| Summary: | <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A) is a prevalent internal modification in eukaryotic mRNA, tRNA, miRNA, and long non-coding RNA. It is also known for its role in plant responses to biotic and abiotic stresses. However, a comprehensive m<sup>6</sup>A transcriptome-wide map for <i>Puccinia striiformis</i> f. sp. <i>tritici</i> (<i>Pst</i>) infections in wheat (<i>Triticum aestivum</i>) is currently unavailable. Our study is the first to profile m<sup>6</sup>A modifications in wheat infected with a virulent <i>Pst</i> race. Analysis of RNA-seq and MeRIP-seq data revealed that the majority of differentially expressed genes are up-regulated and hyper-methylated. Some of these genes are enriched in the plant–pathogen interaction pathway. Notably, genes related to photosynthesis showed significant down-regulation and hypo-methylation, suggesting a potential mechanism facilitating successful <i>Pst</i> invasion by impairing photosynthetic function. The crucial genes, epitomizing the core molecular constituents that fortify plants against pathogenic assaults, were detected with varying expression and methylation levels, together with a newly identified methylation motif. Additionally, m<sup>6</sup>A regulator genes were also influenced by m<sup>6</sup>A modification, and their expression patterns varied at different time points of post-inoculation, with lower expression at early stages of infection. This study provides insights into the role of m<sup>6</sup>A modification regulation in wheat’s response to <i>Pst</i> infection, establishing a foundation for understanding the potential function of m<sup>6</sup>A RNA methylation in plant resistance or susceptibility to pathogens.
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