Cloning And Functional Analysis Of Extracellular Serine Protease Gene In The Mycoparasite Coniothyrium Minitans

Sclerotinia sclerotiorum(Lib.)de Bary is a destructive and cosmopolitan plant pathogen that causes sclerotinia stem rot of oilseed rape.The disease can cause 10-30%seed yield loss on oilseed rape,thus posing a threat for oilseed rape production.Coniothyrium minntans is an obligate parasite fungus on S.sclerotiorum,and has a good potential for biological control Sclerotinia disease of oilseed rape.Large amounts of extracellular serine proteases were secreted during C.minntans mycoparasitism on S.sclerotiorum.However,extracellular serine protease coding gene of C.minntans and its function has not been reported.This study was carried out to clone homologous gene of extracellular serine protease gene in C.minitans(CmSp),to analyze the function of CmSp by protoplast mediated transformation and to understand the roles of CmSp gene in mycoparasitism on S.sclerotiorum.Results are summarized below:Fragment of CmSp gene was obtained via amplifying by degenerate primers,all serine protease genes of C.minitans were obtained via sequence alignment in C.minitans genome database.Finally two extracellular CmSps were confirmed though the signal peptide screening and expression pattern analysis,named CmSp-9856 and CmSp-2653.CmSp-9856 coding sequence was 1335 bp with 4 exons and 3 introns,while CmSp-2653 coding sequence was 1407 bp without intron.Both CmSp were predicted to be subtilisin-like serine protease which contains Asp/Ser/His catalytic triad.CmSp-9856 and CmSp-2653 are upregulated expression when induced by S.sclerotiorum sclerotia extracts.However,expression of CmSp-9856 was significantly higher than CmSp-2653.This result indicated that CmSp-9856 may paly an impoertant role in mycoparasitism on S.sclerotiorum.CmSp-9856 and CmSp-2653 knockout transformants were successfully obtained by Split-Marker method.There was a significant reduce of extracellular protease activity in CmSp-9856 mutants,compared with CmSp-2653 mutants and wild type.This result comfirmed that CmSp-9856 palyed an impoertant role in mycoparasitism on S.sclerotiorum.The biological characteristics of CmSp mutant were no significant differences in mycelial growth and morphogenesis,compared with wild type.The effect of CmSp gene on antifungal substances produced and mycoparasitism of C.miantans were determined.The results showed that CmSp-9856 and CmSp-2653 have no effect on producing antifungal substances.CmSp-9856 knockout mutants showed a decreased capacity in mycoparasitism.But,there was no significant difference between CmSp-2653 mutants and wild type in mycoparasitism.Moreover,prokaryotic expression for CmSp-9856 and CmSp-2653 were conducted.Only CmSp-9856 was successfully induced a 30kDa protein.In conclusion,there are two extracellular serine proteases encoding gene named CmSp-9856 and CmSp-2653 in C.minitans.Both CmSp-9856 and CmSp-2653 have no effect on mycelial growth and morphogenesis.However,lacking CmSp-9856 will results in a significantly decrease of extracellular protease activity and eventually lead to a decreased capacity in mycoparasitism.These results suggested that CmSp-9856 palyed an impoertant role in mycoparasitism on S.sclerotiorum.