Analysis of Non-Coding RNAs and N6-Methyladenosine-Modified Genes in Response to flg22 in Grape Immunity

Grape (Vitis vinifera) is susceptible to fungal pathogens and, consequently, severe yield losses. Investigating the immune mechanisms of the disease-resistant Chinese wild grapes is crucial for developing sustainable disease resistance technologies. Here, we conducted whole-transcriptome and methylated-RNA-immunoprecipitation sequencing to elucidate the immune defence mechanisms underlying grapevine responses to bacterial flagellin 22 (flg22). Certain differentially expressed miRNAs and lncRNAs responsive to flg22 showed greater differences in genotype 'Shanyang' than those in 'Cabernet Sauvignon'. Functional annotation of miRNA target genes revealed that 'Shanyang'-specific pathways were associated with ethylene-activated signalling, etc. Additionally, the cis-target genes of lncRNAs were significantly enriched in the trihydroxystilbene synthase activity, etc. Notably, transient overexpression of lncRNAs TCONS_00015412, TCONS_00070812, and TCONS_00070833 in V. quinquangularis conferred significantly enhanced disease resistance compared to control plants. Significantly different m⁶A peaks were located in the coding sequence and stop-codon regions. Integrated analysis of m⁶A and RNA-seq suggested that m⁶A methylation within the coding sequence generally enhanced mRNA expression. Functional analysis further demonstrated that significantly differentially expressed genes with differential m⁶A modifications were enriched in the plant-pathogen interaction pathway, etc. Furthermore, among m⁶A-modified genes, LRR-RLK in V. quinquangularis was confirmed to enhance grapevine resistance to C. diplodiella through transient overexpression. Altogether, our data strongly indicate that m⁶A methylation and non-coding RNAs regulated immune-related gene expression upon flg22 treatment, thereby modulating grapevine defence signalling pathways against pathogens. These results significantly enhance our understanding of the molecular mechanisms involving non-coding RNAs and m⁶A genes in the grapevine immune-defence responses and provide a valuable theoretical foundation for grapevine resistance breeding.