Functional Analysis Of The Proteins SsNACα And SsPpt1 Regulating The Asexual Development And Pathogenicity Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.） de Bary was one of the most destructive fungi on rapeseed. The sclerotinia stem rot on rapeseed caused by S. sclerotiorum influenced the output and quality of rapeseed badly. In order to provide theoretical and technical support for controlling sclerotinia stem rot effectively, this paper studied the molecular mechanism of biotrophic pathogenicity of S.sclerotiorum, and discussed the function of Nascent Polypeptide Associated Complex Alpha Subunit（NACα） and phosphopantetheinyl transferase, especially their function on the pathogenicity of S.sclerotiorum. The main results of the study were as follows.1. Nascent polypeptide associated Complex Alpha Subunit, Negatively Regulated the Polygalacturonases Expression, Modulates the Pathogenicity of Sclerotinia sclerotiorumS. sclerotiorum is a necrotrophic plant fungus, which produces a compact mass of hardened mycelia playing a major role in disease progression. In this study, a nascent polypeptide associated complex α subunit of S. sclerotiorum（SsNACα;GenBank Accession No. XP₀01593856.1） was cloned and characterized. The full length cDNA of SsNACαcomprised a 615 bp open reading frame（ORF） encoding a 215 residue protein. The expression levels of different morphological stages were analyzed by quantitative RT-PCR.SsNACα expression level was elevated at the sclerotial pigmentation stage and approximately 1.8-fold about the level of hyphal stage. The gene function was investigated using RNA interference, and the results showed that silencing of SsNACα resulted in delayed maturation progress of sclerotial development, the pathogenicity of the silenced mutants was enhanced, and the pectinase activity and the expression levels of polygalacturonases were elevated. Our results suggested that SsNACα silencing could enhance the expression of pectinase enzymes, and induce the pathogenicity of S.sclerotiorum strengthened.2. The bioinformatics prediction of phosphopantetheinyl transferase candidate coding gene in Sclerotinia sclerotiorum.Phosphopantetheinyl transferase（PPTase） was an important class protein superfamily existing in eukaryotes. These PPTase could affect the synthesis of fatty acids or some secondary metabolites involved of acyl carrier proteins. Thus PPTase could involve in theregulation of not only the primary biological metabolism but also the secondary metabolism, and it even eventually affected the growth or pathogenicity of organisms. In this study the candidate coding genes of PPTase in S. sclerotiorum were selected throughing the methods of bioinformatical methods. The results showed that there were three hypothetical protein PPTases in S. sclerotiorum. Their predicted proteins were charactered respectively, and the phylogenetic tree of them and their homologs from different organisms were constructed. This work could provide a cue for further study the function of the phosphopantetheinyl transferase in S. sclerotiorum and other organisms.3. Silencing of a phosphopantetheinyl transferasegene（SsPpt1）affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorumIn the genome sequence of the fungal pathogen S. sclerotiorum, we identified a phosphopantetheinyl transferases gene homologue SsPpt1 with a ACPS modules. And we found that SsPpt1 transcription was accumulated significantly at the sclerotial matuation stage. SsPpt1 gene slienced mutant strains were generated by using RNA interference method. We monitored and characterized the mutant strains, and found that the hyphal growth rate was low down sinificantly and the branch pattern altered. Aberrant sclerotia were produced with reduced about half number. Moreover, the virulence of Ss Ppt1 gene slienced strains was attenuated severely and failed to generate necrotic disease symptoms as the wild type strain. These results indicated that SsPpt1 protein plays an important role in hyphal growth, sclerotial development and pathogenesis of S. sclerotiorum.