Identification Of Key Genes Related To Verticillium Wilt Resistance By Transcriptome-based And Virus-induced Gene Silencing (VIGS) Analyses In Cotton

Cotton(Gossypium spp.)is a globally cultivated economic crop for the importance of its fiber and oil.However,Verticillium wilt,which caused by Verticillium dahliae,has severe detrimental effects to cotton fiber yield and quality in the majority of cotton growing countries.Selection of wheat cultivars resistant to Verticillium wilt is the most economical an effective method for disease control.Study on change of cotton transcripime gene expression after V.dahliae infection,not only provide the mechanism of cotton-Vdahliae interaction,but also provide important candidate resistance genes for cotton breeding.Research on gene cloning related to Verticillium wilt resistance and molecular mechanism of disease resistance is quite limited.To better understand the Verticillium wilt resistance mechanism in cotton,RNA-Seq was developed to analyze difference in gene expression between treatment(inoculation by V991)and control(normal growth).The main results are as follows:In order to study cotton defense mechanisms and reveal multi-pathway networks against pathogen attacks,in this study,we obtained nine RNA samples transcriptome using high-throught RNA sequencing,including four Verticillium dahliae-inoculated and four mock-inoculated G.barbadense cv.H7124 root samples(24h,48h,96h,144h),with Oh sample as control.Each library generated raw tags ranging from 6.90 to 7.50 million.After removing the low quality reads,total tag numbers per lirbrary ranged from 6.60 to 7.30 million and the number of distinct tags up to 19,000.To reveal the molecular events underlying the transcriptome profiles,the sequenced tags of the 9 libraries were mapped to the genome of G.raimondii.More than 57%clean tags can be mapped to 27,000 genes,accounting 73%of all genes in G.raimondii.Further,we performed a comparative transcriptome analysis on mock and V.dahlia-infected roots at various time points.By different pairwise comparisons with statistical test,a total of 14,846 differentially expressed genes(DEGs)involved in the cotton defense response to Verticillium dahlia were detected.Quantitative real-time PCR(qRT-PCR)results revealed that the expression trends of these genes showed significant similarity with the RNA-seq data(R2=82.96%),supporting the validity of RNA-seq data in this study.GO function enrichment analysis revealed that DEGs were enriched mainly in cell signaling,phosphorylation,plant hormones,regulation of physiological process,response to stimulus etc.A great many GO terms,such as cell signaling,phosphorylation and regulation of physiological process,in water-inoculated cotton focused on 48h and 96h,while those in pathogen-inoculated cotton occurred 96h and 144h.,indicating that normal physiological activities of the cotton showed hysteresis after pathogen invasion.Functional and pathway analysis of differentially expressed genes indicated a significant transcriptional complexity.Totally,jasmonic acid(JA)and brassinosteroids(BRs)signaling pathway,receptor-like kinases(RLKs)signaling pathway,reactive oxygen species(ROS),programmed cell death(PCD),and secondary metabolites such as related to lignin biosynthesis,were all involved in mediating cotton defense response.Further,RLKs were identified to modulate the important signals in response to V dahliae.Using virus-induced gene silencing(VIGS),we further verified that three RLKs confer resistance to Verticillium dahliae in cotton.Target gene-silenced plants exhibited typical symptoms of marginal chlorosis or yellowing in the leaves.Data for the rate of diseased seedlings leaves was analyzed to confirm the VIGS results.At last,the control H7124 plants seldom exhibited leaf wilting,with average diseased leaf:healthy leaf ratios of approximately 28%.However,72%of the RLK3-silenced plants were severely infected by V.dahliae,which was similar to the results observed in susceptible control plants(G.hirsutum cv.Junmian-1).Furthermore,65%of the RLK1-silenced plants showed a severe wilting phenotype,while 40%of the RLK2-silenced plants exhibited wilting symptoms on leaves when infected with Vdahliae.These results demonstrate that silencing of the candidate RLK genes compromises the resistance of cotton to this pathogen,and gene-silenced plants exhibited more wilting and etiolated leaves than the vector control plants.In summary,candidate RLK1 and RLK3 genes are important components of resistance to V.dahliae infection in cotton.Combined with transcriptome-based and VIGS analyses,our studies elucidated some key defense-related pathways and genes,providing a useful insight into cotton responses to pathogen attack.Overall,the gene expression profile data will not only help elucidate the mechanism of cotton resistance to V.dahlia,and also provide a resource for cotton improvement to disease resistance.