3.4 m6A methylation was closely associated
with actively expressed genes in BPH-infested plants
We analyzed the Euclidian distance coefficients among gene transcript
profiles based on nanopore DRS. The distance coefficients between the
replicates were lower than those of other objects, and the tile colors
of the two were relatively close (Supporting Information: Figure S7),
suggesting their reproducible patterns. These sequenced genes were
divided into three groups: transcripts per kilobase of exon model per
million mapped reads (TPM) < 1, 1 < TPM <
5, and TPM > 5 (Supporting Information: Table S5). The
proportion of gene numbers in each category was calculated and shown as
a heatmap (Supporting Information: Figure S8). Rice genes that could be
annotated by m6A positions were referred to
‘m6A genes’ and those that could not be annotated were
marked as ‘non-m6A genes’ for subsequent analyses. In
the Nip and Nl-Nip groups, most m6A genes were
distributed in the highly expressed gene category (TPM >
5). In contrast, non-m6A genes were evenly distributed
across the three groups and were biased towards gene categories with low
expression (TPM < 1). Compared to that in Nip, the proportion
of non-m6A genes in low expression category was lower
in Nl-Nip group (Figure 2a, Supporting Information: Figure S8). When the
m6A and non-m6A genes were divided
by gene expression categories—high (TPM ≥ 1) or low (TPM <
1)—the number of transcripts showing high/low expression was recorded
for control Nip and Nl-Nip samples (Figure 2b). We found that most genes
were m6A methylated and distributed in the high
expression category in Nip and Nl-Nip groups; these gene numbers were
enriched upon BPH infestation (Figure 2b). In both Nip and Nl-Nip
groups, m6A-methylated genes were expressed at a
higher level than non-m6A-methylated genes (Figure
2c). Thus, m6A
methylation mainly occurred in highly expressed genes in the control and
BPH-infested samples, while the m6A modified gene
numbers increased in the high expression category with BPH infestation.
3.5 m6A
methylation positively correlated with the transcript expression in
BPH-infested rice
We found 21,718 (76.59%) transcripts that showed no significant
difference (no change), 3,506 (12.36%) were upregulated and 3,131
(11.04%) were downregulated in
BPH-infested plants compared with those in the control plants (Figure
2d, Supporting Information: Figure S9a,
Table S6) . A total of
116,817 methylated positions were
detected among all expressed transcripts. Among the
m6A modifications, 63.99%, 17.43%, and 18.58%
exhibited no change, or fell into the up- or down-m6A
directions, respectively (Supporting Information: Table S6). A large
proportion of m6A modification positions were found in
CDS and 3′-UTR, with most showing no change in expression (Figure 2e,
Supporting Information: Table S6).
Among
up-directed
m6A-methylated transcripts, the number of upregulated
transcripts was higher than that of downregulated transcripts (2,094 vs.
856) in the NI-Nip vs. Nip
comparison; conversely, in down-directed transcripts, the number of
downregulated ones was higher than that of upregulated ones (1,995 vs.
743) (Figure 2e). The correlational heat map showed a strong positive
correlation between the m6A methylation direction and
the corresponding transcript change, with many transcripts and
m6A modifications exhibiting simultaneous up- or
downregulation/direction (Supporting Information: Figure S9b). The
abundance of upregulated transcripts that underwent 5′-UTR methylation
was higher than that of the downregulated transcripts. However, this
tendency was not evident among 3′-UTR methylated transcripts (Figure
2f). Taken together, the
m6A-methylation
differential types in BPH-infested samples were positively correlated
with the corresponding transcript regulation types, with transcripts
containing different m6A methylated functional
elements likely involved in diverse transcript regulation.