Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.
Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.
D000085:Acetates / Q000494:pharmacology; D002087:Butyrates / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000378:metabolism*; D003470:Culture Media; D004790:Enzyme Induction; D007887:Fabaceae / Q000382:microbiology; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D010935:Plant Diseases; D010944:Plants / Q000382:microbiology; D010946:Plants, Medicinal
D000085:Acetates; D002087:Butyrates; D003470:Culture Media; D008563:Membrane Lipids; C033592:4-nitrophenyl butyrate; C000521:cutin; D002265:Carboxylic Ester Hydrolases; C019952:cutinase