Genome-wide analysis of the WRKY family in Nicotiana benthamiana reveals key members regulating lignin synthesis and Bemisia tabaci resistance

WRKY transcription factors (TFs) play a pivotal role in plant resistance against stress. Lignin is a plant component that is of great importance in many industrial and agricultural disciplines. However, how WRKYs regulate plant lignin synthesis pathways and defense against insects is largely unknown. Here, we investigate WRKYs in Nicotiana benthamiana, a model plant and biofactory widely used worldwide, elucidating their responses to the Bemisia tabaci whitefly, a globally significant agricultural pest, and their functions in regulating plant defense by regulating lignin synthesis. Firstly, we identified a total of 118 NbWRKYs in the N. benthamiana genome and performed necessary bioinformatics analyses. Then, we studied nine whitefly-inducible NbWRKYs and found that they were localized in the nucleus, significantly expressed in leaf tissues, and affected the phytohormone pathways. Meanwhile, NbWRKY21, NbWRKY45, NbWRKY81, and NbWRKY117 were proved to be involved in defense against whiteflies. Additionally, through DNA-TFs interaction experiments, we demonstrated that the WRKYs activated the expression of key genes—Cinnamate-4-hydroxymate (C4H), 4-coumarate-CoA ligase (4CL), and Cinnamoyl-CoA reductase (CCR)—in the lignin synthesis pathway. Specifically, NbWRKY81 activated the NbC4H and Nb4CL, while NbWRKY45 promoted NbCCR, by binding to their promoters, affecting whitefly feeding behavior, and therefore enhancing whitefly defense. Our findings contribute to a comprehensive knowledge of the WRKYs in plant defense against herbivore insects by regulating lignin synthesis.