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 Gossypium hirsutum 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, WRKY25 and WRKY33, have been targeted through VIGS in G. hirsutum. VIGS-infiltrated plants were evaluated under drought stress and whitefly infestation. It was observed that GhWRKY33-downregulated plants showed a decrease in whitefly egg and nymph population, and GhWRKY33 was found to be a strong negative regulator of whitefly and drought stress, while GhWRKY25 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 MPK6, WRKY40, HSP, ERF1, and JAZ1, was also analyzed through qRT-PCR. It was found elevated in GhWRKY33-downregulated plants, while GhWRKY25-downregulated plants through VIGS showed the elevated expression of ERF1 and WRKY40, a slightly increased expression of HSP, and a lower expression level of MPK6. Overall, this study provides an important insight into the WRKY TF family and the role of two WRKY TFs in G. hirsutum under drought stress and whitefly infestation. The findings will help to develop crops resilient to drought and whitefly stress.

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