The Functional Analysis Of C2H2 Type Transcription Factor SsSte12 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is one challenging agricultural pathogen to manage and causes large global economic losses annually.The sclerotium and infection cushions are critical for its long-term survival and successful penetration on broad of hosts.The mitogen-activated protein kinase(MAPK)cascades serve as central signalling complexes that are involved in various aspects of sclerotia development and infection.In this work,the putative downstream transcription factor Ss Ste12 of MAPK cascades was analysed in S.sclerotiorum.The Ss Ste12 gene sequence was cloned from S.sclerotiorum c DNA.The gene is 2085 bp in length and contains two conserved domains of STE and Zf-C2H2.Two silencing targets p SD2 target and p SD3 target were designed,and the silencing vectors p SD2-Ss Ste12 and p SD3-Ss Ste12 were successfully constructed.The PEG-Ga Cl2-mediated protoplast transformation of S.sclerotiorum resulted in silencing transformants.The positive Ss Ste12 RNAi mutant strains p SD2-1,p SD2-4,p SD2-11,p SD2-12,p SD3-1,and p SD3-4.,p SD3-7 and p SD3-8 were selected as follow-up studies.To elucidate the function of the Ss Ste12 gene,the following studies were performed on Ss Ste12 RNAi mutant strains and the wild-type UF-1:(1)comparative analysis of colony morphology and growth rate;(2)number of sclerotia formation comparison with sclerotia;(3)Microscopic observation of mycelial morphology and formation of infestation mutants(4)The virulence of RNAi mutants and UF-1 was observed in whole leaf,half leaf and wound infection experiments.RNAi mutants of Ss Ste12 resulted in phenotypes of delayed vegetative growth,reduced size of sclerotia and less appressoria formation.As a consequence,the Ss Ste12 RNAi mutants attenuate pathogenicity on hosts as well.Yeast two-hybrid(Y2H)assay and the bimolecular fluorescence complementation(Bi FC)revealed the Ss Ste12-Ss MCM1 interaction in the nucleus.But Ss MCM1 expressing is independent on the regulation of Ss Ste12 by q RT-PCR testing in Ss Ste12 RNAi mutants.Together with high Ss Ste12 gene transcript accumulation in early development of S.sclerotiorum,our results demonstrate that Ss Ste12 functions as the downstream transcription factor of MAPK in a regulatory role of vegetative mycelial growth,sclerotia development and appressoria formation.And the Ss STE12-Ss MCM1 interaction might play an important role in the regulation of the genes encoding these traits in S.sclerotiorum.