Pathways analysis of Pik-H4 as singleton or pair
To gain insights into the functional pathways governed by Pik-H4as either a singleton or a pair, we conducted transcriptome analyses of
LTH and Pik-H4 NILs exposed to M. oryzae , as well as of
LTH overexpressing Pik1-H4 orPik2-H4 . Notably, as bothPik1-H4 and Pik2-H4 were
located within the nucleus, we focused on identifying changes in the
transcriptomes of these samples through RNA-seq. The data’s quality and
reproducibility were validated through Pearson correlation analysis of R
values among replication groups, indicating robust and consistent
results (Figure S7A-J). Investigation revealed that, at 12 hpi, 86 genes
were significantly up-regulated (Filtered Pik-H4 NILs mock and
LTH at corresponding hpi, P <0.01,
|log2Foldchange|≥2, the same as
below), while 26 genes were significantly down-regulated. At 24 hpi, the
numbers were 268 up-regulated and 126 down-regulated genes (Figure 7A).
Considering that M. oryzae penetration typically occurs around 24
hpi (Li et al., 2023; Yan and Talbot, 2016), these findings suggest thatPik-H4 -mediated resistance induces transcriptome reprogramming
between 12 and 24 hpi post M. oryzae treatment. Through gene
ontology (GO) pathway enrichment analysis of up- or down-regulated genes
(Figure 7B, C), we found that, at 12 hpi, the predominant up-regulated
GO pathways encompassed chloroplast-related molecules such as heme and
tetrapyrrole binding, alongside enzymatic activities such as catalytic
and oxidoreductase functions. In contrast, down-regulated genes were
significantly related to chloroplast-related pathways. At 24 hpi, there
was a conspicuous shift towards chloroplast-related pathways for
up-regulated genes, including chlorophyll binding, photosynthesis, and
thylakoid functions. Concurrently, the primary down-regulated GO
pathways also retained ties to chloroplast functions. Furthermore, our
investigation encompassed several defense-related genes (Figure 7F, S8).
Notably, PBZ1 (Kim et al. , 2008), OsNAC111 (Yokotaniet al. , 2014) and chloroplast immunity genes LHCB5 (Liuet al. , 2019) and OsAPX8 (Jiang et al. , 2016)
exhibited positive involvement in Pik-H4 -mediated immunity. In
line with the findings presented in Figure S6, these results
collectively suggest that Pik-H4 -mediated effector-triggered
immunity (ETI) is closely intertwined with chloroplast-related
processes, indicating a potential role for Pik-H4 in chloroplast
immunity.
We extended our RNA-seq analysis to include
OE-Pik1-H4 /LTH and
OE-Pik2-H4-GFP /LTH plants. The gene counts of
up-regulated and down-regulated genes relative to LTH were 411 and 355,
2521 and 917, and 3357 and 1710 in Pik-H4 NILs,
OE-Pik1-H4 /LTH, and
OE-Pik2-H4-GFP /LTH, respectively (Figure 7D).
Upon GO pathway enrichment analysis, overexpression ofPik1-H4 or Pik2-H4resulted in down-regulation in pathways linked to defense response, heme
binding, tetrapyrrole binding, and transcription regulator activity,
while pathways associated with peroxidase activity, hydrolase activity,
and molecular functions related to the cell skeleton were up-regulated
(Figure S9B, C). Interestingly, most enriched pathways exhibited changes
when Pik1-H4 and Pik2-H4were co-expressed (Figure S9A), indicating the complex interplay betweenPik1-H4 and Pik2-H4 .
Zooming in on the subset of genes that were either up-regulated or
down-regulated across the three types of plants (Figure 7D, red set:
up-regulated; blue set: down-regulated), GO analysis revealed enrichment
primarily in nucleotide binding, transferase activity, metabolic
processes, and catalytic activity (Figure 7E). These pathways may be the
potential mechanism of Pik-H4 in the quiescent stage. A detailed
analysis of immune-related genes unveiled that Pik-H4, acting as
a singleton, reduced the expression of defense-related genes and
transcription factors (TFs, with the exception of OsWRKY13 )
(Figure 7F, S8). However, four genes related to reactive oxygen species
(ROS) were up-regulated, including two chloroplast immunity genes. This
highlights the intricate nature of the regulatory network mediated byPik-H4 .