Comprehensive identification of cotton EPF/EPFL receptors and functional characterization of the GhEPFL1-1-GhER1 module in drought tolerance.

The development of stomatal lineage cells in terrestrial plants is tightly regulated by epidermal patterning factors (EPFs/EPFLs) and their downstream receptors, including ERECTA, TOO MANY MOUTHS (TMM), and SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs). These components form co-receptor complexes that activate the MAPK signaling cascade, playing critical roles in stomatal development, stress responses, and signal transduction. However, the EPF-ERECTA-TMM signaling network remains largely unexplored in cotton (Gossypium spp.). In this study, we performed a genome-wide identification and characterization of the EPF/EPFL, ERECTA, TMM, and SERK gene families in four cotton species (G. hirsutum, G. barbadense, G. arboreum, and G. raimondii), identifying 135 EPF/EPFL, 18 ERECTA, 6 TMM, and 90 SERK genes. Bioinformatics analyses-including gene collinearity, protein domain structure, cis-regulatory elements, and protein-protein interaction predictions-revealed functional divergence and stress-related regulatory potential across these families. Expression profiling in G. hirsutum indicated that several candidate genes, such as GhEPFL1-1, GhER1, and GhSERK17, are responsive to abiotic stresses. To validate these computational predictions, functional assays were conducted. Virus-induced gene silencing (VIGS) of GhEPFL1-1, GhER1, and GhSERK17 led to increased stomatal density and reduced drought tolerance, confirming their roles in stress adaptation. Furthermore, luciferase complementation imaging in Nicotiana benthamiana demonstrated direct interactions between GhEPFL1-1 and GhER1, and co-receptor complex formation with GhSERK17, consistent with molecular docking simulations. Collectively, this study lays a theoretical foundation for further exploration of the EPF/EPFL-mediated peptide-receptor signaling pathway in cotton and its potential application in breeding for stress resilience.