Screening And Funcional Analysis Of Sclerotial Formation Associated Gene In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum (Lib) de Bary is an important plant pathogen fungus with a worldwide distribution. It can infect numerous host plant, including 75 families,450 species. Sclerotia play significant roles in their life cycle. In this study, the gene Ss-MFS which is involved in the formation of sclerotia, was screened from the gene expression profile of S. sclerotiorum and its function was studied. The main results of this study are listed as following,1. Ss-MFS was 2196 bp in length, encoded a 521 aa protein. This protein contained 14 transmembrane helices and conserved motifs of the MFS and efflux-EmrB domains, suggesting that it was the drug resistance transporter of MFS. Real-time PCR results showed that the expression levels of Ss-MFS was very low at 1-2 dpi, but rose quickly at 3 dpi and reached to the peak at 7 dpi when the sclerotia was formation.2. We constructed a RNA silencing vector named sipCH and used it to transform S. sclerotiorum strain Ep-1PNA367 by ATMT technology. In total,22 candidate transformants were screened out. Some Ss-MFS-silenced transformants were confirmed by PCR detection of the hygromycin resistance gene fragment on the T-DNA. Real-time PCR verified that the transcript accumulations of transformants A8, A9 and B49 were reduced to 50%-80%. Biological experiments showed that these transformants generally grew slowly, and the ability of producing sclerotia on PDA and carrot medium was decreased significantly. In addition, the pathogenicity of most candidate transformants was declined when inoculated on rapeseed leaves. Among these, the transformants A8, A9 and B49 not only lost pathogenicity but also the ability of sclerotia formation completely. However, all of them could secrete oxalic acid, suggesting that the incapacity of sclerotia formation and pathogenicity were not related to oxalic acid. The growth of all transformants were inhibited on the mediums composed of different carbon source, indicating that these had no selection for the use of different carbon sources and may be affected by other nutrients.This study confirmed that the ability of sclerotia formation were decreased when Ss-MFS gene expression was inhibited, suggesting that this gene may be associated with the formation of sclerotia.