Genome-Wide Analysis of WRKY Gene Family and Negative Regulation of GhWRKY25 and GhWRKY33 Reveal Their Role in Whitefly and Drought Stress Tolerance in Cotton

Abstract

The WRKY transcription factor family is marked by its significant responsiveness to both biotic and abiotic plant stresses. In the present study, the WRKY family of <i>Gossypium hirsutum</i> has been identified and classified into three groups based on the number of conserved WRKY domains and the type of zinc finger motif. This classification is further validated by conserved domain and phylogenetic analysis. Two members of the WRKY family, <i>WRKY25</i> and <i>WRKY33</i>, have been targeted through VIGS in <i>G. hirsutum</i>. VIGS-infiltrated plants were evaluated under drought stress and whitefly infestation. It was observed that <i>GhWRKY33</i>-downregulated plants showed a decrease in whitefly egg and nymph population, and <i>GhWRKY33</i> was found to be a strong negative regulator of whitefly and drought stress, while <i>GhWRKY25</i> was found to be a moderate negative regulator of whitefly and drought stress. As the targeted genes are transcription factors influencing the expression of other genes, the relative expression of other stress-responsive genes, namely <i>MPK6</i>, <i>WRKY40</i>, <i>HSP</i>, <i>ERF1</i>, and <i>JAZ1</i>, was also analyzed through qRT-PCR. It was found elevated in <i>GhWRKY33</i>-downregulated plants, while <i>GhWRKY25</i>-downregulated plants through VIGS showed the elevated expression of <i>ERF1</i> and <i>WRKY40</i>, a slightly increased expression of <i>HSP</i>, and a lower expression level of <i>MPK6</i>. Overall, this study provides an important insight into the WRKY TF family and the role of two WRKY TFs in <i>G. hirsutum</i> under drought stress and whitefly infestation. The findings will help to develop crops resilient to drought and whitefly stress.