GhCNGC31 is critical for conferring resistance to Verticillium wilt in cotton.

In the past decades, cyclic nucleotide-gated ion channels (CNGCs) have been extensively studied in diploid species Arabidopsis thaliana. However, the functional diversification of CNGCs in crop plants, mostly polyploid, remains poorly understood. In allotetraploid Upland cotton (Gossypium hirsutum), GhCNGC31 is one of the multiple orthologs of AtCNGC2, being present in the plasma membrane, capable of interacting with itself and binding to calmodulins and cyclic nucleotides. GhCNGC31 knockdown plants exhibited slight growth inhibition, and became more susceptible to Verticillium dahliae infection, which was associated with the reduced lignin and flavonoid accumulation, impaired ROS (reactive oxygen species) burst, and down-regulation of defense-related genes PR1, JAZ2, LOX2, and RBOH10. RNA-Seq analysis identified 1817 differentially expressed genes from GhCNGC31 knockdown, of which 1184 (65%) were responsive to V. dahliae infection and accounted for 57% among a total of 2065 V. dahliae-responsive genes identified in this study. These GhCNGC31-regulated genes mainly function with cell wall organization and biogenesis, cellular carbohydrate metabolic or biosynthetic process, cellular component macromolecule biosynthetic process, and rhythmic process. They are significantly enriched in the pathways of plant MAPK signaling, plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. A set of transcription factors (TFs) and resistance (R) genes are among the GhCNGC31-regulated genes, which are significantly over-represented with the TCP and WRKY TFs families, as well as with the R genes of T (TIR) and TNL (TIR-NB-LRR) classes. Together, our results unraveled a critical role of GhCNGC31 for conferring resistance to Verticillium wilt in cotton.