The Effect Of BcKMO Gene On Growth And Development As Well As Pathogenicity In Botrytis Cinerea

A mutant (BCG183) with enhanced pathogenicity was obtained by screening thetransformants of Botrytis cinerea produced by Agrobacterium tumefaciens-mediatedmethod. Surprisingly,the mutant BCG183with slender hyphae showed slower growth rate,neither producing conidia nor sclerotia on PDA. In order to further verifying the impactofBcKMO gene on hyphal growth, conidial and sclerotia production as well as pathogenicityin Botrytis cinerea, Bioinformatics methods were adopted to analyze the BcKMO gene andits encoding production. Base on the BcKMO gene T-DNA insertion mutant BCG183, theBcKMO gene complementing mutant (BCG183/BcKMO) was obtained by using PEGmediated protoplast transformation.Comparative study on hyphal morphology, growth rate,conidial and sclerotia production, pathogenicity as well as major pathogenic factorsincluding cell wall degradation enzyme activity, toxin activity, expression ofpathogenicity-related genes had been done among wild type BC22, the mutant BCG183and the BcKMO gene complementing mutant (BCG183/BcKMO). The main results were asfollow:1. The BcKMO gene encodes kynurenine3-monooxygenase (KMO) havingmonooxygenase FAD conserved domain and four Aromatic-ring hydroxylase-like motifs.The BcKMO protein displayed high homology to Rossmann-fold NAD(P)(+)-bindingprotein (gi156049701) of Sclerotinia sclerotiorum and FAD-dependent oxidoreductase(gi512192943) of Ophiostoma piceae.2. The mutant BCG183with tenuous hyphae revealed slower growth rate, neitherforming conidia nor sclerotia on PDA as well as strengthened pathogenicity on leaves ofkidney bean and cucumber compared with wild type BC22and the BcKMO genecomplementing mutant (BCG183/BcKMO).3. Polymethylgalacturonase (PMG) and endopolygalacturonase (PG) activity ofBCG183was significantly higher than wild-type and BCG183/BcKMO while cellulase(CX), polygalacturonic transeliminase (PGTE) and pectin methyl transelimination enzyme (PMGE) enzyme activity were no significant difference compared with wild-type andBCG183/BcKMO. Toxin activity of BCG183was significantly higher than wild-type andBCG183/BcKMO. The acid production of BCG183significantly decreased compared withwild-type and BCG183/BcKMO. No difference was showed as far as the penetration toonion epiderm was concerned.4. Using Real-Time PCR technology analyzed the expression levels ofpathogenicity-related genes in mutants BCG183and BCG183/BcKMO. Compared withwild-type BC22and BCG183/BcKMO, the expression of pathogenicity-related genes, i.e.,bac, bcg2, bcg3, PkaR, bmp1, Sak1, bcreg1, bos1, bcp1, Ras2, Bcpg1and Sod1weresignificant up-regulated in the mutant BCG183.