Functional Analysis Of The Transcription Factor SsAMS2 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic plant pathogen causing significant financial loss.Over 400 plant species can be infected by S.sclerotiorum.Infection cushion is the important infection structure which plays a decisive role in causing hosts disease outbreak.In the interaction between S.sclerotiorum and hosts,S.sclerotiorum produce a range of pathogenic factors.Oxalic acid is one of the important pathogenic factors,and plays critical roles in pathogenicity,virulence and competition with other fungal species.Eight transcription factor containing GATA zinc finger domains were predicted in S.sclerotiorum.Among these,Ss Nsd1 has been demonstrated to be involved in sclerotia,apothecia and infect cushion development,and negatively control spermospore formation.Ss AMS2 is a GATA transcription factor,which is homologous with Ams2 in Schizosaccharomyces pombe.So we predict Ss AMS2 is involved in transcription initation.In this study,two target sequences of Ss Ams2 was cloned and ligated into p Silent-Dual1.The reconstruction plasmids were transformed into protoplasts of wild type mediated bypolyethylene glycol(PEG)-Ca2+ and Ss Ams2 was silenced.Thus the transformants were obtained.The expression levels of Ss Ams2 in transformants were significantly down regulated.The RNAi mutants exhibited defect in hyphal growth and development,decreased sclerotia weight,curved hyphae.And the numbers of cell nuclei were decreasd.The relatively transcription level of histones and histone variant genes were down regulated,besides,the cell cycle relative factor Cdc6,Cdc28 gene transcription level were also down regulated.These results suggest that Ss AMS2 regulated histone genes transcription and cell division.The resistance to oxidative stress were enhanced when Ss Ams2 was silenced,the results suggest that Ss Ams2 negatively control peroxidase production.There was no significant difference in resistance to osmotic stress between transformants and wild type.The transformants produce decreased infect cushion and oxalic acid,and when inoculated to marrow bean leaves the incidence rate were reduced.The results indicated that Ss Ams2 plays important roles in pathogenicity.The gene function analysis provide theory basis for S.sclerotiorum control.