Stablishment Of Double-stranded RNA-mediated Gen Silencing And Characatezaiton Of CgMEK1in Poplar Anthrasnose Fungus Colletotrichum Gloeosporioides

Poplar anthracnose is one of the most destructive diseases on Poplus sp, whose causal agent is Colletotrichum gloeosporioides. Although the fungus is a broad-host range plant pathogen, only dozens of genes involved in pathogenesis have been identified and characterized. In order to establish a high-throughput platform,at first,we established a polyethylene glycol (PEG)-mediated protoplasts transformation of C.gloeosporioides and obtained the transgenic transformants expressing a green fluorescence protein (GFP).Then,we delivered the double-stranded RNA(dsRNA) expression cassette into protoplasts to trigger silencing for functional genomics research in C. gloeosporioides.PEG-mediated protoplasts transformation. Protoplasts were mixed with the plasmid gGFP containing the hygromycin phosphotransferase (hph) gene and GFP gene and treated with PEG. Screened by the concentration of hygromycin B at300μg·ml-1, transformation frequencies of41transformants per μg DNA were achieved. The results of gDNA PCR amplifications indicated that the hph gene and GFP gene were indeed integrated into the genome of the C1-5-2strain. Fluorescence observation showed clear and strong expression of the green fluorescent protein in fungal structures, and genetic stability of GFP-tagged transformants were stable.Double-stranded RNA(dsRNA)-mediated transient gene silencing. A new silencing vector pSD-SUR1based on RNA-silencing vector (pSD1) with a convergent dual promoter is introduced. As an indicator of gene silencing, GFP fluorescence is used to evaluate efficiency of this silencing system. The fluorescence observation showed GFP fluorescence significantly decreased in some of the silenced strains, comparing with the recipient strain. The GFP mRNA transcript levels in the strains were analyzed using quantitative RT-PCR. Successful dsRNA-induced silencing of GFP expression was achieved, and a silencing efficiency ranged from10%to99%when compare to controls, suggesting that RNA silencing is efficiently promote genetic knockdown.CgMEK1controls appressoia formation and pathogenicity in the fungus CgMEK1gene was inserted into the vector pSD-SUR1and silenced, suggesting that this gene silencing system is effective and is a feasible approach to generate detectable phenotypes in C. gloeosporioides. In conclusion, RNA silencing system opens up new opportunity for exploring gene function in the fungus C. gloeosporioides.