| Cotton wilt caused by Verticillium dahliae is a soil-born disease,which decreases cotton yield and fiber quality seriously,and has become the main obstacle in cotton production.Further mining pathogencity genes related to cotton Verticillium wilt can provide valuable reference for explaining the pathogencity mechanism of V.dahliae and facilitate the disease control.In this study,we explored the genome transcription of highly toxic V.dahliae strain Vd991 at the early stage of infection in upland cotton Coker312 by RNA-seq.Differential expression analysis,gene ontology annotation(GO)and classification,GO enrichment analysis,kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were performed to screen candidate pathogencity genes.Based on the transcriptome anaysis,2 genes were choosen for functional analysis.The main results and conclusions were as follows:1.Screening of candidate pathogencity genes related to cotton Verticillium wilt.1)Differential expression analysis and GO annotation.A total of 979 differentially expressed genes(DEGs)were identified before and after infection,of which 376 were up-regulated and 603 were down-regulated.The result of GO annotation and classification showed that these DEGs were involved in broad biological processes,molecular function,including reproductive process,growth,sporulation,carbohydrate binding,kinase regulator activity,lyase activity and hydrolase activity,et al.There were 33 potential virulence factors in the up regulated DEGs,including 22 secreted protein genes,8 small cysteine-rich protein genes and 6 carbohydrate-active enzyme genes.The role of these potential virulence factors in the pathogenic process of V.dahliae needs further study.2)GO and KEGG enrichment analysis.Enrichment analysis of the up-regulated DEGs were performed,277 GO terms were enriched,including positive regulation of growth rate,cyclin-dependent protein kinases(CDKs)activator activity,purine nucleotide biosynthetic process,pyrimidine nucleobase biosynthetic process,helicase activity,IMP biosynthetic process,protein kinase C binding.A total of 53 KEGG pathways were identified in the up-regulated DEGs,including purine metabolism,pyrimidine metabolism.Genes related to these functions and pathways expressed actively at the early stage of infection.2.Functional analysis of candidate genes related to cotton Verticillium wilt.Based on the transcriptome analysis,pectate lyase gene Vdpl1 and cyclin-dependent kinases(CDKs)regulatory subunit gene Vdcdpk1 were choosen for functional analysis.Deletion mutant Vd⊿pl1 and Vd⊿cdpk1 were obtained successfully by Agrobacterium tumefaciens mediated gene transformation.Pathogenicity assay was performed and the results showed that there were no obvious difference between Vd991 and Vd⊿pl1,the disease index were 90.22% and 91.65%,respectively.While Vd⊿cdpk1 obviously reduced the virulence to cotton,with the disease index is 18.95%.Growth phenotype assay showed that the growth of Vd⊿pl1 on pectin medium was significantly restricted.Moreover,the growth of Vd⊿cdpk1 on sucrose,starch,pectin medium,was significantly restricted.In conclusion,these results indicated that genes related to cell proliferation actively expressed at the early stage of infection.CDKs regulatory subunit gene Vdcdpk1 plays a important role both in the pathogenicity and growth of V.dahliae. |
