Identification of core genes involved in rice defense responses
to M. oryzae
To identify specific or common genes involved in rice defense responses
to M. oryzae at different time point, we performed Venn
diagram analysis of the DEGs from Nipponbare or Hui1586 at all four time
points. The diagrams showed that the majority of rice DEGs were
temporarily up- or down-regulated by M. oryzae at specific
periods. For instance, 2899 of 6725 DEGs from Nipponbare, and 6678 of
9703 DEGs from Hui1586 were specifically regulated by M. oryzaeonly at 12 hpi (Fig. 5A). Relatively small amount of DEGs, 1464 (1119
upregulated and 293 downregulated) and 578 (448 upregulated and 89
downregulated) were overlapped at all time points in Nipponbare or in
Hui1586, respectively (Fig. 5A and Supplemental dataset 6). We then
performed KEGG pathway enrichment analysis on each group of DEGs in Fig.
5A (Supplemental table 2). Among the 2899 DEGs specifically regulated at
12 hpi on Nipponbare, only protein translation related pathways were
significantly enriched (Supplemental table 2), which is consistent with
our previous analysis (Fig. 4). Surprisingly, no KEGG pathway term was
enriched in the 259, 682, 407, 551, 362, 126, 173, 1680, and 832 DEG
groups. Moreover, plant defense related pathways were enriched mainly in
the common 1464 DEGs that were regulated at all four time points on
Nipponbare (Fig 5A and Supplemental table 2). Similarly, in Hui1586,
protein translation pathways were only enriched in 6678 DEGs that
specifically regulated at 12 hpi, and no pathway term was enriched in
711, 501, 284, 221, 523, 197,101, 694, and 186 DEG groups (Fig 5A and
Supplemental table 2). However, different to Nipponbare, defense related
pathways were enriched in all other DEG groups, especially with lowestQ -value in the common 578 DEGs overlapped by all four time points
(Fig. 5A and Supplemental table 2). These results showed that the
expressions of the majority of DEGs from Nipponbare or Hui1586 were
regulated dynamically/temporarily in response to M. oryzae ,
except a common group of defense related genes, most of which were
constitutively upregulated (Fig. 5A). Thus, we hypothesized that
a group of common DEGs might forms the main part of the engine for
immune transcriptional reprogramming.
To test above hypothesis, we focused on the 353 overlapped DEGs between
the 1464 and the 578 common DEGs in Nipponbare and Hui1586 (Fig. 5A and
Supplemental dataset 7). Among these DEGs, 321 were upregulated and 32
were downregulated genes (Fig. 5A). Consistently, hierarchical
clustering of the FPKM values of those 353 genes showed that they fell
into two groups: genes in the large group (A) were upregulated and genes
in the small group (B) were downregulated by M. oryzae in both
cultivars (Fig. 5B). A KEGG pathway analysis of the common 321
upregulated genes showed that four immunity-related pathways were
significantly enriched, including “diterpenoid biosynthesis”,
“flavonoid biosynthesis”, “plant-pathogen interaction” and MAPK
signaling pathways (Fig. 5C and Supplemental dataset 7), suggesting that
these 321 genes were important components in rice resistance againstM. oryzae. The GO enrichment analysis also showed that “defense
response” and “diterpene phytoalexin metabolic” were the most
significantly enriched GO terms (Fig. S2). Furthermore, among the GO
molecular function terms, “carbohydrate binding”, “ion binding”,
“protein kinase activity”, “phosphotransferase activity”,
“oxidoreductase activity”, “kinase activity”, “anion binding”, and
“pattern binding” were significantly enriched (Fig. S2). Carbohydrate
binding proteins are proteins that can interact with sugar chains
(Someya et al., 2010). For example, lectins are carbohydrate-binding
proteins that play numerous roles in biological recognition events.
Several plant lectin receptor kinases are involved in plant innate
immunity (Singh, Chien, Mishra, Tsai, & Zimmerli, 2013; Singh &
Zimmerli, 2013). Among “ion binding” proteins, many different types
are found in plants. Metal ions help stabilize protein and regulate
protein catalytic activity (Lu, Lin, Lin, & Yu, 2012). For example,
calcium, calcium channel and calcium-binding proteins play different and
important roles in activating plant defense responses (J. M. Zhou &
Zhang, 2020). No significant KEGG pathway terms or GO terms were found
to be enriched in the common 32 downregulated genes. Taken together,
these analyses indicated that most of the 321 unregulated genes were
involved in rice immunity and are potential candidates for functional
characterization in further studies. Thus, the 321 genes were termed as
a “core” set of rice immune genes for further analysis.