Introduction
Plants defend against pathogens through effector-triggered immunity (ETI) with Resistance (R) proteins recognition of Avirulence (Avr) proteins. Most R genes belong to the NLRs family. According to the number of functioning proteins, NLRs can be divided into singletons and pairs. The NLR singletons recognize and/or interact with Avrs, resulting in hypersensitive response (HR). The RPP1, ROQ1, and Sr35 directly interact with corresponding effectors and form resistosomes(Maet al. , 2020; Martin et al. , 2020; Zhao et al. , 2022). Rice R proteins Pi-ta and Pi54 also directly recognize Avr-Pita and Avr-Pi54 respectively(Jia et al. , 2000; Ray et al. , 2016). In the case of indirect recognition of effectors, one of the most apparent mechanisms is Arabidopsis Coiled-coil type NLR (CNL) protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1). After pathogen infection, ZAR1 is pentameric to a resistosome together with resistance-related kinase 1 (RKS1) and PBS1-like protein 2 (PBL2)(Wang et al. , 2019). PBL2 acts as a “decoy”(Wang et al. , 2015) and the ZAR1-RKS1-PBL2 resistosome exhibits a plasma membrane localization, function as a Ca2+ channel, and trigger HR(Bi et al. , 2021). After interacting with AVRRpm1 or AVRB, the phosphorylated RIN4 acts as a “guard”, activating the Arabidopsis CNL RPM1 and resulting in cell death(Mackey et al. , 2002). Upon infection, AvrPib competitively binds to the “protector” of Pib, OsSH3P2, and releases Pib from the OsSH3P2-Pib complex, leading to Pib activation(Xieet al. , 2022).
The NLR pairs members play different roles in the integrative decoy model(Cesari, Bernoux, et al. , 2014). The one that recognizes Avr functions as a “sensor” and the other NLR that causes HR and downstream signaling is called a “helper”. As a typical example, the rice CNL pair member RGA5 directly binds to AvrPia, and RGA4 triggers HR(Cesari, Kanzaki, et al. , 2014). In contrast, the PigmR/PigmS pair differs from the decoy model. PigmR recognizes the effector and induces HR, while PigmS inhibits cell death(Deng et al. , 2017). In addition, PigmR and PigmS display low expression levels in all tissues except that PigmS shows a high transcription activity in panicles and pollens. Some NLR pair members are head-to-head (H2H) construction in chromosomes with an interval of a bidirectional promoter (BDP). Bidirectional genes (BDGs) occur widely in nature. InArabidopsis and rice, 5763 and 8742 genes are organized in BDG architectures(Krom and Ramakrishna, 2008). Since sharing transcription factor binding sites and cis elements through BDPs, the BDGs are strongly co-regulated and participated in similar pathways(Williams and Bowles, 2004). The Arabidopsis TNL pairs RPS4/RRS1 and CHS1/SOC3 are organized in a BDP manner and spaced with a ~300 bp promoter region. The PRS4/RRS1 complex is necessary for recognizing the AvrRps4 and Pop2(Williams et al. , 2014). Expression ofSOC3 is promoted in the CHS1 mutant, suggesting that self-regulation through the promoter region of CHS1 /SOC3and the CHS1/SOC3 complex is involved in autoimmunity(Zhang et al. , 2017). The only known NLR BDGs in rice are Pik genes, and 11 of them are reported. The HR assays of Pikh (Zhai et al. , 2014) and Pikp (Zdrzałek et al. , 2020a) indicate that the two members of Pik genes are required for recognition of AvrPik and cell death. The co-function of the NLR gene pair requires regulation and coordination in transcription and protein levels. In rice, most of the NLRs are allelic genes of the model species Nipponbare (Nip). The sequence variation of the allelic NLRs makes RNA-seq-based expression pattern analysis difficult. For example, the coding sequences and promoter regions of Pikh1 /Pikh 2 and the NipponbareLOC_Os11g46200 /LOC_Os11g46210 are apparently different. Consequently, the expression pattern and relation of NLRs, especially the NLR pairs, are still unclear.
The CNL gene pair Pik-H4 is a synonymous mutation of Pikhand is resistant to rice blast fungus(Xiao et al. , 2011). A homeodomain transcription factor OsBIHD1 is found to interact with Pik1-H4 and is required to Pik-H4 induced ETI through regulation of the brassinosteroid-ethylene pathway(Liu et al. , 2017). We report that the NLR gene pairPik1-H4 and Pik2-H4 are positively co-expressed in all tissues, highly expressed in vascular bundles, including mesophyll cells and vascular cells, and induced by rice blast fungus through a bidirectional promoter. OverexpressingPik1-H4 or Pik2-H4promotes the transcription levels of the counterpart Pik-H4 and does not affect the resistance to rice blast. Singleton Pik1-H4 and Pik2-H4 display vesicles and nucleus location respectively during infection, pathway analysis shows that Pik-H4 mediated immunity is involved in transcription reprogramming and Pik-H4 singleton functions in various pathways.