Construction Of High Efficient Gene Knockout System And Functional Analysis Of Secreted Protein In Verticillium Dahliae

Verticillium dahliae is a soil-borne plant pathogen responsible forVerticillium wilt diseases in temperate and subtropical regions, collectively they affect over 200 hosts, including many economically important crops such as cotton. It is reported that V. dahliae produce secreted cell wall degrading enzymes and other extracellular phytotoxins molecules that induce host cell death. So, developing an efficient method of gene knockout system in Verticillium dahliae and perform functional analysis for potential secreted toxin encoding genes by knockout experiment would help clarify the infection mechanism of this fungal pathogen.By using fusion PCR, we constructed gene knockout vectors. By using Agrobacterium tumefaciens-mediated transformation and applying a herpes simplex virus thymidine kinase (HSVtk) gene in T-DNA as a conditional lethal gene to counter-select against ectopic transformants, we developed an efficient method to select gene knockout transformants. Gene knockout frequency for ADE4 and ChsV was 87% and 44%, respectively.The softwares SignalP, TargetP, TMHMM, Big-pi and PROSITE were combined to predict the sectetome of Verticillium dahliae VdLs.17. Finally, we identified 922 possible secreted proteins in Verticillium dahliae VdLs.17 secretome. This study carried out a detailed comparative analysis of genes encoding secreted proteins between Vd114 and Vd991 which show different virulence towards cotton to discover potential Lineage-Specific secreted protein coding genes in Vd991. Finally, we identified 6 possible Vd991 specific secreted proteins. We constructed mutants for each specific proteins in Vd991. But mutants showed similar phenotype with Vd991. One reason is that gene redundancy can mask the phenotype of singal loss-of-function mutant. So, we further analysis the secretion system protein in Vd991.A molecular chaperone of the lumenal hsp seventy gene (VdLHS), which plays a role in the first step of extracellular protein secretion by importing them into the endoplasmic reticulum (ER) and subsequent protein folding, was disrupted via Agrobacterium tumefaciens-mediated transformation in Vd991. The total amount of secreted extracellular phytotoxins is significantly reduced in theâ–³Lhs culture filtrate when culturing the fungus in Czapek's medium to imitate the phytotoxin secreting ability in planta. The mutants were severely impaired in virulence on cotton Junmian 1. Results of this study suggest that VdLHS plays important roles in protein secretion and may provide potential novel targets for controlling the devastating disease.