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 .