Molecular And Biology Charicterization Of Mycoviruses In Sclerotinia Sclerotiorum Hypovirulent Strain SX466

The fungus Sclerotinia sclerotiorum is one of the most devastative plant pathogen which infects rapeseed and other plant species. Currently there is no good way to control this disease, the potential of hypovirulence-associated mycoviruses have attracted much attention because of their potential as biological control agents for combating plant fungal diseases, it is necessary to discovery of new hypovirulent strains to control stem rot of rapeseed.Strain SX466 was isolated from the scleotia in the diseased rapeseed plant stem in Sanxi province. The colony morphologyn was abnormal, slower growth rate,no scleotia production, lower pathogenicity to detached leaves of rapeseed or soybean, exhibiting hypovirulent phenotype. Cloning the full sequence of these ds RNA segments, phylogenetic analysis of Rd Rp domain from ds RNArevealed that there were three mycoviruses. Sclerotinia sclerotiorum megabirna virus 1(Ss MBV1)shared the highest sequence identity with Rn MBV1(44%); Sclerotinia sclerotiorum mitovirus 2 /SX466 was highly similar to another two species of Sclerotinia sclerotiorum mitovirus 2(Ss MV2/SX466)(92%); Sclerotinia sclerotiorum partitivirus /SX466(Ss PV/SX466)was close to Grapevine partitivirus(GPV), identified 60%, we mainly research Ss MBV1 in this paper.Two similarly sized ds RNA segments(L1- and L2-ds RNA), the genome of Ss MBV1, are packaged in rigid spherical particles purified from strain SX466 with approximately 45 nm in diameter. The full-length c DNA sequence of L1-ds RNA/Ss MBV1 contains two large open reading frames(ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerases(Rd Rp), respectively. Although the sequences of ORF1 and ORF2 are non-overlapping, it is notable that there are no in-frame stop codons upstream of ORF2 start codon until the UGA that locates upstream of ORF1 stop codon. There was a heptanucleotideframeshift site and a downstream pseudoknot required for +1 translational frameshift, Rd Rp appears to be expressed as CP/Rd Rp fusion as evinced by the presence on the plus strand of L1-ds RNA. L2-ds RNA/Ss MBV1 comprises two nonoverlapping ORFs(ORFA and ORFB) encoding two hypothetical proteins with unknown functions. Interestingly, a conserved papain-like protease domain, a homologous protein of hypovirus p29 derived from Cryphonectriahypovirus 1, was detected in ORFA-encoded protein of L2-ds RNA/Ss MBV1. Phylogenetic analysis based on protease domain suggests that horizontal gene transfer may have occurred from an ss RNA virus(hypovirus) to a ds RNA virus, Ss MBV1.The 5’-terminal regions of L1 and L2-ds RNA/Ss MBV1 share strictly conserved sequences and form stable stem-loop structures.To further explore whether Ss MBV1 is associated with phenotypic alterations in host fungal strain SX466, horizontal transmission assay and the purified Ss MBV1 containing virus particles fractions were transfected into protoplasts of virus-free strain Ep-1PNA367 via a PEG-mediated protocol. The results indicated that Ss MBV1 successfully transfected by two ways. It is interesting that all tested the derivatives were divided into two different types based on ds RNA content. Some were found to harbor only L1-ds RNA/Ss MBV1 but with loss of L2-ds RNA/Ss MBV1(such as 1980hygV2, A367RV4), whereas the remaining of derivatives was found to harbor both L1 and L2-ds RNA/Ss MBV1(such as isolate 1980hygV1, 1980hygV3, A367RV1 and A367RV2). These results were further confirmed by RT-PCR amplification and Northern blot analysis.Although L2-ds RNA/Ss MBV1 is dispensable for replication, genome packaging, and pathogenicity of Ss MBV1, Our results reveal Ss MBV1 has a slight impact on fundamental biological characteristics of its host regardless of the presence or absence of L2-ds RNA/Ss MBV1. it could enhance transcript accumulationof L1-ds RNA/Ss MBV1 and stability of virus particle. These results were further confirmed by RT-PCR and q PCR.In order to research the function of L2-ds RNA, the silence vector were constructed according to the gene sequence and transformed by ATMT. the silence transformants demonstrated the similar colony morphology and ds RNA land compared to SX466, it was low silence efficiency. The Ep-1PNA367/ A367RV1/A367RV3 high-throughput transcriptome sequencing, there are some differentially expressed Genes, they involved in the metabolic pathway of secondary metabolite synthesis, including the genes, nonribosomal peptide synthetase(NRPSs), cytochrome P450 s and monooxygenases.