Effect Of Root Exudates From Cotton Cultivars With Different Resistance On The Gene Expression In Verticillium Dahliae

Objective:China is the largest cotton consumption and production country in the world.Cotton industry is the main economic income source of most farmers in Xinjiang,one of the economic pillar industries,and the foundation of stability and long-term stability in Xinjiang.Verticillium wilt is the main disease of cotton.Cotton Verticillium Wilt in Xinjiang is becoming more and more serious,which seriously affects the yield and quality of cotton and restricts the production of cotton.V.dahliae is a soil-borne fungus that causes Verticillium wilt disease and is especially difficult to control because of its long-living resting microsclerotia and constant variation of its physiological form in the process of coevolution between V.dahliae and its host.In recent years,with the completion of V.dahliae sequencing and the development of bioinformatics tools,researches have made great progress in the research of pathogenicity related genes of V.dahliae.Due to the complexity of the pathogenic molecular mechanism of V.dahliae,however,our knowledge of the mechanism of pathogenesis of V.dahliae has remained at a preliminary exploratory stage.In this study,we analyzed and compared the transcriptome of V.dahliae induced by root exudates from cotton cultivars with different resistance to V.dahliae,identified the genes that may play an important role in the growth and development,early infection and pathogenicity of V.dahliae and studied their functions.These expression data have advanced our understanding of key molecular events in the V.dahliae interacted with cotton and molecular basis of V.dahliae pathogenicity,and provided a framework for further functional studies of candidate genes to develop better control strategies for the cotton wilt disease.Methods:?1?In this study,RNA-seq analysis was carried out on V.dahliae samples cultured by different root exudates from three cotton cultivars?a susceptible upland cotton cultivar Xinluzao 8,a tolerant upland cotton cultivar Zhongzhimian 2 and a resistant island cotton cultivar Hai 7124?and water for 0 h,6 h,12 h,24 h and 48 h.Each time point had two biological replicates.In total,34 libraries were acquired and used in RNA-seq.Based on the results of sequencing,cluster tree analysis was carried out on the samples,differentially expressed genes?DEGs?were screened and Gene ontology analysis for the DEGs was carried out.?2?According to the amino acid sequence of VdNAT1 gene,the functional domain prediction and evolutionary relationship analysis were carried out.We constructed the knockout vector and expression vector of VdANT1 gene,which were introduced into V.dahliae by PEG-CaCl₂mediated transformation,generated the deletion mutant strains and complemented mutant strain by using,and investigated the colony morphlology,growth rate,hyphae morphlology,conidial yield and germination rate;the pathogenicity test of the wild-type Vd991,VdNAT1 deletion mutant strains and complemented mutant strain was analyzed by injection method in cotton.?3?The HIGS vector of VdNAT1 gene was constructed and transformed cotton by Agrobacterium mediated mathod.The disease index,fungal biomass and target gene expression of cotton plants treated with HIGS were analyzed.Results and conclusions:?1?The results and conclusions of transcriptomics:?1?Using V.dahliae sample cultured for 0 h as a control,statistical analysis of differentially expressed genes revealed that the number of DEGs of samples cultured by root exudates were larger than that of sample cultured by water,and the largest number of DEGs was found in samples cultured by root exudates from susceptible cutivar,suggesting that V.dahliae responded to all kind treatments,but responded more strongly to root exudates from the susceptible cultivar than to those from the tolerant and resistant cultivars.?2?The hierarchical clustering found that 17 samples were classified into two groups?I and II?.The gene expression patterns of all samples within each group were similar,and the gene expression patters between two groups were different;The GO analysis also found that the GO items of upregulated DEGs in group I samples were significantly different from those in group II,suggesting that the samples in group I were at different growth stages compared with samples in group II.Group I contained all the samples cultured by different root exudates and water for 6 h and 12 h,and group II contained all the samples cultured for 48 h.It is notable that the samples cultured by root exudates from two upland cotton cultivars for 24 h were classified into group I,while the samples cultured by island cultivar for 24 h were classified into group II,suggesting that the growth and development of samples cultured by root exudates from two upland cotton cultivars were faster than that of island cultivar.?3?GO analysis revealed the enriched GO terms of up-regulated genes in samples?6 h and 12 h?cultured by three different root exudates were similar.However,the up-regulated genes were quite different in these samples,and only 57 up-regulated genes were found to be common,suggesting that that the molecular mechanism of the response of V.dahliae to different root exudates from three cotton cultivars was different.?4?The GO analysis for up-regulated DEGs in V.dahliae samples cutured by 6 h and 12 h of group I found that hydrolase activity,hydrolyzing O-glycosyl compounds and transmembrane transport were the significantly enriched GO terms,in which some genes have been reported to be closely related to the pathogenicity of fungi,suggusting that 6 h and 12 h were the critical stage of V.dahliae-cotton interaction.?5?31 genes with known functions were found to be up-regulated in V.dahliae samples?6 h or 12 h?cultured by different root exudates,suggesting that these genes may be required for the V.dahliae-cotton interaction.GO analysis for 31 genes found that transmembrane transport was the most significantly enriched GO term.68 genes with known functions were found to be up-regulated only in V.dahliae samples?6 h or 12 h?cultured by root exudates from susceptible cultivar,suggesting that these genes may be related to pathogenesis of V.dahliae.GO analysis for 68 genes found that hydrolase activity,hydrolyzing O-glycosyl compounds and transmembrane transport were the significantly enriched GO terms.26 genes with known functions were found to be up-regulated in V.dahliae samples?6 h or 12 h?cultured by different root exudates and water,suggesting that these genes may be required for the growth and development of V.dahliae.These expression data have advanced our understanding of key molecular events in the V.dahliae interacted with cotton and molecular basis of V.dahliae pathogenicity,and provided a framework for further functional studies of candidate genes to develop better control strategies for the cotton wilt disease.?2?The results and conclusions of functional study of VdNAT1:?1?The deduced protein VdNAT1 belonging to a member of MFS superfamily contains a MFS₁domain.Prediction of transmembrane helices of VdNAT1 indicated that it contains 10 putative transmembrane domains.Phylogenetic tree analysis demonstrated that VdNAT1 has low amino acid identity with the other NAT proteins and MFS proteins in V.dahliae,showing a high amino acid diversity among NAT proteins in V.dahliae.?2?The VdNAT1 deletion mutants showed a lower colony expansion and produced less white hyphae compared to wild type Vd991.In addition,the conidial yield and germination rate of the deletion mutants were lower than that of wild-type V991.Complementation of the mutant strain with VdNAT1 almost completely rescued the attributes described above to wild-type levels.When grown on PDA medium in the presence of nicotinic acid with different concentration?0.025g/L,0.05g/L and 0.1g/L?,the colony of VdNAT1 deletion mutants showed increased growth compared with the growth of it on PDA medium in the absence of nicotinic acid.The deletion mutants produce more white hyphae and dark-colored microsclerotia on PDA medium containing nicotinic acid,indicating that it was able to grow as well as the wild type strain in the presence of exogenous nicotinic acid.The white hyphae was loose on the medium with lower concentration of nicotinic acid,but the texture was dense when the concentration was higher.?3?Compared with the plants inoculated with wild type strain and complemented mutant strain,the cotton plants inoculated with deletion mutant strains showed better growth state,less disease symptoms,lower disease index and higher plant height.Less fungal DNA was amplified from roots of the infected plants inoculated with the deletion mutant strains and fewer fungal colonies were isolated from excised stem tissue sections of cotton seedlings inoculated with deletion mutant strains than with the wild type strain and the complemented mutant strains.?4?Tobacco rattle virus-mediated HIGS in cotton plants silenced VdNAT1 transcripts in invaded V.dahliae strains found that pTRV2-VdNAT1 plants showed mild disease symptoms and formed a smaller biomass in roots of the host compared with pRTV2-00 plants.These results suggest that VdNAT1 was involved in regulating the colony expansion,hyphal growth and spore production and pathogenicity of V.dahliae.