Functional Analysis Of CmAvaB In Development And Conidiation Of Coniothyrium Minitans

Coniothyrium minitans is an important sclerotia mycoparasitic of Sclerotinia sclerotiorum.and could parasite and destroy hyphae and sclerotia of S.sclerotiorum. Thus is an important biocontrol fungus for sclerotinia. In this reserch, a conidiation deficient mutant ZS-1N22225of C. minitans was studied, and CmAvaB gene, encoding the vacuolar morphogenesis protein was cloned. The function of the gene in development of C.minitans was analyzed using gene disrupion and gene complemental techiques. The results were as followed:Compared with the wide type strain ZS-1, the colony morphology of mutant ZS-1N22225was significantly different on potato dextrose agar (PDA), including hyphae tend to grow on culture midia, growth slowed down, and reduced conidiation, The parasitic ability toward to sclerotia of S. sclerotiorum was declined, while the ability to produce anti-bacterial substance was enhanced. showed fragmented vacuolar morphology could been observed with electron microscopy. The conidia production was increased by1%-5%of ZS-1N22225incubated on PDA add with. carrot juice or S. sclerotiorum sclerotia debris.Previously, a genomic DNA fragment flanking in sertional T-DNA in ZS-1N22225was obtained by inverse-PCR. Combining the whole genome sequence of ZS-1, we find that this gene have3315bp, T-DNA inserted in the promoter of the gene CmAvaB, which contains three exons and two introns, encodes a1071aa protein. The deduced amino acid sequence of CmAvaB shared96%,93%and77%identity with vacuolar morphogenesis protein AvaB from Pyrenophora tritici-repentis Pt-1C-BFP, Leptosphaeria maculans JN3and Aspergillus flavus NRRL3357.Inverse-PCR showds that the expression accumulation of CmAvaB in ZS-1N22225was decreased greatly than in ZS-1. In order to study the gene function, CmAvaB in ZS-1was knock-out with Agrobactirium tumfacience mediated-transformation (ATMT). Three CmAvaB knock-out transformants shared similar characteristics with ZS-1N22225, and the resistance to0.1M sorbitol were enhanced, while were more sensitive to sodium chloride and pH stress Complementary test was carried out by transforming the gene into a knock-out transformant, KO20. Two complementarily transformants names C6and C9.shared similar characteristics with ZS-1. The results suggested that CmAvaB gene is involving in growth and development and conidiation.