3.7 BPH infestation regulated the expression of the main m6A modification machinery components
We compared the relative gene expression levels of 5 “WRITER” genes, that is, OsMTA1 , OsMTA2 , OsMTA3 , OsMTA4 , andOsFIP ; 5 “ERASER” genes, namely, ALKBH9B-1 ,ALKBH9B-2 , ALKBH10B , ALKBH10B-L1 , andALKBH10B-L2 ; 12 “READER” genes, that is,OsYTH01–OsYTH12 ; and 5 “DONOR” genes, namely, OsSAM1 , OsSAM1-Like ,OsSAM2 , OsSAM2-Like , and OsSAM3 in Nip and Nl-Nip groups. Based on RT-qPCR analyses, among the five “WRITER” genes, the expression ofOsMTA1 was upregulated while the OsFIP gene was suppressed in BPH-infested samples (Figure 3a). For the “ERASER” genes, the expression of ALKBH9B-1 was slightly increased in the BPH-infested samples, whereas those of ALKBH9B-2 andALKBH10B were downregulated (Figure 3b). In the “READER” genes, the OsYTH02 , OsYTH04 , OsYTH07 , OsYTH08 ,OsYTH11 , and OsYTH12 expression levels were significantly upregulated in BPH-infested samples. Meanwhile, the expression of theOsYTH01 , OsYTH05 ,OsYTH09 , and OsYTH10 genes was significantly suppressed (Figure 3c). Compared to those in control Nip, the expression levels of all five methyl “DONOR” synthesis genes were upregulated in Nl-Nip (Figure 3d). The results of the transcriptome analyses and RT-qPCR were basically consistent, highlighting the reliability of the transcriptome sequencing data (Figure 3).
The dynamic changes in m6A positions in the m6A methylation machinery components were tested. Given that most genes were modified by different m6A sites, we counted the numbers of m6A methylation sites for each gene and further categorized them into three direction types, that is, up-direction (Up), down-direction (Down), and no significant difference (No change) between the Nl-Nip and Nip groups (Supporting Information: Table S9). Among the “DONOR” genes, only OsSAM2exhibited an up-m6A site. For the “WRITER” genes, OsMTA1–OsMTA4 genes contained the up-directed m6A site, while OsFIP showed a down-trend of m6A sites. For the “ERASER” genes, ALKBH9B-1 and ALKBH10B contained the up-directed m6A sites, while ALKBH9B-2 showed a down-trend in m6A sites. Regarding the “READER” genes, OsYTH07 , OsYTH08 , and OsYTH11 only contained up-directed differential m6A sites, whileOsYTH01 , OsYTH06 , OsYTH09 , and OsYTH10 only showed down-directed differential m6A sites. TheOsYTH02 , OsYTH03 ,OsYTH05 , and OsYTH12 genes contained more than 10 differential patterns of m6A positions in the Nl-Nip vs. Nip comparison, including up-, down-, and non-directed m6A sites (Supporting Information: Table S9, S10).
A filtering criteria was established to determine a correlation between the transcription and m6A methylation level of the target genes in the Nl-Nip vs. Nip comparison group as a transcriptional expression fold change < 0.5 or > 2, along with a significant m6A methylation trend p < 0.05, and |meth diff| > 10 (Supporting Information: Table S6, S10). Genes like OsMTA1 was upregulated and showed accompanying upregulated m6A methylation, that is, the number of upregulated m6A sites was higher than that of downregulated m6A sites in this transcript. Genes such asOsFIP was downregulated and exhibited downregulated m6A methylation, that is, the number of downregulated m6A sites was higher than that of the upregulated m6A sites (Figure 3; Supporting Information: Table S9, S10). We identified the OsSAM2 gene in the “DONOR” category,OsMTA1 and OsFIP genes in “WRITER” category,ALKBH9B-2 gene in the “ERASER” category, and OsYTH01 ,OsYTH05 , OsYTH07–OsYTH12 genes in the “READER” category. These genes not only differentially expressed but also included the differential m6A methylation sites with the same regulatory trend (Figure 3e). This indicated that m6A modification may positively regulate the transcription of target transcripts. m6A modifications were observed in genes responsible for plant m6A methylation machinery, highlighting their potential role in regulating target gene expression dynamics. This mechanism may be an important post-translational regulatory strategy in rice, particularly in response to BPH infestation.