3.10 BPH infestation activated defense-related phytohormones pathways though dynamic m6A modifications
We investigated whether anti-herbivore defense hormones JA and SA metabolic pathways are regulated by m6A modification and whether the expression profiles of their metabolic genes are altered by BPH infestation in rice. All 9 selected biosynthesis genes in the JA pathway, except downregulatedOsLOX5 , were upregulated in BPH-infested plants (Figure 5a). Expression levels of all the JA-responsive genes, OsPR5-1 , OsPR10 , and OsRbohC-F , were downregulated in BPH-infested samples; the remaining genes, apart from non-regulated OsWRKY10 , were significantly upregulated (Figure 5b,c). Among the JA biosynthesis genes,OsAOS2 , OsJMT1 , and OsLOX8only exhibited upregulated differential m6A sites; OsAOS1 , OsLOX2 , and OsLOX5 exhibited both up-, and down-directed m6A sites (Supporting Information: Table S15, S16). For JA-responsive genes, OsPR2 , OsPR5-2 ,OsPR5-3 , OsPR5-4 , OsMYB2 , and OsWRKY28 only contained up-directed differential m6A sites, while OsPR5-1 , OsPR10 ,OsRbohD , and OsRbohF only showed down-directed differential m6A sites. OsRbohB ,OsRbohC , and OsRbohE contained both up- and down-directed m6A sites (Supporting Information: Table S15, S17). Most upregulated genes showed a positive correlation with the up-directed m6A modification positions, such asOsJMT1 , OsAOS1 , OsAOS2 , and OsLOX8 in biosynthesis genes, andOsPR2 , OsPR5-2 ,OsPR5-3 , OsPR5-4 ,OsMYB2 , and OsWRKY28 in responsive genes. Both m6A modification and the relative expression ofOsPR5-1 , OsPR10 , OsLOX5 , and OsRbohC-F were significantly downregulated in BPH-infested plants (Figure 5a-c; Supporting Information: Table S15-17).
In the SA pathway, all candidate biosynthesis-related genes were upregulated in BPH-infested samples (Figure S12a). Among the response-related genes, OsPR1-12 , OsPR1-51 , OsPR1a ,OsPR1b , and OsWRKY4 5 were upregulated, whileOsPR1-21 , andOsPR1-101 were downregulated in BPH-infested samples (Figure S12b). Regarding the m6A position direction, OsCM andOsWRKY45 only exhibited up-directed differential m6A sites; OsICS1 and OsPR1-101contained down-directed differential m6A sites; andOsEDS1 , OsPAL , and OsPAL1 showed both up- and down-directed m6A sites (Supporting Information: Table S15, S18, S19). The m6A modification level of SA pathway-related genes also showed a positive correlation with transcriptional regulation. The genes that followed such pattern included OsPAL1 and OsCM in the biosynthesis genes andOsPR1-101 and OsWRKY45 in the responsive genes (Supporting Information: Figure S12; Table S15, S18, S19). However, the proportion of m6A modified genes was lower than that of the JA pathway-related genes.
BPH infestation successfully induced phytohormones accumulation in plants. This degree of induction was more pronounced for JA production than for SA (Figure 5d,e; Supporting Information: Figure S12c), which validated the RT-qPCR and transcriptome data of the related transcripts. These were accompanied by dynamic m6A methylation modification and gene expression changes in major hormonal pathway-related genes, especially in genes related to the JA metabolic pathway.