u n c t i o n of sole c a r b o n source i n the g r o w t h m e d i u m . T h e T - 8 s t r a i n of F. solani was g r o w n o n 0 . 5 % acetate (o), 1.5% acetate ( Δ ) , or 1.5% glucose ( · ) as the sole c a r b o n source. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for Microbiology.)
1
2 DAYS
3
F i g u r e 2. Specific a c t i v i t y of cutinase r e s u l t i n g f r o m g r o w t h i n c u t i n c o n t a i n i n g m e d i u m w i t h the a d d i t i o n of a second c a r b o n source. T h e T - 8 s t r a i n of F. solani was grown on m e d i u m c o n t a i n i n g 200 m g of apple c u t i n a n d glucose ( A ) or acetate ( B ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )
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defective i n cutinase; the procedure for m u t a n t selection was described i n Ref. 13 a n d is s u m m a r i z e d i n F i g u r e 3. I n i t i a l l y , Fusarium was g r o w n o n m e d i u m c o n t a i n i n g peanut m e a l - p e p t o n e - S t a d e x II t o p r o d u c e m i c r o c o n i d i a needed for genetic studies (13). T h e c o n i d i a were uv i r r a d i a t e d to i n t r o d u c e m u t a t i o n s , r e g r o w n to p e r m i t expression of a n y m u t a t i o n , a n d p l a t e d o n g r o w t h m e d i u m c o n t a i n i n g agarose a n d 0 . 5 % acetate as the sole c a r b o n source. T h e r e s u l t i n g colonies were t h e n overlaid w i t h agarose c o n t a i n i n g P N B . W i t h i n 30 m i n the p a r e n t a l type h y d r o l y z e d the s u b s t r a t e a n d t u r n e d y e l l o w , a n d the p r e s u m p t i v e m u t a n t s r e m a i n e d w h i t e . T h e r e s u l t i n g PNB1 m u t a n t was one out of 4 , 3 0 0 colonies screened. T h e properties of t h i s m u t a n t were further characterized as described below. P r e l i m i n a r y e v a l u a t i o n of the m u t a n t i n d i c a t e d t h a t i t h a d p r o d u c e d low levels of the cutinase w h e n g r o w n o n either acetate or c u t i n as the sole c a r b o n source. A t i m e course of the p r o d u c t i o n of cutinase was e x a m i n e d for the T - 8 p a r e n t a l s t r a i n a n d the PNB-1 m u t a n t s t r a i n when g r o w n o n c u t i n . A s s h o w n i n F i g u r e 4, the m u t a n t e x h i b i t e d a r e d u c t i o n i n a c t i v i t y of 8 0 - 9 0 % over the 15-day g r o w t h p e r i o d , w h e n e n z y m e a c t i v i t y was assayed u s i n g the a r t i f i c i a l substrate P N B ( P a n e l A ) or w h e n assayed u s i n g [ C ] l a b e l l e d n a t u r a l s u b s t r a t e c u t i n ( P a n e l B ) . These d a t a suggested t h a t the m u t a n t was defective i n cutinase b u t not i n general esterase. 1 4
N e x t the parent a n d m u t a n t were c o m p a r e d for t h e i r a b i l i t y to i n d u c e cutinase b y c u t i n a n d h y d r o l y z e d c u t i n (consisting of s m a l l m o l e c u l a r weight inducers) after g r o w t h on glucose. A s s h o w n i n F i g u r e 5, cutinase a c t i v i t y increased over the three-day i n d u c t i o n p e r i o d for b o t h s t r a i n s i n the presence of c u t i n ( P a n e l A ) or h y d r o l y z e d c u t i n ( P a n e l B ) ; however, cutinase was i n d u c e d less effectively i n the m u t a n t s t r a i n , as evidenced by a n 8 0 - 9 0 % r e d u c t i o n . H y d r o l y z e d c u t i n was a 10-fold less effective inducer t h a n c u t i n i n b o t h s t r a i n s . These d a t a i n d i c a t e d t h a t lack of i n d u c t i o n i n the m u t a n t was not related to its i n a b i l i t y to h y d r o l y z e c u t i n to s m a l l m o l e c u l a r weight inducers. C o n s e q u e n t l y the defect observed was not related to the i n a b i l i t y of the m u t a n t to produce the i n d u c e r . T o g a i n f u r t h e r insight i n t o the n a t u r e of the defect i n PNB-1 m u t a n t , the c o n c e n t r a t i o n dependence of the cutinase enzyme a c t i v i t y was e x a m i n e d over a wide s u b s t r a t e range a n d c o m p a r e d w i t h t h a t of the p a r e n t a l s t r a i n ( F i g u r e 6). I n b o t h s t r a i n s , the enzyme a c t i v i t y was s a t u r a t e d w i t h i n creasing substrate c o n c e n t r a t i o n , a n d gave s i m p l e M i c h a e l i s - M e n t e n curves w h i c h were subsequently fitted by c o m p u t e r to a single M i c h a e l i s t e r m . T h e K for P N B was d e t e r m i n e d to be 0 . 9 7 ± 0 . 2 0 a n d 0 . 6 4 ± 0 . 0 7 m M , respectively, i n the p a r e n t a l a n d m u t a n t s t r a i n s , i n d i c a t i n g t h a t l i t t l e change i n the affinity for the substrate h a d o c c u r r e d . B y c o n t r a s t , the V was reduced by 9 2 % f r o m 38.86 ± 2.68 to 2.62 ± 0 . 0 9 / i m o l / m i n per m g p r o t e i n i n the p a r e n t a l a n d m u t a n t s t r a i n s , respectively. T h e large r e d u c t i o n i n V m i g h t result f r o m a q u a n t i t a t i v e r e d u c t i o n of a n o r m a l enzyme or f r o m a n aberrant enzyme being produced i n n o r m a l quantities. m
m
a
x
m
a
r
T o find out i f the m u t a n t was m a k i n g less e n z y m e , the two s t r a i n s were i n d u c e d to produce cutinase (as i l l u s t r a t e d i n F i g u r e 5) i n the presence of [ S ] m e t h i o n i n e , a n d the e x t r a c e l l u l a r proteins were separated b y 35
29.
Cutinase-Defective Mutant of Fusarium solani
DANTZIG
Ο Ο
UV O
Mutagenize
T-8 parent
Grow colonies on agarose medium containing 0.5% acetate
Grow in fresh medium for 3 days
Assay for cutinase production
403
ο ο PNB-1 mutant
F i g u r e 3. S u m m a r y o f the selection procedure for cutinase-defective m u t a n t . T h e T - 8 s t r a i n o f F. solani was m u t a g e n i z e d b y u l t r a v i o l e t i r r a d i a t i o n , g r o w n for 3 days, a n d p l a t e d o n m e d i u m c o n t a i n i n g 0 . 5 % acetate a n d agarose for 5-7 days t o p e r m i t colony f o r m a t i o n . Subsequently, t h e colonies were overlaid w i t h a n agarose s o l u t i o n c o n t a i n i n g 1.26 m M P N B . T h e p a r e n t a l colonies h y d r o l y z e d the substrate a n d t u r n e d yellow w h i l e the p r e s u m p t i v e m u t a n t colonies r e m a i n e d w h i t e a n d were selected for a n a l y s i s . F u r t h e r details are given i n Ref. 13.
404
PLANT C E L L W A L L POLYMERS
τ
1
Γ
DAYS F i g u r e 4. Specific a c t i v i t y of cutinase for the T - 8 p a r e n t a l s t r a i n a n d PNB-1 m u t a n t s t r a i n after g r o w t h o n m e d i u m c o n t a i n i n g 200 m g c u t i n . E n z y m e a c t i v i t y was assayed w i t h the a r t i f i c i a l substrate P N B ( P a n e l A ) a n d w i t h the n a t u r a l substrate, [ C ] - l a b e l l e d c u t i n ( P a n e l B ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. (c) 1986, A m e r i c a n Society for M i c r o b i o l o g y . ) 14
29.
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Cutinase-Defective Mutant of Fusarium solani
405
F i g u r e 5. C o m p a r i s o n of effect of a d d i t i o n of c u t i n or h y d r o l y z e d c u t i n o n the i n d u c t i o n of cutinase. T h e two strains were g r o w n o n m e d i u m c o n t a i n i n g 0 . 1 % glucose for 3 d a y s , a n d then 200 m g of c u t i n ( A ) or 8 m g of h y d r o l y z e d c u t i n ( B ) was a d d e d to the g r o w t h m e d i u m o n day zero. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )
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POLYMERS
F i g u r e 6. K i n e t i c s of the specific a c t i v i t y of cutinase p r o d u c e d b y the p a r e n t a l T - 8 s t r a i n a n d the PNB-1 m u t a n t s t r a i n . T h e d a t a were fitted to a M i c h a e l i s - M e n t e n e q u a t i o n (13). ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )
29.
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407
electrophoresis o n a 1 5 % s o d i u m d o d e c y l sulfate p o l y a c r y l a m i d e gel. A fluo r o g r a p h of the gel i n d i c a t e d t h a t b o t h s t r a i n s possessed a p r o t e i n b a n d of a b o u t 22,000 i n the same m o l e c u l a r weight range as the cutinase isozymes; however, by densitometer t r a c i n g the m u t a n t e x h i b i t e d about a n 8 0 % red u c t i o n i n this b a n d . I m m u n o b l o t s were conducted w i t h r a b b i t a n t i c u t i n a s e s e r u m w h i c h confirmed t h a t t h i s b a n d corresponded to cutinase ( F i g u r e 7). T h u s , the PNB-1 m u t a n t a p p e a r e d to make less of a " n o r m a l " e n z y m e . A r e d u c t i o n i n cutinase p r o d u c t i o n s h o u l d result i n the PNB-1 m u t a n t b e i n g less v i r u l e n t . T h e pathogenesis of the two s t r a i n s were e v a l u a t e d i n a p e a s t e m bioassay developed b y K o l a t t u k u d y a n d coworkers i n w h i c h i n fection by Fusanum solani results i n w o u n d f o r m a t i o n w i t h i n three days o n the e p i c o t y l of pea sedlings (15). T h e v i r u l e n c e of T - 8 h a d p r e v i o u s l y been s h o w n t o be reduced i n t h i s assay b y the a d d i t i o n of i n h i b i t o r s of cutinase or b y r a b b i t a n t i c u t i n a s e antibodies (15-18), i n d i c a t i n g t h a t cutinase played a n i m p o r t a n t role i n pathogenesis. W h e n the cutinase-defective m u t a n t was e v a l u a t e d i n the bioassay, the m u t a n t e x h i b i t e d a 5 5 % r e d u c t i o n (p < 0.05) i n v i r u l e n c e c o m p a r e d w i t h the T - 8 p a r e n t a l s t r a i n a n d the a d d i t i o n of purified cutinase at 1 m g / m l to the m u t a n t enhanced w o u n d f o r m a t i o n to 8 0 % of t h a t of the parent (p > 0.5). These d a t a further s u p p o r t the n o t i o n t h a t the m u t a n t was defective i n cutinase. Discussion T a k e n together, these d a t a i n d i c a t e t h a t Fusarium solani p r o d u c e d low levels of cutinase when g r o w n o n acetate as the sole c a r b o n source a n d t h a t t h i s c a r b o n source was less repressive t h a n glucose. T h e finding t h a t a cutinase-defective m u t a n t c o u l d be isolated u s i n g t h i s g r o w t h c o n d i t i o n p r o v i d e d a d d i t i o n a l s u p p o r t t h a t cutinase was b e i n g p r o d u c e d w h e n g r o w n o n acetate. T h u s there were three discrete g r o w t h c o n d i t i o n s w h i c h affected the synthesis of cutinase b y the m i c r o o r g a n i s m : one i n w h i c h the e n z y m e was repressed (such as glucose); one i n w h i c h the e n z y m e was i n d u c e d to h i g h levels (such as w i t h c u t i n or its h y d r o l y z e d p r o d u c t s ) ; a n d one i n w h i c h i t was neither i n d u c e d nor repressed (such as a c e t a t e ) — r e s u l t i n g i n the p r o d u c t i o n of basal levels of enzyme. Therefore, cutinase p r o d u c t i o n b y a fungus present i n the field i n the presence of a g o o d c a r b o n source w o u l d be expected to be repressed. O n c e t h a t c a r b o n source was depleted, t h e n b a s a l levels of the e n z y m e m a y be s y n t h e s i z e d . If the fungus was i n the p r o x i m i t y of the p l a n t c u t i c l e , t h e n c u t i n m a y be h y d r o l y z e d , r e s u l t i n g i n i n d u c e r f o r m a t i o n a n d r a p i d i n d u c t i o n o f cutinase synthesis a i d i n g the fungus i n the p e n e t r a t i o n of the p l a n t (12). T h e PNB-1 m u t a n t is a " l e a k y " m u t a n t t h a t is p a r t i a l l y defective i n cutinase p r o d u c t i o n , a n d a p p a r e n t l y p r o d u c e d 80 to 9 0 % less " n o r m a l " e n z y m e . T h e r e g u l a t i o n of the r e s i d u a l e n z y m e i n the m u t a n t was u n changed. T h e e n z y m e was repressed by glucose, a n d was i n d u c e d b y c u t i n or h y d r o l y z e d c u t i n after d e p l e t i o n of glucose. C u t i n a s e a c t i v i t y increased over a 15-day t i m e p e r i o d when the m u t a n t was g r o w n o n c u t i n as the sole c a r b o n source. T h e m u t a n t was also less v i r u l e n t t h a n the p a r e n t a l s t r a i n . T h e n a t u r e of the PNB-1 m u t a t i o n r e m a i n s to be e l u c i d a t e d a n d
PLANT C E L L W A L L POLYMERS
408
F i g u r e 7. C o m p a r i s o n of separated [ S ] - l a b e l l e d e x t r a c e l l u l a r proteins f r o m the c u t i n i n d u c t i o n m e d i u m of the p a r e n t a l a n d m u t a n t s t r a i n s o n 1 5 % s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gels b y fluorography a n d W e s t e r n b l o t t i n g . M y c e l i a were g r o w n a n d i n d u c e d w i t h c u t i n (as i l l u s t r a t e d i n F i g ure 5) w i t h the a d d i t i o n of 115 / i C i of [ S ] m e t h i o n i n e . A 20-fold concen t r a t e d s a m p l e c o n t a i n i n g 100,000 c p m for the parent (lanes 1 a n d 3) or the m u t a n t (lanes 2 a n d 4) was a p p l i e d to p a r a l l e l gels. T o t a l p r o t e i n synthesis was a n a l y z e d by fluorography of the gel c o n t a i n i n g lanes 1 a n d 2. I m m u n o r e a c t i v e m a t e r i a l i n the gel c o n t a i n i n g lanes 3 a n d 4 was detected w i t h r a b b i t a n t i c u t i n a s e s e r u m after e l e c t r o b l o t t i n g . M o l e c u l a r weight s t a n d a r d s ( χ 1 0 ) are i n d i c a t e d o n the left. T h e b a n d c o r r e s p o n d i n g to the m o l e c u l a r weight of cutinase is i n d i c a t e d w i t h the a r r o w . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . ) 35
35
3
29.
DANTZIG
Cutinase-Defeetive Mutant of Fusarium solani
409
m a y reside i n a p r o m o t e r for the s t r u c t u r a l gene o f cutinase or a n as yet u n i d e n t i f i e d r e g u l a t o r y gene. A l t e r n a t i v e l y , the m u t a n t m a y be defective i n t h e secretion o f cutinase. F u r t h e r s t u d y o f t h e PNB-1 m u t a n t s h o u l d p r o v i d e i n s i g h t i n t o the c o m p l e x r e g u l a t i o n o r secretion o f this b i o p o l y m e r h y d r o l y z i n g e n z y m e . W i t h a better u n d e r s t a n d i n g o f these processes, i t m a y be possible t o design agents t h a t c a n intervene i n cutinase p r o d u c t i o n r e s u l t i n g i n the c o n t r o l o f f u n g a l pathogenesis i n t h e field. Acknowledgments S p e c i a l t h a n k s are given t o D r . K o l a t t u k u d y for p r o v i d i n g the r a b b i t c u t i nase a n t i s e r u m a n d purified cutinase used i n t h i s s t u d y . Literature Cited
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