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.