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.