Sclerotinia Sclerotiorum Pathogenicity-related Effectors Screening And Mechanism Of Action Study

Sclerotinia sclerotiorum is a widespread necrotrophic fungus that causes substantial economic losses every year.Sclerotinia has a particularly serious impact on rapeseed,causing not only massive yield reduction or even crop failure but also affecting the quality of rapeseed oil.At present,no germplasm resources in Brassica napus with full S.sclerotiorum resistance have been found and the key resistance genes are not clear.The key unknown pathogenic factors of S.sclerotiorum can be used as probes to find new resistance genes and provide new ideas for rapeseed resistance breeding.With the development of sequencing technology,a large number of potential effectors in the S.sclerotiorum genome have been predicted.However,most of the effectors predicted by the bioinformatics studies lack in-depth experimental validation.In this study,we systemically cloned 84 potential S.sclerotiorum effectors.By transiently overexpressing these effectors in tobacco leaves through Agroinfiltration and then inoculating S.sclerotiorum on the agroinfiltrated leaves,we identified five effectors(S251,S275,S288,S45,and S201)that could promote S.sclerotiorum infection.To further investigate the effectors,we constructed S.sclerotiorum mutants of these five effectors and successfully obtained homozygous knockout mutants of two genes(S275 and S45,renamed as SsERP1 and SsMIP1 subsequently).We further explored the mechanisms by which SsMIP1 and SsERP1 affect the pathogenicity of S.sclerotiorum.1.SsERP1 promotes S.sclerotiorum infection by inhibiting the ethylene pathwayProtein sequence analysis revealed that SsERP1 belongs to the Egh16 family and contains a secretory signal peptide.Transient expression of SsERP1 fused to GFP in tobacco resulted in fluorescent signals observed in both cytoplasm and nucleus,and the subcellular localization of SsERP1 was not altered after removal of the signal peptide.Ectopic overexpression of SsERP1 in plant leaves promoted S.sclerotiorum infection.The pathogenicity of SsERP1 knockout mutants was reduced,while their acid production,mycelial growth,and sclerotium formation were normal,suggesting that SsERP1 specifically modulates the pathogenicity,but not the growth and development,of S.sclerotiorum.By lactophenol cotton blue staining of B.napus leaves inoculated with Sserp1 or wild-type strain,we observed that Sserp1 developed less appressorium than wildtype at the early infection stage.Manually pre-piercing B.napus leaves before inoculation was able to rescue the pathogenicity of Sserpl mutant.Transcriptome analysis indicated that SsERP1 promotes S.sclerotiorum infection by inhibiting the plant ethylene signaling pathway.Knocking down SsERPl expression by in vitro synthesized double-stranded RNA was able to effectively suppress S.sclerotiorum infection,which confirms the role of SsERP1 in S.sclerotiorum pathogenesis and further suggests a potential strategy for S.sclerotiorum control.2.SsMIPl interacts with MORF2 to affect the pathogenicity of S.sclerotiorumHomologous sequence analysis showed that the homologous protein of SsMIP1 is only found in Ascomycota.Signal peptide prediction analysis showed that SsMIPl contains a secretory signal peptide.YTK12 yeast secretion assay verified the function of SsMIP1 signal peptide,suggesting that SsMIP1 is a secretory protein.When transiently expressed in tobacco leaves,the fluorescence of GFP-fused SsMIP1 was observed in both cytoplasm and nucleus,and similar subcelluar localization was also detected for truncated SsMIP1 without the signal peptide.Ectopic overexpression of SsMIP1 in either tobacco or Arabidopsis thaliana promoted S.sclerotiorum infection,consistent with the reduced pathogenicity of SsMIP1 knockout mutants,suggesting that SsMIP1 positively modulates S.sclerotiorum pathogenicity.However,acid production,mycelial growth,and sclerotium formation of SsMIP1 knockout mutants were similar to that of wildtype strain,indicating that SsMIP1 did not affect the growth and development of S.sclerotiorum.Transcriptome analysis of Ssmipland wild-type strains inoculated oilseed rape leaves indicated that SsMIPl promotes S.sclerotiorum infection by inhibiting the plant ethylene signaling pathway.Y2H experiments and BiFC experiments showed that SsMIPl interacts with MORF2,an RNA editing factor for chloroplast genes.Inducible expression of SsMIP1 in pER8-SsMIP1 transgenic A.thaliana reduced the RNA editing efficiency of MORF2.Silencing plant MORF2 gene using VIGS suppressed the plant ethylene signaling pathway and reduced plant resistance to S.sclerotiorum,indicating that the MORF2 gene can positively regulate plant resistance to S.sclerotiorum through the ethylene pathway.In summary,in this study,we systemically cloned and screened putative S.sclerotiorum effectors,and identified five novel effectors that contribute to S.sclerotiorum pathogenicity.Action mechanisms for two of these five effectors,SsMIP1 and SsERP1,were further investigated in depth.Our work adds new insights into the pathogenesis of S.sclerotiorum,and provides mechanistic basis and gene targets for S.sclerotiorum control.