Functional Analysis Of VdNOP12 And VdLHS1 Required For Virulence In Verticillium Dahliae

Verticillium dahliae Kleb.belongs to the fungal genus Vericillium of class Deuteromycetes,which has a wide range of hosts and can cause verticillium wilt of more than 200 dicotyledonous plants.Cotton verticillium wilt caused by it is called "cancer" of cotton.This disease is one of the main restricting factors of cotton production in China.There are currently no effective means to control it although the infection cycle of this disease is clear.The reason is that microsclerotia can survive for many years in the soil as well as lack of disease-resistant cultivars and effective and environmental-friendly fungicides.Therefore,to study the pathogenesis of V.dahliae is the basic work of designing effective control strategies for this disease.Using the T-DNA insertional mutant library of V.dahliae defoliating strain Vd991 as the material,9 pathogenicity-defective mutants were obtained from screening virulence of 294 T-DNA mutants on cotton.Southern blot assay showed that only one mutant had twoT-DNA insertions and the remaining eight mutants harbored a single insert of T-DNA.The single copy insertion rate was 88.9%.Analysis of biological characteristics found that growth rate and conidia production of these mutants comparing with wild type Vd991 were impaired by the T-DNA insertion at varying degrees.DNA sequences flanking T-DNA insertion were isolated by hiTAIL-PCR(high-efficiency thermal asymmetric interlaced PCR),and these sequences were aligned to genome sequences of the VdLs.17 strain to identify the genomic position of insertion.Furthermore,these pathogenicity related genes were cloned from the wildtype strain Vd991.These results will lay the foundation for further studying mechanisms of these genes involved in pathogenicity.Based on the above results,the mutant of M11H01 was selected for further study.Flanking sequence analysis showed that the T-DNA of the mutant destroyed the expression of a hypothetical gene.Sequence analysis found that the protein sequences encoded by this gene had some similarity to the low temperature-sensitive NOP12 in yeast.Therefore,this hypothetical gene was named VdNOP12.Temperature sensitivity assay showed that the growth rate of T-DNA mutant and ?VdNOP12 was significantly lower than the wildtype strain,indicating that VdNOP12 was involved in the low temperature stress response of V.dahliae.Conidia production assay showed that destroying VdNOP12 significantly reduced the sporulation of V.dahliae even though at 25 ?.Subcellular localization analysis showed that VdNOP12 was located in the nucleus.The disease index of cotton inoculated with ?VdNOP12 mutant was significantly lower than that of wildtype strain at normal growth temperature.The results indicated that the gene was not only related to the low temperature sensitivity of V.dahliae but also to the sporulation and pathogenicity of V.dahliae.Consequently,it is speculated that this gene was not only related to the low temperature sensitivity but also to the pathogenicity and conidia production in V.dahliae.Cell wall-degrading enzymes(CWDE)secreted by V.dahliae have been considered to play important roles in pathogenesis.However,little is known about the secretion mechanisms of CWDE used by this pathogen.In this study,we investigated the role of the V.dahliae LHS1(VdLHS1),the homolog of which in Magnaporthe oryzae is an endoplasmic reticulum(ER)chaperone protein that was previously implicated in pathogenicity and expression of extracellular enzymes.We showed that VdLHS1 colocalized with ER-tracker dye Blue-White DPX in ER.The ?VdLHS1 mutant showed defects in radial growth and asexual conidiospores.The disease index of cotton inoculated with ?VdLHS1 mutant was significantly lower than that of wildtype strain throughout the assay up to 30 days post-inoculation.On complete medium(CM)plates supplemented with hydrogen peroxide,sodium chloride,sorbitol and SDS,the growth rates of the ?VdLHS1 mutant and the wildtype were not significantly different from those on CM.Furthermore,the activities of several extracellular enzymes in culture filtrates from the ?VdLHS1 mutant and the wildtype were compared using different test plate assay and enzyme linked immunosorbent assay.The activities of celluase,ligninase,laccase,pectinase and xylanase were significantly reduced in the ?VdLHS1 mutant,and those of amylase and beta-glucosidase did not change markedly in the mutant.However,activities of protease increased remarkably in the ?VdLHS1 mutant.Together,our results suggest that the VdLHS1 is requisite for pathogenicity,conidiation and secretion of extracellular enzymes.