Molecular Mechanism In Cotton Defense Response To Verticillium Dahliae And Functional Characterization Of Gb WRKY1

Cotton Verticillium wilt caused by Verticillium dahliae is a soil-borne vascular disease. The representative symptoms caused by V. dahliae in the susceptible cotton include leaf curl, necrosis and defoliation. The disease has become the most economically important disease of cotton and severe outbreak of this disease causes reduction in fibre quality and significant yield losses. Through research on the molecular mechanism during the cotton defense response to V. dahliae, and identification of defense related genes for the use of genetic engineering methods to cultivate resistant cotton varieties will be the main direction of future cotton breeding. In this study, a suppression subtractive hybridization (SSH) based cDNA library constructed from V. dahliae inoculated Gossypium barbadense variety’Hai7124’and deep-sequencing technology based RNA-Seq were developed to better understand the molecular mechanism in cotton defense response to V. dahliae. Through gene expression analysis, a number of defense related genes were identified. Detailed functional identification of a defense related WRKY-type transcription factor isolated from the SSH cDNA library was studied in Arabidopsis. The main results of this study were as follow:A suppression subtractive hybridization (SSH) based cDNA library was constructed from V. dahliae inoculated G. barbadense variety’Hai7124’. A total of211unique genes were differentially identified and classified into11functional categories. The largest groups contain genes involved in metabolism, stress/defence response, cell structure and signal transduction. More than one-third of the genes (38%) were identified as unknown classification or function. This study identified a set of disease-related genes involved in the process of the response, including pathogenesis-related genes of various classes, oxidative burst-related genes and secondary metabolism-related genes, which were better understand in the disease resistance. The characterization of some transcription factors and kinases enabled us to better understand the defence mechanisms. Northern blot analysis and quantitative real-time PCR (qPCR) were performed to investigate the expression patterns of some representative genes and characterize the role of some signal molecules (H2O2, ethylene, jasmonic acid and salicylic acid) in the cotton defence response. Our results suggested that a complicated and concerted mechanism involving multiple pathways including salicylic acid, jasmonic acid and ethylene was responsible for the cotton defence response to V. dahliae. The results suggested that ethylene play a putative role in the resistant response as a signal molecule in the cotton defense response.The incompatible pathosystem between resistant cotton (Gossypium barbadense cv.’Hai7124’) and V. dahliae strain V991was used to study the cotton transcriptome changes after pathogen inoculation by RNA-Seq. Of32(?)774genes detected by mapping the tags to assembly cotton contigs,3442defence-responsive genes were identified. Gene cluster analyses and functional assignments of differentially expressed genes indicated a significant transcriptional complexity. Quantitative real-time PCR (qPCR) was performed on selected genes with different expression levels and functional assignments to demonstrate the utility of RNA-Seq for gene expression profiles during the cotton defence response. Detailed elucidation of responses of leucine-rich repeat receptor-like kinases (LRR-RLKs), phytohormone signalling-related genes, and transcription factors described the interplay of signals that allowed the plant to fine-tune defence responses. On the basis of global gene regulation of phenylpropanoid metabolism-related genes, phenylpropanoid metabolism was deduced to be involved in the cotton defence response. A closer look at the expression of these genes, enzyme activity, and lignin levels revealed differences between’Hai7124’ and ’YZ-1’. Both types of plants showed an increased level of expression of lignin synthesis-related genes and increased phenylalanine-ammonia lyase (PAL) and (POD) enzyme activity after inoculation with V. dahliae, but the increase was greater and faster in the’Hai7124’. Histochemical analysis of lignin revealed that’Hai7124’not only retains its vascular structure, but also accumulates high levels of lignin. Furthermore, quantitative analysis demonstrated increased lignification and cross-linking of lignin in stem of’Hai7124’. Overall, a critical role for lignin was believed to contribute to the cotton disease resistance to V. dahliae.GbWRKY1is a cloned WRKY transcription factor from Gossypium barbadense cv.’Hai7124’, which was firstly identified as defense related gene and showed highest similarity with AtWRKY75in Arabidopsis thaliana which was assigned to class Ⅱc of WRKY family. Overexpression of GbWRKYl in Arabidopsis resulted of attenuated Pi starvation stress response, including reduced accumulation of anthocyanin and more biological yield. Moreover, overexpression plants exhibited relative high level of total P and Pi as well as enhanced accumulation of acid phosphatases ativity. Genes encoding highaffinity Pi transporters (PT1, PT2, and PHT1;8) and phosphatases (PS1, ACP5, and RNS1) were upregulated in overexpression plants compared to WT plants under Pi-sufficient condition. The result demonstrated that GbWRKY1may be act as a positive regulator involved in the global Pi starvation response. Together with the fact that AtWRKY75was only induced upon Pi starvation, the results suggested that the overexpression of GbWRKY1caused plants exhibited a Pi starvation response in the Pi-sufficient condition, including enhanced number of LR and accmulation more Pi, which may attenuate Pi starvation stress response of overexpression plants. To explore whether the alterations of root system architecture and enhanced Pi signaling response in the overexpression plants by modulating the auxin signaling response, the response to exogenous IAA treatment was investigated. Together with the result of internal IAA concentration and gene expression level of TIR1and ARF19, it seemed that the overexpression plants was more sensitivity to exogenous auxin under Pi-sufficient conditions compared with the WT. Changes in the auxin sensitivity of the root seemed to be involved in the developmental response of the Arabidopsis root system to Pi starvation. Overall, the involvement of GbWRKY1in the modification of the root system by changed auxin sensitivity to regulate Pi starvation responses. The expression of GbWRKY1was induced by V. dahliae inoculation in cotton. The putative role of GbWRKY1in the cotton defense response still need further study.