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.