Isolation And Functional Analysis Of Acetolactate Synthetase Gene VdILV2 And VdILV6 From Verticillium Dahliae

Verticillium wilt of cotton caused by Verticillium dahliae(V.dahliae),named “cotton cancer”,is a soil-borne vascular fungal disease.According to the complex pathogenic molecular mechanism of V.dahliae and its long-term survival of dormancy microsclerotium,there is no practical method to control this disease.To study the function of essential genes in V.dahliae pathogenic process can promote to further reveal the pathogenic molecular mechanism of V.dahliaeand has great significance for the prevention of cotton verticillium wilt.Host-induced gene silencing(HIGS)is a practical and effective technology in crop disease prevention or gene function verification by silencing virulence genes of pathogens.In this study,the virulence functions of VdILV2 and VdILV6 were analyzed and identified by using HIGS technology through constructing a series of tobacco rattle virus(TRV)interfering vectors targeting V.dahliaeacetolactate synthase(VdALS)gene.VdILV2 gene knockout vector was also constructed and gene knockout mutant was screened to further analyze the function of this gene in the growth and development process.The main results of this research were listed as follows:1.The comparison of VdALS amino acid sequence with that of in Magnaportheoryzae.Weidentified VdALSCatalytic subunits and regulatory subunits,named VdILV2 and VdILV6,respectively.The results of bioinformatics analysis showed that ALS in V.dahliae may be as correlated with its pathogenicity as ALS in Magnaportheoryzae.VdILV2 and VdILV6 showed 81.07% and 78.64%amino acid identitywith MoILV2 and MoILV6 in Magnaportheoryzae,respectively.The protein structure prediction finds that VdILV2 has three conservative function domains TPP_enzyme_N,TPP_enzyme_M,and TPP_enzyme_C.VdILV6 has two conservative function domains,ACT(aspartate kinase,chorismate mutase and TyrA)at the N-terminal and ALS at the C-terminal.2.HIGS system was established and the resistance identification results showed that HIGS targeting VdILV2 and VdILV6 could significantly improve upland cotton resistance to verticillium wilt.After inoculation with Vd991 spore solution for 16 days,the vascular bundles of TRV:00 and CK plants showed marked accumulation of V.dahliae melanin and microsclerotia.However,TRV: VdILV2-1-,TRV: VdILV2-2-,TRV: VdILV6-1-and TRV: VdILV6-2-treated plants exhibited a smaller accumulation of melanin and microsclerotia,and the Verticillium wilt symptom was also relatively light.The HIGS-treated plants CK and TRV:00 seedlings showed markedly wilted leaves,and the percentage of sick leaves reached approximately 85% at 20 days after inoculation(dpi),but only approximately 45% in TRV2: VdILV2-2-and TRV2: VdILV6-1-treated seedlings.Approximately 56% and 72% in TRV2: VdILV6-2-and TRV2: VdILV2-1 silenced plants separately displayed wilting or defoliation at 20 dpi.3.Results of reverse transcription PCR(RT-PCR)and quantitative real-time PCR(qRT-PCR)analysis showed that The expression levels of VdILV2 and VdILV6 were decreased in TRV: VdILV2-1-,TRV: VdILV2-2-,TRV: VdILV6-1-and TRV: VdILV6-2-treated cotton plants.The results indicated that the improvement of resistance was related to HIGS' targeted inhibition of the expression of VdILV2 and VdILV6,which were related to the pathogenicity of V.dahliae.4.The knockout vectorpGKO-VdILV2 of the disease-related gene VdILV2 was successfully constructed,and two VdILV2 knockout mutants were preliminarily abtained by PCR analysis.