Analysis Of Upland Cotton In Response To Verticillium Dahliae Infection By ITRAQ And Functional Identification Of Resistance Gene

Verticillium wilt,one of the main diseases in the growth and development of cotton,seriously affects cotton yield and fiber quality.In this study,iTRAQ(isobaric tags for relative and absolute quantitation)technology was used to detect the proteomic changes of upland cotton cultivar ND601 under the stress of Verticillium wilt,which will lay a foundation for the molecular mechanism underlying upland cotton resistance to Verticillium wilt.In addition,a CRVW(cotton resistance to Verticillium wilt)gene from upland cotton was cloned,and its function of resistance to Verticillium wilt was verified by VIGS(virus-induced gene silencing)technology,and its mechanism which participating in resistance to Verticillium dahliae through SA(salicylic acid)signaling pathway was preliminarily proved.The main results were as follows:1?A total of 5654 proteins were identified in upland cotton leaf tissue using iTRAQ,among which 298 proteins were differentially expressed,including 140 up-regulated proteins and 158 down-regulated proteins.2?qRT-PCR was used to detect the transcription levels of six differentially expressed proteins(DEPs),including APX(ascorbate peroxidase)?RNP1(RNA-binding family protein1)?IMPL1(inositol monophosphatase like 1)?BAHCC1(BAH and coiled-coil domain-containing protein 1)?RABB1C(RAB GTPase homolog B1C)and EXOS3(exosome complex component 3).The transcription level of EXOS3 was different from its protein expression,which might be caused by post-translation regulation and modification.3?All DEPs were divided into 12 functional categories,including signal transduction,transcription,lipid metabolism,cell wall and phenylalanine metabolism,protein metabolism,energy and carbohydrate metabolism,nucleic acid metabolism,ROS(reactive oxygen species)related proteins,hormone regulation,photosynthesis,cell transport,and other functions.And the possible mechanisms of part pathways involved in the interaction between cotton and Verticillium wilt were discussed.4?CRVW was cloned,which mainly existed in plant cell membrane and cytoplasm.CRVW was expressed in the root,stem and leaf of cotton,indicating that CRVW had tissue expression specificity.5?After SA spraying,CRVW expression was significantly upregulated.After inoculated with V.dahliae,CRVW was significantly up-regulated in both resistant variety ND601 and susceptible variety CCRI8,indicating that CRVW was induced by SA and V.dahliae.6?After treatment with V.dahliae for 20 days,the disease index of CRVW silencing group was significantly higher than that of CK group,indicating that CRVW silencing significantly reduced the resistance of cotton seedlings to Verticillium wilt.7?After silencing CRVW,SA content in cotton seedlings decreased significantly.ICS1(isochorismate synthase 1)?EDS1(enhanced disease susceptibility 1)?PAD4(phytoalexin deficient 4)? NPR1(nonexpresser of PR gene 1)and PR1(pathogenesis-related protein 1)related to SA accumulation and signal regulation were significantly down-regulated,indicating that CRVW might participated in the process of cotton resistance to Verticillium wilt through SA signal pathway.In summary,298 differentially expressed proteins were identified in upland cotton leaves,mainly involved in protein metabolism,energy and carbohydrate metabolism,nucleic acid metabolism and other metabolic processes.CRVW located in cytoplasm and cell membrane and could be induced by V.dahliae and SA.After inoculation with V.dahliae,the expression level of SA signal pathway marker genes and SA content in CRVW silencing plants significantly decreased,indicating that CRVW might participate in cotton resistance to Verticillium wilt through the SA signaling pathway.