Identification And Analysis Of Sclerotinia Sclerotiorum Genes Suppressed By The Infection Of Mycovirus

Sclerotinia sclerotiorum is a major pathogen of the important oil crop- rapeseed and causes serious damage to rapeseed production, both on yield and quality. The infection of mycovirus alters many of its characteristics, including reduced-virulence (hypovirulent), slow growth and colony development. To understand the molecular basis for those changes, a cDNA library of virulent strain EP-1PNAa (single-ascospore-isolation of hypovirulent strain EP-1PN) with mRNA extracted from hyphae after one-day, two-day and three-day was constructed, and then probed with cDNA reverse-transcripted from mRNA extracted after two days, three days and four days of hypovirulent stain EP-1PN. 1116 clones with no hybridization signal were obtained.210 cDNA clones from 1116 were selected for sequencing and the database searches were performed using the NCBI non-redundant GenBank database. 139 of the cDNAs had significant homology to sequences in the database while 12 clones had no homology to sequences examined. Sequences were then grouped into MIPS functional categories according to their putative BLASTX identification. The groups include Metabolism, Energy, Transcription, Protein synthesis, Protein destination, Transport facilitation, Vesicular transport, Cytoskeleton biogenesis, Signal transduction, Cell defense/stress, Organization and Unclassified function.Expression of 210 clones was examined using dot-blot cDNA array and virtual Northern blots. The result of cDNA array exhibited those clones on membrane probed with mRNA of EP-lPNAa had a hybridization signal clearly stronger than that on membrane probed with mRNA of EP-1PN, which proved the reliability of differential selection and suggested the mycovirus affected the expression of its host extensively. According to the result of the cDNA array dot-blots, 18 clones were selected for northern blotting to check their expression at RNA level in EP-lPNAa and EP-1PN. 12 clones (66%) showed different expression pattern between EP-lPNAa and EP-lPN,namely having a stronger hybridization signal in strong-virulence stain RNA. The results confirmed the result of dot-blot cDNA array. Putative proteins of those confirmed genes included pathogenic relevant factor- endopolygalacturonase and Cyclophilin; pathogenic relevant signal conduction factor-Inorganic pyrophosphatase; the protein regulary factor-zinc-finger protein; energy synthesis relevant product Phosphoglycerate kinase and Enolase (2-phosphoglycerate dehydratase) and protein synthesis precursor 40S ribosomalprotein.Further research of clone MVSG530showed that MVSG530 enoded cyclophilin, which belongs to a large gene family. The gene family is reported often to involve in growth, development and virulence formation of pathogens. The mycium of S. sclerotiorum was very sensitive to the immnosuppressive drug Cyclosporin A, which means there exists the active site of Cyclosporin A -cyclophilin in 5. sclerotiorum and cyclophilin is very important for the growth , development and virulence formation.All those genes including Cyclophilin may get involved in pathogenesis of S. sclerotiorum and contribute to its pathogenesis, and this need further study in the future. Our study provides not only theory basis for the mechanism of pathgenesis of S. sclerotiorum, but also molecular basis of mycovirus-fungi interrelations.