Study On A Novel DsRNA Virus Detected In Verticillium Dahliae Kleb Yue-fen Cao

As one of the most devastating harm in cotton production, Cotton Verticillium wilt disease is caused by the fungal agent Verticillium dahliae Kleb. Making studies on the virus infecting the V. dahliae has a potential significance both for the interaction of virus- V. dahliae- Cotton and for the biological control of Cotton V. wilt disease.Total 11 V.dahliae isolates originally collected from cotton fields of three different places in Shaanxi Province, China were screened for the presence of dsRNA. The result showed that only one isolate V. dahliae 0-21, which is of the lowest pathogenicity among the 11 V. dahliae isolates, carried with it 4 dsRNAs.The dsRNA segments were cloned with modified single primer amplification technique (M-SPAT). Four sequences were determined by cDNA sequencing and termed VcR1 (3594 bp), VcR2 (3313 bp), VcR3 (2983bp) and VcR4 (2932 bp) respectively according to their sizes. The alignment of the four nucleartide sequences showed that their 5′- and 3′-UTRs shared highly internal similarities and had conserved terminal stretches of UGAUAAAAAA and UUUACUACU, respectively. The deduced protein of 1108 aa (molecular mass 127.5 kDa), encoded by VcR1, had high homology to RNA-dependent RNA polymerases of viruses from genus Chrysovirus. Eight motifs characteristic of RdRPs in mycoviruses were identified. VcR2 - VcR4 potentially encoding the major capsid protein (1016 aa; 113.5 kDa), a unknown function protein (764 aa; 84.1 kDa) and a putative ovarian tumor (OTU) proteinase (823 aa; 90.2 kDa), respectively, had the properties of the other components of a chrysovirus. Four motifs conserved in the superfamily of OTU proteinase were identified in the putative protein of VcR4. All of these four sequences had high similarity to 3 members in genus Chrysovirus, Cryphonectria nitschkei chrysovirus 1 (CnV-1), Penicillium chrysogenum virus (PcV) and Aspergillus fumigatus chrysovirus (AfuCV). Phylogenetic analyses based on the amino acid sequences encoded by the four dsRNA formed subgroups with members from genus Chrysovirus, respectively, which showed more close to CnV-1, PcV and AfuCV in evolution. The results based on the genome organization, sequence alignments and phylogenetic analysis supported that the four dsRNA segments belonged to the genome of a new species of the genus Chrysovirus. Virus-like particles (VLP) were spherical diameter of about 35nm. The result of SDS-PAGE detection of VLP confirmed that VcR2 encoded the CP of a virus. The genome of VLP had same size with the dsRNAs from fungus. Viral genomic hybridization verified that these four dsRNAs were origined from one virus. And we coined it Verticillium dahliae chrysovirus 1.In summary, the complete sequences of the genome of a new chrysovirus were first identified and the view on viral mediated hypovirulence of plant-pathogenic fungus was discussed.