Objective Pine wilt disease has posed a serious threat to the Pinus tabuliformis forest ecosystem in China. Currently, the epitranscriptomic regulatory mechanisms of conifers in response to pathogen infection remain unclear. This study aimed to investigate the dynamic changes of the m⁶A methylation landscape in P. tabuliformis upon B. xylophilus infection, screen and validate key m⁶A-regulated R genes, elucidate the molecular mechanism of m⁶A-mediated R gene regulation, and construct a “methylase-m⁶A-R gene-resistance” regulatory model, thereby providing theoretical foundations and genetic resources for molecular breeding of disease-resistant conifers.

Method Three-year-old P. tabuliformis seedlings were used as materials. Fourteen days after inoculation with B. xylophilus, the stem segments were subjected to whole-transcriptome and m⁶A modification profiling using Oxford Nanopore direct RNA sequencing. Multi-omics integrative analysis was performed to screen the key candidate gene PtLRR-XI1, which was then heterologously transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. Its disease resistance function was validated by assessing infection incidence and antioxidant physiological indicators. The methylation modification was validated by co-expression in tobacco.

Result After B. xylophilus infection, a total of 4 527 differentially expressed genes were identified in the stem segments of P. tabuliformis, with downregulated expression of methyltransferase genes and upregulated expression of demethylase genes. A total of 62 787 differentially methylated sites were identified at the genome-wide level, predominantly enriched in the 3′UTR regions, exhibiting a conserved “RRACH” motif. Integrative analysis identified 268 transcripts with simultaneous significant changes in expression and methylation, which were enriched in the plant-pathogen interaction and MAPK signaling pathways. Among these, PtLRR-XI1, a gene belonging to the LRR-RLK family, was identified. Its promoter contains multiple hormone- and stress-responsive elements, and it is highly conserved in conifers. Heterologous overexpression of this gene reduced the incidence of pine wilt disease in transgenic A. thaliana from 72.9% in the wild type to 25.0%−39.6%. Furthermore, after site-directed mutagenesis of two differential m⁶A modification sites in the 3′UTR of PtLRR-XI1, transient transformation in Nicotiana benthamiana leaves showed that the GFP fluorescence intensity of the mutated sequence was significantly weaker than that of the unmutated control, indicating that the expression of PtLRR-XI1 is regulated by m⁶A modification.

Conclusion B. xylophilus infection induces reprogramming of the m⁶A methylation modification system in P. tabuliformis. PtLRR-XI1 is a key resistance gene regulated by this modification, and its overexpression enhances the resistance of A. thaliana to B. xylophilus. The m⁶A modification sites in the 3′UTR region of this gene positively regulate its expression. This study offers a potential gene target for molecular breeding of disease resistance in P. tabuliformis.