The Function Of GATA Transcription Factor In The Growth Development And Pathogenic Process Of Sclerotinia Sclerotiorum (Lib.) De Bary

The Leotiomycetes fungus Sclerotinia sclerotiorum?Lib.?de Bary is a notorious plant pathogen,it infects over 600 plant species and incites rapid host tissue maceration in a non-discriminant manner.Because of the broad host range and caused heavy crop loss.S.sclerotiorum are white filamentous fungi with a complete life cycle.It produces hardened multicellular sclerotium in the form of aggregation of vegetative hyphae,Sclerotia as a crucial structure in survivability and pathogenicity of S.sclerotiorum can differentiate either into vegetative hyphae or into apothecia which could release a lot of airborne ascospores to infect host plant and start a new disease cycle.Therefore,the prevention and control of S.sclerotiorum is very difficult.In order to better understand the gene expression during the different morphological development of S.sclerotiorum and further improve the information of the S.sclerotiorum genome database.We performed comparative transcriptome analysis of hyphae,sclerotia and apothecia at three developmental stages to obtain new genes and annotate them and to make reasonable predictive analysis of alternative splicing and SNP loci for S.sclerotiorum.Combined with the changes in expression levels,the functional modules were predicted by the annotation and classification of GO,KEGG and COG.For the data obtained by transcriptome sequencing,a large class of zinc finger protein transcription factor family genes were selected for in-depth analysis,and their expression in different morphological developments in S.sclerotiorum was statistically found.During the process,the transcription factors of this family were mobilized more,and the number of genes that were differentially expressed were significantly increased.Among them,GATA transcription factors,one of the components of the C4 zinc finger protein subfamily,were found to be relatively conserved in fungi,and were widely involved in the growth and development process.The expression levels of mRNA in the growth and development stages of different forms have changed greatly.Therefore,we selected three GATA transcription factor genes for further functional verification.The GATA transcription factor SsSFH1,as one of the well conserved TFs in eukaryotic organisms,little is known about the roles of SFH1 protein in the filamentous fungi,and the homologous proteins in yeast,as components of the chromatin remodeling complex RSC,are involved in the regulation of gene transcription and chromosome repair.Here,we characterized the roles of Sssfh1 in the developmental process and fungal pathogenicity by using RNA interference?RNAi?-based gene silencing in S.sclerotiorum.RNA-silenced strains with significantly reduced Sssfh1 RNA levels exhibited slower hyphal growth and decreased reactive oxygen species?ROS?accumulation in hyphae compared to the wild-type?WT?strain.Yeast two-hybrid?Y2H?and bimolecular fluorescence complementation?BiFC?assays demonstrated that SsSFH1 interacts with SsMSG5,a MAPK phosphatase in S.sclerotiorum.Furthermore,Sssfh1-silenced strains exhibited enhanced tolerance to NaCl and H₂O₂.Results of infection assays on soybean and common bean?Phaseolus vulgaris?leaves indicated that Sssfh1 is required for full virulence of S.sclerotiorum during infection in the susceptible host plants.Collectively,our results suggest that the TF SsSFH1 is involved in growth,ROS accumulation and virulence in S.sclerotiorum.The typical GATA transcription factor SsAREA contains a conserved GATA domain.In fungi,AREA transcription factors are involved in nitrogen metabolism and are key regulators of nitrogen uptake.In this study,the RNAi gene silencing strategy was used to reduce the expression of SsareA in varying degrees.The results showed that SsareA was involved in the growth of mycelium,and the mycelial growth rate of RNAi-silenced strains was greatly affected.At the same time,the melanin was more accumulated.The melanin deposition of the silenced transformants is significantly higher than that of the WT strain,and the morphology and distribution of the sclerotia after gene down-regulation are also significantly changed.The pathogenicity was significantly reduced.The SsSRE transcription factor contains two conserved GATA domains whose homologous proteins verify that the gene is involved in the synthesis of the fungal iron vector.The results of gene function verification in this study indicated that the RNAi transformants expressed by down-regulation of Sssre gene had no sclerotia formation,no melanin accumulation,and hyphae could not form mature infection structure,and no oxalic acid was produced.QRT-PCR showed that the expression level of Ssoah1,a key gene for oxalic acid formation in Sssre gene silencing transformants,was significantly reduced.Protein network prediction of SsAREA and SsSRE transcription factors,double validation of Y2H and BiFC showed that SsAREA and SsSRE transcription factors interacted with SsSTE12 and SsMCM1which found in MAPK signaling pathway.In this study,RNA-Seq analysis was carried out at the growth and development level of S.sclerotiorum.To analyze the changes of mRNA levels in different morphological processes of polymorphic pathogenic fungi from the transcriptome level.The evolutionarily conserved GATA zinc finger transcription factors were selected.Functional verification and protein interaction analysis of the family proteins SsSFH1,SsAREA and SsSRE provide molecular basis for sclerotia development,infection cushion formation and pathogenicity.In addition,this study revealed the relationship between GATA transcription factors SsAREA and SsSRE and MAPK signaling pathway,which laid a foundation for the study of the regulation mechanism of sclerotia and infection cushion formation.