The small GTP-binding protein GhARAC3 is involved in cotton resistance to Verticillium dahliae by regulating lignin metabolism.

Plant Rho/ROP GTPases act as essential molecular switches in disease resistance. However, how their protein stability is regulated and integrated with lignin metabolism remains poorly understood. Here, we demonstrate that the cotton small GTPase GhARAC3 serves as a positive regulator of resistance against the vascular pathogen Verticillium dahliae (V. dahliae). Genetic analyses reveal that overexpression of GhARAC3 enhances defense responses in cotton and Arabidopsis, whereas its silencing significantly compromises plant immunity. Under normal physiological conditions, the E3 ubiquitin ligase GhKEG acts as a key regulator that targets GhARAC3 for proteasomal degradation by ubiquitinating multiple lysine residues. Notably, the lysine 161 (K161) residue within the conserved G5 motif is identified as an in vivo ubiquitination site. Mutation of this residue (K161R) impairs GTPase activity and compromises disease resistance, without altering its subcellular localization at the plasma membrane and nucleus. Pathogen infection attenuates this ubiquitination to promote the stabilization of GhARAC3. Genetic epistasis analysis indicates that GhARAC3 requires GhLAC14, a transmembrane laccase, to confer effective immunity.Rather than altering GhLAC14 protein stability, GhARAC3 physically interacts with and stimulates the enzymatic activity of the laccase. Additionally, GhARAC3 promotes reactive oxygen species (ROS) production. This dual activation of laccase activity and the ROS burst drives the rapid deposition of guaiacyl lignin to fortify the plant cell wall. Furthermore, transcriptomic profiling reveals that GhARAC3 broadly upregulates downstream defense-related pathways. Collectively, our findings uncover a regulatory module linking ubiquitin-mediated GTPase turnover to laccase activation, providing a mechanistic framework for improving cotton disease resistance.