Function Of Transmembrane Protein VdSho1 In Regulating Penetration And Melanin Biosynthesis In Verticillium Dahliae

Verticillium dahliae is an important pathogen causing Verticillium wilt,which has a wide host range and can infect hundreds of dicotyledons including cotton,tomato,lettuce and other important cash crops.Sho1 is a membrane sensor protein with four transmembrane domains,which plays important regulatory roles in a variety of fungi.But the specific regulatory mechanism of Sho1 in Verticillium dahliae has not been reported.In this paper,functions of VdSho1 and its downstream MAPK signaling cascades in regulating the penetration of cellophane membrane and melanin synthesis by Verticillium dahliae were analyzed.Main results and conclusion are listed below:1.Cellophane membrane penetration assay revealed that VdSho1 regulate the penetration ability of Verticillium dahliae.The transcriptome analysis showed that VdSho1 positively regulate expression level of melanin synthesis related genes.Meanwhile,penetration ability of Vd8 was significantly inhibited by applying of melanin biosynthesis inhibitors,meaning that melanin could promote the mycelial infection of Verticillium dahliae into cellophane membrane.These results indicated that the cellophane membrane penetration ability depends on melanin production in Verticillium dahliae,and VdSho1 affected the penetration ability by regulating the melanin synthesis in Verticillium dahliae.2.Using yeast two hybrid,we found that cytoplasmic part of VdSho1 could interact with Vst50.RNA-seq results showed that genes regulated by Vst50 and VdSho1 were highly consistent,indicating that VdSho1 and Vst50 located in the same signal transduction pathway.Analysing genetic relationship between VdSho1 and Vst50,the results suggested that VdSho1 lies upstream of Vst50 in signal transduction during membrane penetration process in Verticillium dahliae.In addition,deletion of both MAPK signaling cascade pathway elements Vst7 and Vst11 dramatically reduced penetration ability and melanin levels.The results of yeast two-hybrid showed that Vst50 interacted with Vst7 and Vst11 respectively.Mutations in the domain of Vst50 interacting with Vst7 and Vst11,respectively,resulted in significant decreases in penetration ability and melanin synthesis of pathogens,suggesting that Vst50-Vst7-Vst11,a MAPK cascade pathway,played an important role in invasion of cellophane membranes.3.The cellophane membrane penetration assay showed that mutation of any transmembrane domain seriously affected the melanin level during cellophane membrane penetration in Verticillium dahliae.By applying high concentration glycerol,function of VdSho1 in face of cell osmotic pressure change was analyzed.The results showed that the penetration of cellophane membrane of Vd8 strain decreased with the increase of extracellular glycerol,suggesting that the penetration of mycelium required a certain osmotic pressure to be maintained within the hyphopodium.1 M glycerol treatment slightly increased level of melanin synthesis related genes in Vd8 strain,while those in ?Sho1 mutant were significantly declined.Using membrane permeability regulator nystatin,the results showed that VdSho1 participate in response to nystatin signal,regulating penetration level and melanin content.To analyse role of VdSho1 transmembrane domain respond to cell membrane permeability,cellophane membrane penetration assay show that VdSho1 transmembrane domain were essential for melanin level and penetration ability regulation caused by membrane permeability changes.4.Pathogenicity assay showed the mutation of VdSho1 dramatically reduced the virulence and colonization level of V.dahliae.Scanning Electron Microscope showed that ?Sho1 mutant entering host root was significantly lower than that of wild type.In addition,VdSho1 gene was induced to express at early stage of infection.Results above showed that VdSho1 is a pathogenic gene.In conclusion,VdSho1,as a membrane sensor,participates in the induction of intracellular and extracellular osmotic pressure changes,and regulates the pathogenicity of Verticillium dahliae by regulating the level of melanin in mycelium,improving the penetration ability of Verticillium dahliae.