Functional Analysis Of C2H2 Zinc Finger Protein SsZFH1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is a broad host range plant pathogen and causes lots of loss in the worldwide scale every year.Sclerotinia sclerotiorum can form the infection structure,named comprounded appressorium,in order to adhere and penetrate the host plant during pathogenic processes.In addition,Sclerotinia sclerotiorum is able to synthesize oxalic acid and cell wall degrading enzymes,which play important role in resisting host defense reaction and accelerating pathogenicity.C2H2 zinc finger protein make up one of the largest sequence-specific transcriptional factor superfamilies in eukaryotes but are poorly explored in fungi.The function of the C2H2 zinc finger protein has been implicated in growth and development,conidiation,sexual reproduction,pathogenicity,drug resistance and abiotic stress response.The Sszfh1 protein of Sclerotinia sclerotiorum has a C2H2 zinc finger domain in the C-terminal thus is belonged to C2H2 zinc finger protein family.In this study,944 bp upstream and 929 bp downstream flanking fragments of Sszfh1 were amplified from the genomic DNA of Sclerotinia sclerotiorum strain UF-1 and were sequentially cloned into the same pXEH vector to obtain a Sszfh1 gene replacement vector,pXEH-L-R.The Sszfh1 gene replacement vector was used to replace the Sszfh1 sequence with the hygromycin resistance cassette during PEG-mediated transformation.After purification by transferring the edge of the colony to PDA media supplemented with hygromycin,two independent knock-out mutants were obtained.In this study,the Sszfh1 gene complemented vector pNDH-OGG-Sszfh1 was constructed and was transformed into the protoplast of the Sszfh1 gene-knock-out mutant.After purification and PCR detection,one Sszfh1 complemented strain which showed same phenotype as wild type was obtained.The difference between the wild type and the knock-out mutants were mainly as followed:(1)The knock-out mutants showed a significant reduction in vegetative hyphal growth and abnormal hyphae branch,which tend to aggregate together.The colonial morphology and the site of the formation of sclerotia were quite different from the wild type.In the knock-out mutants,the number of the sclerotia increased but the dry weight decreased.(2)The knock-out mutants have the ability to synthesize oxalic acid and form the comprounded appressorium,and showed decreased pathogenicity on detached leaves with the ability to penetrate the host.(3)Disruption of Sszfh1 resulted in resistance to oxidative stress and increased sensitivity to salt,hyperosmotic stress,fungicide and sodium dodecyl sulfonate.The expression level of Sssop1 as well as Ssnox1 and Ssnox2,which is concerned with stress response,is significantly decreased in the knock-out mutants.These results pointed that Sszfh1 is involved in the regulation of various stress responses.This study demonstrates,in Sclerotinia sclerotiorum,the function role of Sszfh1,makes a contribution to the explanation of development and pathogenicity at the molecular level,lay a foundation of the research about C2H2 zinc finger proteins and provide a new perspective on integrated management of sclerotiniose.