Identification And Function Analysis Of GhWRKY40in Cotton(Gossypium Hirsutum L.)

During the whole life of plants, they are constantly affected by their environmental conditions in the form of different abiotic and biotic stresses, such as drought, water, low temperature, hot, pathogens and so on. Stress is perceived and transduced through a series of signaling molecules that ultimately affect the regulation of stress-inducible genes to initiate the synthesis of different types of proteins, including transcription factors, enzymes, molecular chaperones, ion channels, and transporters, or to alter their activities. Among these proteins, transcription factors (TFs) play key roles in eliciting stress responses by modulating the expression of specific target genes in a temporal and spatial manner; they are also necessary for normal development and proper responses to physiological or environmental stimuli. WRKY proteins are a class of zinc finger-containing TFs that are encoded by large gene families in all higher plants. There are reports that WRKY TFs play a pivotal role in many physiological processes. Although lots of researches have revealed the role of WRKY TFs in these aspects, the mechanisms and crosstalk underlying the ways that WRKY members act their role still need to be further investigated; and their functions perhaps differ from each other under different genetic backgrounds. Moreover, most studies are focused on model plants, studies of WRKYs in crops are limited. Cotton is one of the most important commercial crops worldwide and is an excellent source of fiber and oil. The study of cotton WRKYs will provide new insight that may aid in creating cotton plants that are better able to adapt to environmental challenges. In the present study, we selected the economically important species cotton (Gossypium hirsutum L.) as the experiment material, a multiple stress-responsive cotton WRKY genes, namely GhWRKY40, were identified and characterized.The main results are as follows:(1) According to multiple alignment and phylogenetic analyses of the WRKY proteins, GhWRKY40was placed into group Ⅱa of the WRKY superfamily. Subcellular localization indicated that the gene was localized in the nucleus. Thus, it may function in the nucleus. Transcriptional activation analysis in yeast showed that the GhWRKY40protein may serve as a transcriptional activator.(2) The expression patterns of GhWRKY40measured by qPCR indicated that it can be induced by various abiotic and biotic stimuli, such as wounding, bacterial pathogen Ralstonia solanacearum, and diverse signalling molecules, such as salicylic acid (SA), jasmonate (JA), ethylene (ET). These results indicated that GhWRKY40may be involved in diverse signalling pathways. To further confirm the GhWRKY40gene is induced by stress, the upstream region of the GhWRKY40gene was isolated by I-PCR. Analysis of this region using the PlantCARE and PLACE databases revealed many putative cis-elements, suggesting that GhWRKY40plays a role in the plant response to environmental stress.(3) Ectopic expression of GhWRKY40in Nicotiana benthamiana enhanced the wounding tolerance and increased the susceptibility to R. solanacearum. Moreover, the overexpression of GhWRKY40represses the expression of the JA-responsive genes JAZ1and JAZ3, and the SA-dependent genes PR1a, PR2and PR4upon wounding and R. solanacearum treatments.(4) GhWRKY40can interact with upstream MAPK GhMPK20and interacts with GhMPK20in the nucleus as evidenced by yeast two-hybrid system and bimolecular fluorescence complementation (BiFC) system, providing valuable information that aids our understanding of the relationship between MAPKs and the WRKY family proteins in cotton.