Functional Analysis Of Specific Secreted Proteins In The High Virulent Verticillium Dahliae Strain

Verticillium dahliae(VD) is a causal agent of Verticillium wilt disease in over 600 plant species,184 of which are economically important crops. Verticillium wilt causes billions of dollars in annuallosses worldwide. Verticillium wilt on cotton was called “cotton’s cancer”. It is the first major diseaseon cotton production in China and seriously impaired cotton yields. Previous studies showed thatextracellular secreted proteins play an important role in inducing typical symptoms in cotton plants.Therefore, to extensively study of extracellular secreted proteins would provide a theoretical basis forcontrol of this disease.The whole genome of the high and low virulent VD strain, VDG1 and VDG2 were previouslysequenced by our lab. Through comparative genomics analysis, we obtained 11 specific fragments inVDG1 in comparison with VDG2. Secreted proteins on these big fragments were predicted by signal P3.0, TMHMM 2.0, Wo LF PSORT, Phobius and Y-Loc webserver were predicted. 6 specific secretedproteins were identified and named as High Virulent Specific Secreted Protein1(HSSP1), HSSP2,HSSP3 HSSP4, HSSP5 and HSSP6 respectively.According to the genome information, the primer pair were designed to amplify the 6 genes and toverify the speicificity in the VDG1. The signal peptide sequences of the 6 gene were respectivelypredicted by Signal P 3.0 and cloned into the yeast signal trap vector to validate the secreted property.The results showed that these signal peptide sequences are active, i.e., the 6 proteins are all secretedproteins. Based on these results, the 6 genes expression pattern were analyzed at different time afterinoculation with VDG1 at Czapek’s agar plates with a diverse carbon sources including xylose,starch,cellulose,pectin,xylan,and galactose. The results indicated that these genes were induced to expressionat the early infection stage.In order to evaluate the pathogenicities of the 6 HSSP genes, we firstly developed PEG-mediatedprotoplast transformation method. Using this method, we successfully obtained 5 HSSP knockoutmutants excluded the HSSP4. These mutants were reduced the mycelia and spores yeilds in comparisonwith VDG1. Although the colony morphology of the mutants were not changed, the different carbonbioavailability of the mutants were changed. Furthermore, the fungal pathogenicity assay on susceptiblecotton variety Junmian 1 showed that disease index of the mutants(38.39±2.30) was considerablydecreased compared to wild type(50.27±1.34). In conclusion, 6 specific genes encoded 6 activelysecreted proteins and the HSSPs as specific secreted proteins played important roles in the VDG1 pathogenicity.