Genome-wide identification of GhRLCK-VII subfamily genes in Gossypium hirsutum and investigation of their functions in resistance to Verticillium wilt.

BACKGROUND: The receptor-like cytoplasmic kinases subfamily VII (RLCK-VII) is critical in regulating plant growth, development, and pattern-triggered immunity. However, a comprehensive exploration of these genes in the allotetraploid Gossypium hirsutum is still lacking. This study aimed to identify RLCK-VII genes in G. hirsutum and investigate their evolutionary history, structural features, expression patterns, and role in plant defense. RESULTS: Seventy-two RLCK-VII genes in the G. hirsutum genome were unveiled and classified into nine groups following their phylogenetic analysis with Arabidopsis thaliana. Group VII-1 was the largest, accounting for 28%, while Groups VII-2 and VII-3 had only one member each. The analysis using MCScanX revealed that these 72 genes formed 166 collinear gene pairs and were resided on 26 chromosomes of G. hirsutum, suggesting that they were derived from whole genome segmental duplication events. Their calculated Ka/Ks values were below one, implying the occurrence of purification selection during the evolution and inhibition of gene function differentiation/loss. All members of the RLCK-VII subfamily possessed two conserved domains, PKinase-Tyr and PKinase, and several conserved PBS1 kinase subdomains, individually included in one of the ten motifs identified using MEME. The RNA-Seq results showed that RLCK-VII genes exhibited different spatiotemporal expression, indicating their involvement in cotton growth, development, and defense responses to Verticillium dahliae. The transcription patterns of RLCK-VII genes found by RNA-Seq were further validated using qRT-PCR assays after inoculating "20B12" (cotton cultivar) with "V991" (V. dahliae). The virus-induced gene silencing (VIGS) assays uncovered that two RLCK-VII genes (Gohir.A13G227248 and Gohir.A10G219900) were essential to G. hirsutum resistance to Verticillium wilt. CONCLUSIONS: These observations offer valuable insight into the attributes and roles of RLCK-VII genes in G. hirsutum, potentially enable the breeding of new cotton cultivars with enhanced resistance to Verticillium wilt.

A Phytophthora capsici RXLR effector targets and inhibits the central immune kinases to suppress plant immunity.

Receptor-like cytoplasmic kinase subfamily VII (RLCK-VII) proteins are the central immune kinases in plant pattern-recognition receptor (PRR) complexes, and they orchestrate a complex array of defense responses against bacterial and fungal pathogens. However, the role of RLCK-VII in plant-oomycete pathogen interactions has not been established. Phytophthora capsici is a notorious oomycete pathogen that infects many agriculturally important vegetables. Here, we report the identification of RXLR25, an RXLR effector that is required for the virulence of P. capsici. In planta expression of RXLR25 significantly enhanced plants' susceptibility to Phytophthora pathogens. Microbial pattern-induced immune activation in Arabidopsis was severely impaired by RXLR25. We further showed that RXLR25 interacts with RLCK-VII proteins. Using nine rlck-vii high-order mutants, we observed that RLCK-VII-6 and RLCK-VII-8 members are required for resistance to P. capsici. The RLCK-VII-6 members are specifically required for Phytophthora culture filtrate (CF)-induced immune responses. RXLR25 directly targets RLCK-VII proteins such as BIK1, PBL8, and PBL17 and inhibits pattern-induced phosphorylation of RLCK-VIIs to suppress downstream immune responses. This study identified a key virulence factor for P. capsici, and the results revealed the importance of RLCK-VII proteins in plant-oomycete interactions.

Genome-Wide Identification and Expression Analysis of RLCK-VII Subfamily Genes Reveal Their Roles in Stress Responses of Upland Cotton.

Receptor-like cytoplasmic kinase VII (RLCK-VII) subfamily members are vital players in plant innate immunity and are also involved in plant development and abiotic stress tolerance. As a widely cultivated textile crop, upland cotton (Gossypium hirsutum) attaches great importance to the cotton industry worldwide. To obtain details of the composition, phylogeny, and putative function of RLCK-VII genes in upland cotton, genome-wide identification, evolutionary event analysis, and expression pattern examination of RLCK-VII subfamily genes in G. hirsutum were performed. There are 129 RLCK-VII members in upland cotton (GhRLCKs) and they were divided into nine groups based on their phylogenetic relationships. The gene structure and sequence features are relatively conserved within each group, which were divided based on their phylogenetic relationships, and consistent with those in Arabidopsis. The phylogenetic analysis results showed that RLCK-VII subfamily genes evolved in plants before the speciation of Arabidopsis and cotton, and segmental duplication was the major factor that caused the expansion of GhRLCKs. The diverse expression patterns of GhRLCKs in response to abiotic stresses (temperature, salt, and drought) and V. dahliae infection were observed. The candidates that may be involved in cotton's response to these stresses are highlighted. GhRLCK7 (GhRLCK7A and D), which is notably induced by V. dahliae infection, was demonstrated to positively regulate cotton defense against V. dahliae by the loss-of-function assay in cotton. These findings shed light on the details of the RLCK-VII subfamily in cotton and provide a scaffold for the further function elucidation and application of GhRLCKs for the germplasm innovation of cotton.