Interactors Screening Of Ss-FoxE2 Gene And Functional Analysis Of Ss-Chk2 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum?Lib.?de Bary is a very important plant pathogenic fungus.It infects over 500 species of plants worldwide and cause a disease called white mold.And it causes a lot of economic losses each year.At present,the main method to control this fungus is the chemical pesticides.But there are many problems about these pesticide,such as environmental pollution and increased resistance in fungus.Studying the pathogenicity and growth of S.sclerotiorum from the molecular level can provide us with new ideas to control it.Previous studies have shown that the sclerotium and apothecium have a key role in life cycle and infection cycle.Sclerotium not only can survive in adversity,but also produce mycelium that cause primary infection.Apothecium that germinate from sclerotium can produce ascospore.And these ascospores can cause epidemic of S.sclerotiorum by infection.In the previous research some transcription factors with a Forkhead domain were associated with the development of sclerotia and apothecium.Ss-FoxE2,on of these forkhead proteins,can influence sclerotia germination.The deletion mutant of Ss-FoxE2 gene can not develop apothecium.In order to clarify the upstream regulatory mechanism of Ss-FoxE2 gene that mediate apothecium development,yeast one hybrid technique was used to screen the interacting proteins with Ss-FoxE2 in this study.In the S.sclerotiorum,a protein Ss-Chk2 was found to have a Forkhead-associated domain and a serine/threonine kinases domain.Meanwhile,this study also carried out an analysis of Ss-Chk2 gene function,because it is related to the growth and development of S.sclerotiorum as predicted.Seven proteins interacting with Ss-FoxE2 gene were screened out from the cDNA library of S.sclerotiorum by Y1 H,and numbered from SsE2-1 to SsE2-7.A false positive SsE2-7 was verified by yeast cotransformation with bait and AD vectors which have full-length c DNA of these proteins.Through the prokaryotic expression technique,all the proteins except SsE2-5 were successfully expressed and were soluble proteins except for SsE2-2.And some proteins were purified with Ni-NTA Agarose.The EMSA result show that SsE2-1-2 and SsE2-1-3 fusion protein can bind to the probe 2 and probe 3,but this result can not eliminate TF protein interference.A deletion mutant of SsChk2 had created through homologous recombination.Phenotypic analysis showed that the gene had little effect on the infection structure and pathogenicity,and the mycelial growth became faster after knockout in normal culture medium.For different stress conditions,the phenotype is different.The mutant was more susceptible to 1 M KCl,1 M NaCl and 0.02% SDS,and the mycelium grew slower than the wild type.On 1 M sorbitol medium,mycelium of mutant and wild type was little difference.The mutant was more sensitive for different concentrations of H₂O₂ and can not grow on 15 m M H₂O₂,but wild type can grow on it.These results showed that Ss-Chk2 gene had a role in S.sclerotiorum adaptation to adversity.The results of this study laid the foundation for the further study of Ss-FoxE2 regulation of apothecium development and Ss-Chk2 regulation of growth and development.