Cloning And Functional Analysis Of A Specific Secreted Protein Gene VdSSP1in The High Virulent Verticillium Dahliae Strain

Verticillium dahliae is a soil-borne fungus causing destructive and widespread vascular wiltdiseases in over200dicotyledonous plant species, including cotton, eggplant, cucumber, etc. Verticiliumwilt disease causes enormous economic losses in cotton production. The virulence of V. dahliaeresponds differently depending on the effect of pathogen and host co-evolution, heterocaryosis or theenvironment, leading to novel pathogenic type generation. Recently, most of the V. dahliaepathogenicity studies focused on the secreted proteins that act as a virulence factor and considered asthe main reason for Verticillium wilt.Previous comparative genomic analysis between two V. dahliae isolates, the high virulent VDG1and low virulent VDG2disclosed a VDG1-specific fragment, named SCF73, which spans27.1kb,encoding five genes （VDG1₀1014-VDG1₀1018）. Knocking-out of this fragment in VDG1wild-typestain caused significant decrease in virulence, revealing that SCF73played an important role in thevirulence of V. dahliae.In this study, the basic information of the five genes in SCF73was further analyzed, VDG1₀1014encodes an adoxidoreductase, VDG1₀1016is similar to glycosyl hydrolase family18, VDG1₀1018issimilar to glycosidase，VDG1₀1015and VDG1₀1017encode two hypothetical proteins. Genesecretion analysis indicated that VDG1₀1016is a potential secreted protein, so named VdSSP1（V.dahliae specific secreted protein1）. The full length cDNA of VdSSP1is1708bp, containing85bp5’UTR,102bp3’UTR, and1521bp open reading frame, which encodes506deduced amino acidresidues. Two introns with110bp and116bp were identified. The prediction of encoded proteinshowed that VdSSP1belongs to typical secreted proteins. The transcripts of VdSSP1were not detectedin normal culture condition when using sucrose as a carbon source. However, the gene was induced3days when grew on starch as a carbon source medium or5days on pectin ones, and the expression alsoup-regulated in the V. dahliae infected cotton stems5days after inoculation. These results suggestedthat the function of VdSSP1responds to the plant cell wall components utilization and closely related tothe V. dahliae infection progress.The yeast signal trap system based on the vector pSUC2carried a truncated invertase gene wasused to identify the secretion activity of VdSSP1. Yeast contains pSUC2::VdSSP1grew well inCMD-W medium （with sucrose） and in the absence of tryptophane （Trp）, or in YPRAA media （withraffinose instead of sucrose）. In addition,2,3,5-triphenyltetrazolium chloride （TTC） can be reduced tored1,3,5-triphenylformazan. These results showed that the signal peptide of VdSSP1can guideinvertase to extracellular successfully, strongly suggested that VdSPP1is a secreted protein in V.dahliae.Knock-out vector of pGKO2::VdSSP1was constructed and the deletion mutant of VdSSP1（ΔVdSSP1） was obtained by Agrobacterium tumefaciens-mediated transformation method.Pathogenicity assay showed that ΔVdSSP1significantly reduced the V. dahliae virulence to cotton,disease index values is35.92±0.81compared to the wild type of49.16±0.68. Moreover, the growth of the knockout mutant was restricted on the different carbon source media, especially the pectin andstarch medium. The growth diameter was only28.39%and57.67%of the control, respectively.Functional complementation of ΔVdSSP1indicated that the virulence of complementary transformantswas enhanced on cotton, and the disease index values is44.91±0.74which is similar to the wild type.The growth ability was partially recovered in the pectin and starch medium. Similar results were alsofound on the complementation assay to the low virulent strain VDG2which is lack VdSSP1. Therefore,it is strongly suggested that the virulence function of VdSSP1is associated with pectin and starchdegradation.In conclusion, VdSSP1is a specific secreted protein gene in highly virulent strain VDG1comparedwith the low virulent strain VDG2, which contributes to the virulence of V. dahliae and participates inpectin and starch degradation. It is indicated that VdSSP1act as a secretion virulence factor in the V.dahliae towards host （cotton） invasion.