Different roles of Pik1-H4 andPik2-H4 as singletons and pair
Most of the NLR proteins are localized in the cytoplasm and nucleus. For instance, the Arabidopsis RPS4(Heidrich et al. , 2011) and RRS1(Deslandes et al. , 2003), rice Pikh-1/Pikh-2 pair are localized in cytoplasm and nucleus(Zhai et al. , 2014), while the RGA4/RGA5 pair is cytoplasmic(Cesari, Kanzaki, et al. , 2014). Pik1-H4 and Pik2-H4 were observed to be localized in the plasma membrane and chloroplasts in planta(Figure 6A, E, Figure S6). Combined with the top enriched GO pathways at 24 hpi (Figure 7C), our results indicated that Pik genes might play roles in membrane and chloroplast immunity. Notably, Pik1-H4 as a singleton was found in vesicles around rice blast fungus, while Pik2-H4 singleton appeared in the nuclei of peripheral cells surrounding the infected cells (Figure 6D, H). The enrichment pathways of overexpression of singleton Pik-H4varied (Figure S9B, C). This suggests that the functions of thePik-H4 gene pair might involve distinct pathways when it comes to recognizing Avr proteins and inducing HR in response to the invasion of rice blast fungus (Figure 5G).
Singleton Pik1-H4 orPik2-H4 promoted expression of ROS-related genesOsRBOHB , OsRAC1 , LHCB5 and OsAPX8 (Figure 7F) but down-regulated defense response pathway (Figure S9B, C), defense-related genes PR1a , PR1b , PR10 andPBZ1 (Figure 7F), as well as multiple WRKY TFs (Table S2). Interestingly, when Pik1-H4 andPik2-H4 are coupled together, no suppression of WRKY TFs was observed. Since both Pik1-H4 and Pik2-H4 are localized in the nucleus, it implies thatPik-H4 may play a role in transcriptional regulation. Specifically, Pik1-H4 has been found to interact with the TF OsBIHD1 and contribute to disease resistance(Liu et al. , 2017), although the precise function of Pik2-H4 in the nucleus is yet to be fully understood. The GO enrichment changes between 12 hpi and 24 hpi imply that Pik-H4 functions in transcription during ETI (Figure 7B, C). The findings indicate that singletonPik-H4 is susceptible based on transcriptome profile. It is plausible that Pik-H4 singletons or the pair may function as transcriptional cofactors, leading to transcriptional reprogramming in the context of immune responses.