The Function Of SsNR Gene In Regulating The Growth And Development And Pathogenesis Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic,homothallic,fungal pathogen that causes disease in a wide range of plants. It can cause a disease called sclerotinia rot in rapeseed,soybean, sunflower and other oil crops, resulting in great losses to agricultural production. The completion of whole-genome sequencing of S. sclerotiorum, and the popularization of DNA sequencing, RNA interference (RNAi), high-throughput proteomics and metabolomics and other techniques can greatly facilitate the research on the functional genes of S. sclerotiorum.Nitrate is a major source of inorganic nitrogen for higher plants, algae, fungi, yeasts and bacteria. Nitrate reductase (NR), a key enzyme for the assimilation of nitrate, has been widely found in a variety of bacteria, fungi and higher plants, and its activity determines the rate of nitrate assimilation. As one of the rate-limiting enzymes during the metabolism of biological nitrogen,NR is inextricably related to the biological absorption and utilization of nitrogen.In the present study, SsNR, the NR-encoding gene in S. sclerotiorum, was cloned, and then SsNR-pSilent-1 expression vector was constructed by gene silencing. NR-silenced mutant of S.sclerotiorum was obtained by PEG-mediated protoplast transformation. Moreover,two mutant strains NR12 and NR66 were isolated based on HygB resistance, phenotypic identification and RT-PCR to evaluate the function of SsNR gene in regulating the growth, development and pathogenesis of S. sclerotiorum.The results revealed that compared with wild type, SsNR-silenced mutants grew slowly, with many short branches at the tips of mycelia, and deformed infection cushion, and sclerotium formation in mutants was inhibited, and oxalic acid production is weakened. In addition, the mutants were highly sensitive to Congo red, SDS, H2O2 and NaCl, but insensitive to sorbitol. The pathogenicity of the mutants was weakened. By analyzing the expression of pathogenicity-related genes, we found that Ggt1, Sac1 and Smk3 were down-regulated, while Ubq and CYP were up-regulated.In summary, SsNR plays an important role in the formation of sclerotia, appressorium,infection cushion, and the vegetative growth and pathogenesis of S. sclerotiorum. The results provide a theoretical basis for studying the pathogenic factors and molecular mechanism of S.sclerotiorum.