PhWRKY23 Positively Contributes to Herbivore Resistance and Is Associated with Phytohormone and Defense-Related Responses in Populus hopeiensis

Populus hopeiensis is an important native poplar species in northern China, but herbivorous insect damage seriously affects its growth and ecological function. WRKY transcription factors play important roles in plant stress responses; however, the function of WRKY23 homologs in woody plant resistance to chewing herbivores remains unclear. In this study, a herbivore-responsive WRKY transcription factor gene, PhWRKY23, was identified from P. hopeiensis. PhWRKY23 expression was significantly induced by Spodoptera litura feeding, with a maximum increase of approximately 69.71-fold the control level at the highest damage level, and the encoded protein was predominantly localized in the nucleus. To investigate its function, PhWRKY23-overexpressing and RNA interference transgenic lines were generated. In the choice feeding assay, the consumed leaf area of PhWRKY23-overexpressing plants was approximately 85.5% lower than that of WT plants after 8 h. In the no-choice feeding assay, the total larval mass after 6 d was approximately 38.8% lower in larvae fed on overexpression plants and 51.0% higher in larvae fed on RNAi plants than in those fed on WT plants. Physiological analysis showed that RNAi plants accumulated significantly more MDA than WT and overexpression plants, whereas overexpression plants had higher chlorophyll a, chlorophyll b, and carotenoid contents than the other genotypes. Phytohormone analysis further showed that PhWRKY23-overexpressing plants accumulated higher levels of jasmonic acid, jasmonoyl-L-isoleucine, and salicylic acid, whereas abscisic acid showed no significant difference among genotypes. Yeast two-hybrid screening identified several candidate PhWRKY23-interacting proteins, and pairwise validation confirmed that PhWRKY23 interacted with PhDOX1 in yeast. These results indicate that PhWRKY23 positively contributes to herbivore resistance in P. hopeiensis and that this resistance phenotype is associated with changes in JA, JA-Ile, and SA accumulation and defense-related physiological traits.