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\begin{document}

\title{Co-expression and function of head-to-head NLR gene pair Pik-H4}



\author[1]{Fengwei Gu}%
\author[1]{Huabin Xie}%
\author[1]{Qiwei Huang}%
\author[1]{Shang Gao}%
\author[1]{Tao Guo}%
\author[1]{Jiafeng Wang}%
\author[1]{Hui Wang }%
\affil[1]{South China Agricultural University}%


\vspace{-1em}



  
  \date{May 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Nucleotide-binding, leucine-rich repeat (NLR) genes play a pivotal role
in shaping plant effector-triggered immunity in response to pathogen
invasions. However, the mechanisms governing the expression and behavior
of NLRs, particularly in the context of head-to-head NLR gene pairs, in
the presence of pathogens, remain uncovered. In this study, we dissected
the \emph{Pik-H4} promoter (P \emph{\textsubscript{Pik-H4}}) at the TATA
boxes and conducted an in-depth investigation into split promoter
activity using Agro-infiltration assays. The segments spanning 593-1232
bp and 2016-2492 bp (starting from -1 bp of
\emph{Pik\textsubscript{1}-H4}) within P \emph{\textsubscript{Pik-H4}}
emerged as core regions for expressing \emph{Pik\textsubscript{1}-H4}
and \emph{Pik\textsubscript{1}-H4} respectively. Nevertheless, merging
these two core fragments failed to recover the promoter activity in both
directions. Employing Gus staining, promoter activity assays and
qRT-PCR, we unveiled the co-expression of \emph{Pik\textsubscript{1}-H4}
and \emph{Pik\textsubscript{2}-H4} throughout the whole plant.
Additionally, in the presence of the rice blast fungus, their
co-amplification was observed in leaves and leaf sheaths. Strikingly,
\emph{Pik-H4} exhibited heightened expression within vascular bundles.
Moreover, perturbing the \emph{Pik\textsubscript{1}-H4} and
\emph{Pik\textsubscript{2}-H4} co-expression relationship via
overexpression in rice or \emph{Nicotiana} did not disrupt the immune
response. Upon infection, the singleton Pik \textsubscript{1}-H4
localized within vesicles, while Pik \textsubscript{2}-H4 predominantly
occupied the nucleus within leaf sheath cells. Transcriptome analysis
highlighted \emph{Pik-H4}-mediated resistance triggering a transcriptome
reprogramming between 12 and 24 hours post-inoculation. Notably,
overexpression of \emph{Pik\textsubscript{1}-H4} or
\emph{Pik\textsubscript{2}-H4} enriches various pathways compared to the
\emph{Pik-H4} Lijiangheituanxingu near-isogenic lines. In summary, these
findings unravel the intricate dynamics of co-expression and singular
functionality within NLR bidirectional gene pairs upon pathogen
invasion.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Plants defend against pathogens through effector-triggered immunity
(ETI) with Resistance (R) proteins recognition of Avirulence (Avr)
proteins. Most \emph{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(Ma\emph{et al.} , 2020; Martin \emph{et al.} , 2020; Zhao
\emph{et al.} , 2022). Rice R proteins Pi-ta and Pi54 also directly
recognize Avr-Pita and Avr-Pi54 respectively(Jia \emph{et al.} , 2000;
Ray \emph{et al.} , 2016). In the case of indirect recognition of
effectors, one of the most apparent mechanisms is \emph{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 \emph{et al.} , 2019). PBL2 acts as a ``decoy''(Wang
\emph{et al.} , 2015) and the ZAR1-RKS1-PBL2 resistosome exhibits a
plasma membrane localization, function as a Ca\textsuperscript{2+}
channel, and trigger HR(Bi \emph{et al.} , 2021). After interacting with
AVRRpm1 or AVRB, the phosphorylated RIN4 acts as a ``guard'', activating
the \emph{Arabidopsis} CNL RPM1 and resulting in cell death(Mackey
\emph{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(Xie\emph{et al.} , 2022).

The NLR pairs members play different roles in the integrative decoy
model(Cesari, Bernoux, \emph{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, \emph{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 \emph{et al.} , 2017).
In addition, \emph{PigmR} and \emph{PigmS} display low expression levels
in all tissues except that \emph{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.
In\emph{Arabidopsis} and rice, 5763 and 8742 genes are organized in BDG
architectures(Krom and Ramakrishna, 2008). Since sharing transcription
factor binding sites and \emph{cis} elements through BDPs, the BDGs are
strongly co-regulated and participated in similar pathways(Williams and
Bowles, 2004). The \emph{Arabidopsis} TNL pairs RPS4/RRS1 and CHS1/SOC3
are organized in a BDP manner and spaced with a \textasciitilde{}300 bp
promoter region. The PRS4/RRS1 complex is necessary for recognizing the
AvrRps4 and Pop2(Williams \emph{et al.} , 2014). Expression
of\emph{SOC3} is promoted in the \emph{CHS1} mutant, suggesting that
self-regulation through the promoter region of \emph{CHS1}
/\emph{SOC3}and the CHS1/SOC3 complex is involved in autoimmunity(Zhang
\emph{et al.} , 2017). The only known NLR BDGs in rice are \emph{Pik}
genes, and 11 of them are reported. The HR assays of \emph{Pikh} (Zhai
\emph{et al.} , 2014) and \emph{Pikp} (Zdrza\selectlanguage{polish}ł\selectlanguage{english}ek \emph{et al.} , 2020a)
indicate that the two members of \emph{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 \emph{Pikh1} /\emph{Pikh} 2 and
the Nipponbare\emph{LOC\_Os11g46200} /\emph{LOC\_Os11g46210} are
apparently different. Consequently, the expression pattern and relation
of NLRs, especially the NLR pairs, are still unclear.

The CNL gene pair \emph{Pik-H4} is a synonymous mutation of
\emph{Pikh}and is resistant to rice blast fungus(Xiao \emph{et al.} ,
2011). A homeodomain transcription factor OsBIHD1 is found to interact
with Pik\textsubscript{1}-H4 and is required to \emph{Pik-H4} induced
ETI through regulation of the brassinosteroid-ethylene pathway(Liu
\emph{et al.} , 2017). We report that the NLR gene
pair\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-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.
Overexpressing\emph{Pik\textsubscript{1}-H4} or
\emph{Pik\textsubscript{2}-H4}promotes the transcription levels of the
counterpart \emph{Pik-H4} and does not affect the resistance to rice
blast. Singleton Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4
display vesicles and nucleus location respectively during infection,
pathway analysis shows that \emph{Pik-H4} mediated immunity is involved
in transcription reprogramming and \emph{Pik-H4} singleton functions in
various pathways.

\textbf{Results}

\textbf{The characterization of \emph{Pik-H4} promoter}

The \emph{Pik} genes, such as \emph{Pik-H4} (Figure 1A), consist of two
adjacent and H2H NLRs sharing a promoter region. The promoter region
of\emph{Pik-H4} (P\emph{\textsubscript{Pik-H4}} ) was cloned and aligned
to that of \emph{Pik} -\emph{Nip} and \emph{Pikh} (Figure S1). The
sequence similarity of P\emph{\textsubscript{Pik-H4}} (2492 bp) and
P\emph{\textsubscript{Pikh}} (2542 bp) is 97.44\%, and the major
sequence differences are located between 1010 bp to 1400 bp of
P\emph{\textsubscript{Pik-H4}} , which results in lacking 8 TFBSs
compared to P\emph{\textsubscript{Pikh}} .
P\emph{\textsubscript{Pik-H4}}and P\emph{\textsubscript{Pik-Nip}} (1099
bp) only share 25.41\% similarity, and the promoter sequences of the
\emph{Pik-1} direction are highly dissimilar. Interestingly, there are
two types of promoter length of available promoter sequences of reported
\emph{Pik} genes.\emph{Pik\textsuperscript{*}} , \emph{Pi1} ,
\emph{Pik-h} , and\emph{Pik-m} promoter regions are 2542 bp in length,
while those of\emph{Pi7} , \emph{Pik-H4} , and \emph{Pikp} are
approximately 2492 bp. All these promoters are highly conserved even
though these \emph{Pik}genes were cloned from different donors (Table
S1). Then, the functional motifs of P\emph{\textsubscript{Pik-H4}} were
detected (Figure 1B). Ten putative TATA boxes were predicted in
P\emph{\textsubscript{Pik-H4}} , and 3 of them were bidirectional TATA
boxes. The bidirectional TATA box (5'-TATATAT-3') confers bidirectional
transcriptional activity and is highly active (Xu et al., 1991).
Therefore, the bidirectional TATA box may enable the
P\emph{\textsubscript{Pik-H4}} transcript flanking genes. Besides, TFBSs
of well-known immunity-related TFs (ERF, WRKY and Dof) were also found
in P\emph{\textsubscript{Pik-H4}} , indicating the pathogen-induced
possibility of \emph{Pik-H4} .

\textbf{Functional analysis of \emph{Pik-H4} promoter}

To identify the crucial region of P\emph{\textsubscript{Pik-H4}} ,
P\emph{\textsubscript{Pik-H4}} was fused with
\emph{RFP}(\emph{Pik\textsubscript{1}-H4} direction) and
\emph{GFP}(\emph{Pik\textsubscript{2}-H4} direction) for further study
(Figure S2A). These constructs were subsequently introduced into rice
protoplasts and tobacco mesophyll cells via plasmid transformation, and
the bidirectional promoter activity was observed in both cell types
(Figure S2B). Interestingly, the fluorescence intensities of RFP and GFP
exhibited synchronized trends and intensities (Figure S2C).
Consequently, we focused on conducting promoter activity assays in
tobacco mesophyll cells for further investigations.

Given the essential role of the TATA box in eukaryotic RNA polymerase
function and transcription specificity, the
P\emph{\textsubscript{Pik-H4}} was split according to the position of
TATA boxes. In the \emph{Pik\textsubscript{1}-H4} direction, the
recovery of RFP expression to native promoter (NP) levels occurred until
the 7\textsuperscript{th} TATA box (1-7T) was included (Figure S2D),
indicating the criticality of the 593-1232 bp region
for\emph{Pik\textsubscript{1}-H4} transcription. As the following
promoter fragments were added, the promoter activity
towards\emph{Pik\textsubscript{1}-H4} direction decreased, suggesting
that the\emph{cis} elements in the following region might negatively
regulate\emph{Pik\textsubscript{1}-H4} expression. Interestingly, all
split promoters with no transcription start site (TSS) drove GFP
expression at levels similar to the NP.

In the direction of \emph{Pik\textsubscript{2}-H4} , the 2-1T sequence
(2016-2492 bp) was sufficient to restore the
P\emph{\textsubscript{Pik-H4}} activity
towards\emph{Pik\textsubscript{2}-H4} direction (Figure S2I). The region
spanning from the 5\textsuperscript{th} to the
10\textsuperscript{th}TATA box upstream \emph{Pik\textsubscript{2}-H4}
enhanced the GFP expression, suggesting a positive regulatory role
in\emph{Pik\textsubscript{2}-H4} transcription. Notably, this region
overlapped with the critical sequence of \emph{Pik\textsubscript{1}-H4}
, establishing P\emph{\textsubscript{Pik-H4}} as a bidirectional
promoter configuration. In summary, the 593-1232 bp and 2016-2492 bp of
P\emph{\textsubscript{Pik-H4}} were core regions
for\emph{Pik\textsubscript{1}-H4} and
\emph{Pik\textsubscript{2}-H4}expression respectively.

Considering the bidirectional manner and examination of the core
promoter region of P\emph{\textsubscript{Pik-H4}} , a series of
synthetic bidirectional promoters were constructed based on
P\emph{\textsubscript{Pik-H4}} . Surprisingly, while B1, B2, and B4
promoters failed to restore GFP expression levels (Figure 2C), the B3
promoter, comprising the core promoter regions of
both\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4} ,
augmented the GFP signal. Our findings underscore the complexity of
regulatory interactions among distinct \emph{cis} elements within the
promoter region, particularly when integrating two directional core
regions.

A symmetric integrated TATA box, comprising two bidirectional TATA
boxes, was identified within P\emph{\textsubscript{Pik-H4}} , prompting
a functional analysis. Firstly, the 100 bp fragment including three
bidirectional TATA boxes (Figure 2B, BT1) conferred promoter activity in
both orientations and this transcriptional activity was recovered to NP
levels in the presence of TSSs of the downstream genes (BT2). Notably,
the loss of function of the integrated bidirectional TATA box by
mutating it from 5'-TATATATA-3' to 5'-AAAAAAAA-3' (M1) significantly
compromised promoter activity in both directions, while the normal
bidirectional TATA box (M2, M3) had comparatively milder effects on
bidirectional promoter activity. Modifying the regular TATA box into the
integrated TATA box (M4, M5) demonstrated that the promoter activity
towards the \emph{Pik\textsubscript{2}-H4} direction increased as the
integrated TATA box shifted, while the activity of the opposite
orientation remained intact. This suggests that the function of the
integrated bidirectional TATA box in the
\emph{Pik\textsubscript{2}-H4}direction may be position-dependent within
the \emph{Pik-H4} promoter region. Our findings were consistent with a
previous report that the symmetry 5'-TATATATA-3' is highly active in
both directions(Xu, Thali and Schaffner, 1991).

\emph{Pik\textsubscript{1}-H4} \textbf{and\emph{Pik\textsubscript{2}-H4}
co-express \emph{in planta}}

The expression patterns of BDGs can be classified as co-regulated,
anti-regulated or with one direction exclusively
regulated(Trinklein\emph{et al.} , 2004). Given the cooperative function
of\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4} , it
is logical to infer that the \emph{Pik-H4} gene pair is co-regulated. To
validate this hypothesis, P\emph{\textsubscript{Pik1-H4}} ::\emph{GUS}
/Nip, P\emph{\textsubscript{Pik2-H4}} ::\emph{GUS} /Nip and\emph{RFP}
::P\emph{\textsubscript{Pik-H4}} ::\emph{GFP} /Nip plants were generated
to examine tissue specificity and promoter activity of\emph{Pik-H4} . As
shown in Figure 3A and Figure S3A, P\emph{\textsubscript{Pik-H4}}
displayed activity in both directions in roots, stems, leaf sheaths,
leaves, spikelets, and seeds in both GUS staining assay and fluorescent
plants, exhibiting constitutive expression patterns. Detecting the
fluorescence intensity of RFP and GFP and relative expression levels of
\emph{Pik-H4} in \emph{Pik-H4}Lijiangheituanxingu (LTH) near-isogenic
lines (NILs, hereafter,\emph{Pik-H4} NILs) showed similar expression
trends of \emph{Pik-H4}(Figure 3B, 3D). In accordance with the tobacco
filtration assay, the activity of P\emph{\textsubscript{Pik1-H4}} was
stronger than P\emph{\textsubscript{Pik2-H4}} . Employing the Pearson's
correlation test, the fluorescence intensity, representing the
transcription levels of \emph{Pik\textsubscript{1}-H4} and
\emph{Pik\textsubscript{2}-H4} , demonstrated a significantly positive
co-regulation \emph{in planta}(Figure 3C) and across all tissues (Figure
S3B-G). In addition, RFP and GFP signals were particularly robust within
vascular bundles in roots, stems, leaf sheaths, and leaves, revealing
high P\emph{\textsubscript{Pik-H4}} activity in vascular bundles
compared to mesophyll (Figure 3E). Zooming into the cellular level
(Figure 3F), the\emph{Pik-H4} expression patterns were also measured.
Since the leaf sheaths from rice seedlings are thin enough for laser
confocal microscopy, we used leaf sheaths for further research. The
transcription activity of P\emph{\textsubscript{Pik-H4}} in both
directions exhibited a linear correlation in both mesophyll cells
(Figure 3G) and vascular cells (Figure 3H) in leaf sheaths. Importantly,
the fluorescence intensity of P\emph{\textsubscript{Pik-H4}} was higher
in vascular cells (Figure 3I), aligning with the observed trends at the
tissue level.

\textbf{Rice blast fungus up-regulates the expression of \emph{Pik-H4}}

As a resistant gene pair, \emph{Pik\textsubscript{1}-H4} collaborates
with \emph{Pik\textsubscript{2}-H4} to participate in rice blast
immunity. The presence of W-boxes within the
P\emph{\textsubscript{Pik-H4}} , which has been documented to confer
promoter responsiveness to fungal or bacterial infections(In \emph{et
al.} , 2020), suggests a potential pathogen-induced expression mechanism
for \emph{Pik-H4} . To identify whether \emph{Pik-H4} responds to rice
blast fungus, the \emph{Pik-H4} NILs leaves were spray inoculated with
rice blast fungus carrying AvrPik-E. We found that the \emph{Pik-H} 4
expression levels peaked at 24 hours post-inoculation (hpi) through
qRT-PCR analysis (Figure S4A, C), a pattern distinct from the mock
treatment (Figure S4B). Notably, this peak coincided with the onset
of\emph{M. oryzae} invasion, wherein the penetration peg of the fungus
invades rice cells around 24 hpi, followed by the subsequent spread of
invasive hyphae between cells(Yan and Talbot, 2016). The temporal
alignment of \emph{Pik-H4} expression peak with \emph{M. oryzae}invasion
highlights its up-regulation at the initiation of ETI.

Since Nip is infectious to AvrPik-E, the\emph{RFP}
::P\emph{\textsubscript{Pik-H4}} ::\emph{GFP} /Nip plants were also
inoculated and observed \emph{in planta} . Visible lesion areas emerged
on 2 days post-inoculation (dpi) in leaves, and our observations
continued as these lesions evolved. As the lesions expanded, the leaf
mesophyll became increasingly transparent, enabling laser penetration.
As a result, fluorescence intensity data from 4 dpi onward was not
collected for this study. The P\emph{\textsubscript{Pik-H4}} activity
was measured from the lesion area to the peripheral area along the
dotted line in Figure 4A. Within the lesion area, the promoter activity
of \emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4} was
lower than the average levels but rose at the edge of the lesion (Figure
4B). The RFP signal was higher than the average levels of leaves from
the lesion edge to the lesion peripheral region while the GFP signal
dropped to the normal state. These findings suggest that \emph{Pik-H4}is
responsive to rice blast fungus and a putative signaling cascade
operates from the lesion area to its periphery. Similar trends were
observed at the lesion edge as the lesion expanded in the following days
post-inoculation (Data not shown). We observed the promoter activity
changes of \emph{M. oryzae} at the biotrophic and necrotrophic stages
within vascular bundles (Figure 4C). During the early stage of hypha
growth, \emph{Pik-H4} displayed no responsiveness (Figure 4D). However,
as the hyphae advanced along the vascular bundles,
P\emph{\textsubscript{Pik-H4}} activity exhibited up-regulation.
Conversely, at the distal end of lesions by 4 dpi, the transcription
levels of P\emph{\textsubscript{Pik-H4}} demonstrated no significant
difference compared to the mock treatment. Interestingly, the
fluorescence intensity of spreading hyphae showed no difference between
the hyphal region and its surroundings, while at the biotrophic stage,
the intensity was significantly higher than the peripheral area (Figure
4E). As for cell scale, the activity of P\emph{\textsubscript{Pik-H4}}in
both mesophyll cells and vascular cells markedly increased upon the
appearance of \emph{M. oryzae} (Figure 4F-I), although the linear
correlation between \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} promoter activity was lost (Figure 4J,
K). Notably, no discernible P\emph{\textsubscript{Pik-H4}} activity
difference was observed in cells harboring \emph{M. oryzae} spores or
hyphae and their adjacent cells (Figure S4G).

Given that the \emph{Pik-H4} gene pair demonstrated co-expression in
various tissues, rice blast was inoculated in leaf sheaths for
P\emph{\textsubscript{Pik-H4}} activity analysis. In \emph{Pik-H4}
NILs,\emph{Pik-H4} conferred rice blast resistance in leaf sheaths
(Figure S4D). P\emph{\textsubscript{Pik-H4}} exhibited responsiveness to
the rice blast infection, leading to the up-regulation of \emph{Pik-H4}
in leaf sheaths at 24 hpi in contrast to the mock treatment (Figure S4E,
F). Taken together, the \emph{Pik-H4} gene pair was rice blast
inducible.

\textbf{Disequilibration of \emph{Pik-H4} expression did not affect rice
blast resistance}

The \emph{Pik} gene pairs trigger HR together facing Avr and form a
complex (Zdrza\selectlanguage{polish}ł\selectlanguage{english}ek et al., 2020a). Considering the co-regulation of
the\emph{Pik-H4} gene pair via the BDP, we investigated whether the
disequilibration of \emph{Pik-H4} expression had any influence on rice
blast resistance. Remarkably, the overexpression of
either\emph{Pik\textsubscript{1}-H4} or \emph{Pik\textsubscript{2}-H4}
in LTH did not confer disease resistance (Figure 5A-C). Conversely,
within\emph{Pik-H4} NILs, overexpression of
either\emph{Pik\textsubscript{1}-H4} or
\emph{Pik\textsubscript{2}-H4}sustained rice blast resistance.
Interestingly, overexpression of either\emph{Pik-H4} in LTH led to an
enhancement in the expression of the other \emph{Pik} gene in LTH
(Figure 5D). The relative expression levels analysis showed
overexpressing one of the \emph{Pik-H4} in \emph{Pik-H4}NILs
up-regulated the counterpart (Figure 5E). This intriguing regulatory
relationship was successfully replicated in tobacco, yielding analogous
outcomes (Figure 5F). These findings collectively suggest that,
facilitated by the P\emph{\textsubscript{Pik-H4}} , the expression
levels of \emph{Pik-H4} are self- and co-regulated. To eliminate the
innate regulation of \emph{Pik-H4} in rice, agroinfiltrations of
different ratios of \emph{Pik-H4} with \emph{35S} promoter were
performed in tobacco (Figure 5G). At the protein level, combinations of
Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4 in 1:1, 1:2, or 2:1
ratios failed to induce HR in the absence of AvrPik-E. However, upon the
introduction of AvrPik-E, these combinations exhibited activity in ion
leakage assays, with similar conductivity levels recorded at 3 dpi.
These results suggested that altering the disequilibration
of\emph{Pik-H4} in protein levels did not compromise rice blast
resistance.

\textbf{Subcellular localization of Pik\textsubscript{1}-H4 and
Pik\textsubscript{2}-H4 \emph{in planta}}

As NLR pairs, Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4
collaborate to counter rice blast, invoking HR. In the previous study,
we found that \emph{Pik-H4} maintained some expression levels and was
blast-induced. To clarify how \emph{Pik-H4} changes between the resting
and active stages, our focus turned to observing the subcellular
localization of Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4
singletons or as a pair within leaf sheaths of LTH or \emph{Pik-H4} NILs
plants. Interestingly, Pik\textsubscript{1}-H4-GFP (Figure 6A, B) and
Pik\textsubscript{2}-H4-GFP (Figure 6E, F) exhibited varying subcellular
localization patterns across different cells.
Pik\textsubscript{1}-H4-GFP and Pik\textsubscript{2}-H4-GFP were
observed in the plasma membrane and cytoplasm, with the GFP signals
partially coinciding. In OE-\emph{Pik\textsubscript{1}-H4} -\emph{GFP}
/LTH plants, Pik\textsubscript{1}-H4-GFP localization extended to the
nucleus and vesicle-like particles (Figure 6C), while the
Pik\textsubscript{2}-H4-GFP singleton predominantly resided within the
nucleus. Upon \emph{M. oryzae} invasion of leaf sheaths,
Pik\textsubscript{1}-H4-GFP relocated to vesicles (Figure 6D), whereas
Pik\textsubscript{2}-H4-GFP translocated to the nucleus (Figure 6H).
Notably, as a sensor, Pik\textsubscript{1}-H4 singleton may be
transported towards \emph{M. oryzae} via vesicles (Figure S5A, B). Our
observations revealed Pik\textsubscript{1}-H4 within numerous minute
vesicles (Figure S5C-E) in cells neighboring blast-invaded or normal
cells. These findings suggest that Pik\textsubscript{1}-H4 plays a vital
role in recognizing Avr via vesicle transportation, possibly originating
from smaller particles in healthier cells and converging into larger
vesicles in the presence of rice blast fungus.

Upon overexpression of \emph{Pik\textsubscript{1}-H4}
or\emph{Pik\textsubscript{2}-H4} in \emph{Pik-H4} NILs, their
subcellular localization was similar to \emph{Pik-H4} singletons in the
absence of rice blast fungus (Figure 6I, K), with fewer instances of
Pik\textsubscript{1}-H4-GFP residing in vesicles. Given the resistance
of \emph{Pik-H4} NILs against rice blast, we did not find severe
invasion but only several cells with a single spore or short hypha
(Figure 6J, L). The appearance of \emph{M. oryzae} significantly
attenuated the Pik\textsubscript{1}-H4-GFP or
Pik\textsubscript{2}-H4-GFP and chlorophyll signals in cells inhabited
by the fungus and their neighboring cells, indicative of cell death.
Although the GFP signals were weak, the \emph{Pik-H4} were mainly found
in the membrane or cytoplasm. Interestingly, Pik\textsubscript{1}-H4 and
Pik\textsubscript{2}-H4 were found in chloroplasts with weak GFP signals
(Figure S6A-H) \emph{in planta} .

\textbf{Pathways analysis of \emph{Pik-H4} as singleton or pair}

To gain insights into the functional pathways governed by
\emph{Pik-H4}as either a singleton or a pair, we conducted transcriptome
analyses of LTH and \emph{Pik-H4} NILs exposed to \emph{M. oryzae} , as
well as of LTH overexpressing \emph{Pik\textsubscript{1}-H4}
or\emph{Pik\textsubscript{2}-H4} . Notably, as
both\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-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 \emph{Pik-H4} NILs mock and
LTH at corresponding hpi, \emph{P} \textless{}0.01,
\textbar{}log\textsubscript{2}Foldchange\textbar{}[?]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 \emph{M. oryzae} penetration typically occurs around 24
hpi (Li et al., 2023; Yan and Talbot, 2016), these findings suggest
that\emph{Pik-H4} -mediated resistance induces transcriptome
reprogramming between 12 and 24 hpi post \emph{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, \emph{PBZ1} (Kim
\emph{et al.} , 2008), \emph{OsNAC111} (Yokotani\emph{et al.} , 2014)
and chloroplast immunity genes \emph{LHCB5} (Liu\emph{et al.} , 2019)
and \emph{OsAPX8} (Jiang \emph{et al.} , 2016) exhibited positive
involvement in \emph{Pik-H4} -mediated immunity. In line with the
findings presented in Figure S6, these results collectively suggest that
\emph{Pik-H4} -mediated effector-triggered immunity (ETI) is closely
intertwined with chloroplast-related processes, indicating a potential
role for \emph{Pik-H4} in chloroplast immunity.

We extended our RNA-seq analysis to include
OE\emph{-Pik\textsubscript{1}-H4} /LTH and
OE\emph{-Pik\textsubscript{2}-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 \emph{Pik-H4} NILs,
OE\emph{-Pik\textsubscript{1}-H4} /LTH, and
OE\emph{-Pik\textsubscript{2}-H4-GFP} /LTH, respectively (Figure 7D).
Upon GO pathway enrichment analysis, overexpression
of\emph{Pik\textsubscript{1}-H4} or
\emph{Pik\textsubscript{2}-H4}resulted 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
\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4}were
co-expressed (Figure S9A), indicating the complex interplay
between\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-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 \emph{Pik-H4} in the quiescent stage. A detailed
analysis of immune-related genes unveiled that \emph{Pik-H4,} acting as
a singleton, reduced the expression of defense-related genes and
transcription factors (TFs, with the exception of \emph{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
by\emph{Pik-H4} .

\textbf{Discussion}

\textbf{The complexity in the long BDP mechanism}

The TATA box is a fundamental promoter element within eukaryotes, which
is crucial in initiating transcription for all three RNA polymerases.
The canonical TATA box sequence is typically recognized as
5'-TATAWAW-3'(Kwak \emph{et al.} , 2013). However, a bidirectional TATA
box (5'-TATATAT-3') has been identified to possess transcriptional
capabilities in both directions, demonstrating heightened
transcriptional activity(Xu, Thali and Schaffner, 1991). Analyzing split
tests for BDPs lacks specific guiding principles. In this study, we
employed the TATA box as a pivotal marker for segmenting the
P\emph{\textsubscript{Pik-H4}} . The TATA box sequence at 462-469 bp in
the \emph{Pik-H4} promoter is a bidirectional TATA box, comprising two
bidirectional TATA boxes situated on both the sense strand and the
antisense strand, respectively (Figure 2E). Mutation of this segment
resulted in a significant reduction in downstream gene expression on
both sides of the promoter. Examining the promoter activity of a 100 bp
sequence encompassing the integrated bidirectional TATA box, along with
an adjacent bidirectional TATA box, revealed that the sequence indeed
possessed bidirectional activity. Nevertheless, it could not fully
restore the transcriptional activity of
\emph{Pik\textsubscript{1}-H4}and \emph{Pik\textsubscript{2}-H4} in both
directions back to the levels of NP (Figure 2D-F, BT1). Moreover, the
recovery of expression intensity in downstream reporter genes was
observed after adding the TSSs for both genes (Figure 2D-F, BT2),
indicating that the 100 bp sequence primarily functions as a
bidirectional regulatory element, with TSSs being indispensable for gene
expression.

Transcriptional regulation is a complex process involving a multitude of
factors, including TFBSs, \emph{cis} elements, SNPs(Liu \emph{et al.} ,
2019), neighboring transcription units(Lee \emph{et al.} , 2014) and
various epigenetic modifications (Chen et al., 2014). In contrast to the
human genome, where approximately 67\% of BDPs are shorter than 300 bp
(Trinklein et al., 2004), plant BDPs tend to be longer (Krom and
Ramakrishna, 2008), introducing greater complexity to their regulatory
mechanisms. Some reports on split BDPs focus on segments within 1500 bp
(Banerjee et al., 2013; Rao and Virupapuram, 2021), and identify crucial
promoter regions that contribute to the basal expression of BDGs under
specific experimental conditions. However, these studies often overlook
the intricate \emph{cis} regulatory elements for gene regulation or
specificity. In our research, even by combining the minimal core
promoter regions of \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} , we could not achieve BDP activity
equivalent to NP levels (Figure 2A-C, B3). Intriguingly, this
combination even increased GFP expression, indicating the intricate
interplay of \emph{cis} elements within P\emph{\textsubscript{Pik-H4}} .
Given that \emph{Pik} BDPs encompass lengths of 2492 bp or 2542 bp,
further assays to split and analyze promoters are necessary to unravel
the underlying regulatory mechanisms of bidirectional NLR gene pairs.
Unveiling the functionality of BDPs holds significant implications for
synthetic biology, but it's evident that more extensive research is
needed to understand these processes comprehensively.

\textbf{NLR gene pair \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} co-express in a BDG manner}

NLR genes can be classified into singletons and pairs, depending on the
number of genes involved in plant immunity. The Arabidopsis TNL gene
pair \emph{RRS1} and \emph{SOC3} was reported to be organized in a BDG
manner(Zhang \emph{et al.} , 2017). It is well-known that the NLR gene
pairs in the \emph{Pik} locus are H2H genes. Despite this, there is
limited knowledge about regulating NLR gene expression. In this study,
we conducted a comprehensive analysis of the expression pattern
of\emph{Pik-H4} and found that under the regulatory control of
P\emph{\textsubscript{Pik-H4}} , \emph{Pik-H4} exhibited significantly
positive co-expression across various tissues. Furthermore, the
infection caused by the rice blast fungus induced its expression in
leaves and leaf sheaths. Based on these findings, we hypothesize
that\emph{Pik-H4} , functioning as a pair of resistance genes, maintains
a basal expression level throughout the plant during the resting phase.
This basal expression level acts as a surveillance system to detect the
presence of effector proteins promptly, thereby initiating the disease
resistance pathway upon pathogen invasion. Interestingly,
while\emph{Pikh-1} maintains constitutive transcription, \emph{Pikh-2}
is up-regulated in response to the rice blast fungus
challenge(Zhai\emph{et al.} , 2014). Identifying ten W-boxes in
P\emph{\textsubscript{Pik-H4}} (Figure 1A) highlights the significance
of the W-box, a binding site for WRKY TFs, in pathogen-induced
expression(In \emph{et al.} , 2020). Notably, the observed divergence in
expression between \emph{Pik-H4} and \emph{Pikh} might stem from
differences in their BDP sequences (Figure S1). Furthermore, our
investigation revealed robust promoter activity of \emph{Pik-H4} within
the vascular bundles of roots, stems, and leaf sheaths. One possible
explanation for this pattern is that the hyphae of \emph{M. oryzae} find
a more accessible propagation route through vascular bundles, thereby
necessitating heightened expression in these regions to provide a
specific defense response. As a sensor protein, Pik\textsubscript{1}-H4
exhibited an expression level approximately 0.6 times higher than that
of Pik\textsubscript{2}-H4, and a noticeable divergence in expression
was observed between the two at 24 hpi (Figure S4C). This finding was
consistent with the results of promoter activity analyses following
injection (Figure 4). Moreover, the appearance of rice blast fungus
disrupted the linear expression relationship of \emph{Pik-H4} (Figure
4J, K), suggesting that \emph{M. oryzae} might stimulate
\emph{Pik-H4}expression in invaded and peripheral cells. This phenomenon
prompts further exploration into the intricate mechanisms underlying
signal transduction and NLR expression, which is vital for gaining
deeper insights into the plant immune system's response to disease
challenges.

It is well known that NLRs play a crucial role in triggering ROS
production as part of the plant's defense response. For H2H NLR pairs,
Pikp and Pikh are essential for initiating HR when confronted with the
AvrPik effector(Zhai \emph{et al.} , 2014; Zdrza\selectlanguage{polish}ł\selectlanguage{english}ek \emph{et al.} ,
2020b). This mechanism contrasts with non-H2H NLR pairs like\emph{Pigm}
(Deng \emph{et al.} , 2017) and\emph{RGA4} /\emph{RGA5} (Cesari,
Kanzaki, \emph{et al.} , 2014), which function differently regarding HR
induction. We broke the expression relationship between
\emph{Pik\textsubscript{1}-H4} and\emph{Pik\textsubscript{2}-H4} under
the control of P\emph{\textsubscript{Pik-H4}} by overexpressing one of
them in\emph{Pik-H4} NILs and expressing different ratios of
\emph{Pik-H4} in tobacco, the rice blast resistance and conductivity in
ion leakage assays were not affected (Figure 5A, G). This intriguing
result suggests that the expression levels of \emph{Pik-H4} do not
significantly influence the functional integrity of
Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4 proteins involved in
immunity. It further indicates that the coordinated action of the
Pik\textsubscript{1}-H4/Pik\textsubscript{2}-H4/AvrPik-E unit is
sufficient to trigger HR, indicating the robustness of the plant's
immune response machinery.

\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4} are
organized in a BDG manner, functioning together to trigger rice blast
resistance. This collaborative action is facilitated by the
co-regulation of \emph{Pik-H4} through bidirectional promoters, which
are influenced by the presence of rice blast fungus \emph{in
planta.}Notably, a feedback regulatory mechanism exists
between\emph{Pik\textsubscript{1}-H4} and
\emph{Pik\textsubscript{2}-H4}(Figure 5E, F), further highlighting the
intricate nature of their coordinated function. The gene structure of
this BDG is well-suited to facilitate the controlled up-regulation of
\emph{Pik-H4} expression during immune responses, while also ensuring a
consistent baseline expression in the absence of pathogen infection.
This inherent readiness to counteract pathogens at any moment indicates
the BDG structure's adaptive advantage in enhancing the plant's overall
immune preparedness. Through the structure of BDG, NLR gene pairs
establish a direct transcriptional regulatory link, which offers a more
resource-efficient way to execute immune responses.

\textbf{Different roles of \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} as singletons and pair}

Most of the NLR proteins are localized in the cytoplasm and nucleus. For
instance, the \emph{Arabidopsis} RPS4(Heidrich \emph{et al.} , 2011) and
RRS1(Deslandes \emph{et al.} , 2003), rice Pikh-1/Pikh-2 pair are
localized in cytoplasm and nucleus(Zhai \emph{et al.} , 2014), while the
RGA4/RGA5 pair is cytoplasmic(Cesari, Kanzaki, \emph{et al.} , 2014).
Pik\textsubscript{1}-H4 and Pik\textsubscript{2}-H4 were observed to be
localized in the plasma membrane and chloroplasts \emph{in
planta}(Figure 6A, E, Figure S6). Combined with the top enriched GO
pathways at 24 hpi (Figure 7C), our results indicated that \emph{Pik}
genes might play roles in membrane and chloroplast immunity. Notably,
Pik\textsubscript{1}-H4 as a singleton was found in vesicles around rice
blast fungus, while Pik\textsubscript{2}-H4 singleton appeared in the
nuclei of peripheral cells surrounding the infected cells (Figure 6D,
H). The enrichment pathways of overexpression of singleton
\emph{Pik-H4}varied (Figure S9B, C). This suggests that the functions of
the\emph{Pik-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 \emph{Pik\textsubscript{1}-H4}
or\emph{Pik\textsubscript{2}-H4} promoted expression of ROS-related
genes\emph{OsRBOHB} , \emph{OsRAC1} , \emph{LHCB5} and \emph{OsAPX8}
(Figure 7F) but down-regulated defense response pathway (Figure S9B, C),
defense-related genes \emph{PR1a} , \emph{PR1b} , \emph{PR10}
and\emph{PBZ1} (Figure 7F), as well as multiple WRKY TFs (Table S2).
Interestingly, when \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} are coupled together, no suppression
of WRKY TFs was observed. Since both Pik\textsubscript{1}-H4 and
Pik\textsubscript{2}-H4 are localized in the nucleus, it implies
that\emph{Pik-H4} may play a role in transcriptional regulation.
Specifically, Pik\textsubscript{1}-H4 has been found to interact with
the TF OsBIHD1 and contribute to disease resistance(Liu \emph{et al.} ,
2017), although the precise function of Pik\textsubscript{2}-H4 in the
nucleus is yet to be fully understood. The GO enrichment changes between
12 hpi and 24 hpi imply that \emph{Pik-H4} functions in transcription
during ETI (Figure 7B, C). The findings indicate that
singleton\emph{Pik-H4} is susceptible based on transcriptome profile. It
is plausible that \emph{Pik-H4} singletons or the pair may function as
transcriptional cofactors, leading to transcriptional reprogramming in
the context of immune responses.

\textbf{Materials and methods}

\textbf{Motif analysis and sequence alignment}

The motif analysis for promoters was performed with
PlantRegMap(Tian\emph{et al.} , 2020). The promoter sequences
of\emph{Pik\textsuperscript{*}} (Zhai \emph{et al.} , 2011), \emph{Pi1}
(Hua\emph{et al.} , 2012), \emph{Pi7} (Campbell, Chen and Ronald,
2004),\emph{Pikh} (Zhai \emph{et al.} , 2014), \emph{Pikm} (Ashikawa
\emph{et al.} , 2008), and \emph{Pikp} (Zdrza\selectlanguage{polish}ł\selectlanguage{english}ek \emph{et al.} , 2020a)
were collected as reported. Sequence alignment was performed with
MAFFT(Rozewicki \emph{et al.} , 2019) and was visualized with ESPript
3(Robert and Gouet, 2014).

\textbf{Plasmid construction and rice transformation}

To evaluate the P\emph{\textsubscript{Pik-H4}} activity, the promoter
region of \emph{Pik-H4} was cloned from \emph{Pik-H4} NILs leaves with
CTAB extraction and was inserted into a binary vector pGR\emph{RFP}
::\emph{GFP} . Then, the pGR vector was introduced into Nipponbare via
\emph{Agrobacterium} -mediated (EHA105) transformation. For promoter
split and mutation assay, series plasmids were constructed with
multi-fragment homologous recombination PCR. For GUS staining,
P\emph{\textsubscript{Pik1-H4}} and P\emph{\textsubscript{Pik2-H4}} were
cloned to pCAMBIA1305.1 and induced into Nipponbare. The coding
sequences of \emph{Pik\textsubscript{1}-H4}
and\emph{Pik\textsubscript{2}-H4} were inserted into the
pOX-\emph{GFP}P\emph{\textsubscript{Ubi}} ::GFP vector and transformed
into LTH or\emph{Pik-H4} NILs. \emph{Pik\textsubscript{1}-H4}
,\emph{Pik\textsubscript{2}-H4} and \emph{AvrPik-E} coding sequences
were cloned to pEAQ(Sainsbury, Thuenemann and Lomonossoff, 2009) with
reconstruction: pEAQ-\emph{Flag}P\emph{\textsubscript{35S}}
::\emph{Flag} , pEAQ-\emph{HA}P\emph{\textsubscript{35S}} ::\emph{HA}
and pEAQ-\emph{Myc}P\emph{\textsubscript{35S}} ::\emph{Myc} respectively
for tobacco transient expression.

\textbf{Plant materials and growth conditions}

The rice cultivar Nip, rice blast susceptible cultivar LTH,\emph{Pik-H4}
NILs in LTH background were used for constructing transgenic plants and
inoculation assays. The\emph{RFP} ::\emph{P\textsubscript{Pik-H}4}
::\emph{GFP} /Nip, P\emph{\textsubscript{Pik1-H4}} ::GUS/Nip and
P\emph{\textsubscript{Pik2-H4}} ::GUS/Nip were used for expression
pattern analysis of \emph{Pik-H4} . The
OE-\emph{Pik\textsubscript{1}-H4} -\emph{GFP} /\emph{Pik} and
OE-\emph{Pik\textsubscript{2}-H4} -\emph{GFP} /\emph{Pik} were generated
by crossing OE-\emph{Pik\textsubscript{1}-H4} -\emph{GFP} /LTH and
OE-\emph{Pik\textsubscript{2}-H4} -\emph{GFP} /LTH with \emph{Pik-H4}
NILs respectively. Nip was also used for protoplast transformation to
identify bidirectional promoter activity. \emph{Nicotiana
benthamiana}was used for agroinfiltration to evaluate promoter activity
and HR assays.

Rice cultivars grew on soil in a greenhouse with a natural light cycle
and 30\selectlanguage{ngerman}°C/light, 26°C/dark. Tobacco seeds were germinated in MS medium
and transferred to the soil after germination in a growth chamber with a
16-h/8-h light/dark cycle at 24°C.

\emph{M. oryzae} \textbf{materials and inoculation assay}

The \emph{M. oryzae} isolate GDYJ7 harboring \emph{AvrPik-E} was used in
this study. Spores were produced by growing the hypha in a complete
medium with a 12-h/12-h light/dark cycle at 28°C for 10-14 days. Spray
inoculation was performed as previously reported (Bonman, 1986) with
modification to evaluate gene expression levels. Spores were collected
with 0.02\% Tween-20, and spore concentration was adjusted to
5×10\textsuperscript{5} spores/mL. The spore suspension was sprayed onto
the surface of two-week-old rice seedling leaves evenly until visible
droplets appeared with a spray gun. Samples were collected in 0, 6, 12,
24, 36, 48, 60, and 72 hpi after incubation in the dark for 12 h for
quantitative real-time PCR (qRT-PCR). For phenotyping, leaves of 6- to
8-week-old seedlings were punch inoculated as described(Park \emph{et
al.} , 2012). Disease symptom was observed at 7 dpi. The lesion length
was calculated using the software ImageJ. The sporulation rate on
lesions was determined as described(Park \emph{et al.} , 2012). Leaf
sheath inoculation was performed as described(Koga \emph{et al.} , 2004)
with modification. In brief, the leaf sheaths of 2-week-old seedlings
were carefully peeled. The spore suspension was adjusted to
5×10\textsuperscript{5} spores/mL and was injected with a syringe onto
the inner surface of leaf sheaths. The seedlings were laid on a 20 cm
plate with water and were grown with a 12-h/12-h light/dark cycle at
28°C for 7 days.

\textbf{Transient expression, promoter activity analysis and cell death
assay in \emph{N. benthamiana}}

Expression vectors (pGR\emph{RFP} ::P\emph{\textsubscript{Pik-H4}}
::\emph{GFP} and a series of pGR-based promoter split or mutation
vectors, pEAQ P\emph{\textsubscript{35S}}
::\emph{Pik\textsubscript{1}-H4} ::\emph{Flag} , pEAQ
P\emph{\textsubscript{35S}} ::\emph{Pik\textsubscript{2}-H4} ::\emph{HA}
, pEAQ P\emph{\textsubscript{35S}} ::\emph{AvrPik-E} ::\emph{Myc} ) were
transformed into \emph{Agrobacterium tumefaciens} GV3103. Each strain
was set to OD\textsubscript{600}=0.2 and incubated in an induction
buffer as described(Lapin \emph{et al.} , 2019) in the dark at room
temperature for 1 h. Then, the suspension was injected into 3- to
4-week-old tobacco leaves with a syringe. Data were collected after 2-3
days of infiltration. For promoter activity analysis, fifteen tobacco
mesophyll cells from three seedlings were imaged with a confocal
microscope (CarlZeiss LSM 750), and the fluorescence intensity was
measured via the software ZEN. The excitation/emission wavelengths were
488 nm/490-560 nm for GFP, 543 nm/580-660 nm for RFP. Five 8-10 mm
leaves with three biological replications were cut and put in tubes with
10 mL milliQ water for 30 min, then transferred to a new tube with 1 mL
milliQ water at room temperature. Conductivity was measured at 0 h and 6
h with a conductivity meter (Lei-ci DDBJ-350).

\textbf{Rice protoplast isolation and transformation}

Rice protoplast isolation from 1-week-old etiolated seedling sheaths and
PEG-mediated transformation were performed as described(Zhang \emph{et
al.} , 2011). After incubation in the dark for 12-16 h at room
temperature, the transformed rice protoplasts were observed with a
confocal microscope.

\textbf{RNA extraction and qRT-PCR}

Total RNA was extracted from inoculated leaves using TRIzol reagent
(Invitrogen) following the manufacturer's protocol. RNA samples were
reverse transcripted to cDNA (Vazyme HiScript III All-in-one RT SuperMix
Perfect for qPCR). The qRT-PCR reaction systems were prepared as
manufacturer's instruction (Vazyme AceQ Universal SYBR qPCR Master Mix)
using a Real-Time PCR System (Applied Biosystems StepOnePlus\selectlanguage{english}). The
2\selectlanguage{greek}\textsuperscript{-ΔΔCt}\selectlanguage{english} method was used to evaluate the gene expression
levels. The rice \selectlanguage{greek}\emph{β-actin}\selectlanguage{english} (\emph{LOC\_Os03g50885} ) was used as
the control gene and three biological repeats were performed for
qRT-PCR. The results were visualized with Prism (v8.0.1) or ChiPlot(Xie
\emph{et al.} , 2023).

\textbf{Transcriptome profiling and statistical analysis}

To elucidate the putative transcriptional function of \emph{Pik-H4} ,
transcriptome profiling was performed by RNA-seq. For pathways analysis
post-inoculation, three- to four-week-old LTH and \emph{Pik-H4} NILs
seedlings were spray inoculated with \emph{M. oryzae} isolate GDYJ7 as
previously described. Up to 0.1 g shoots were sampled at 0 (as mock
treatment), 12 and 24 hpi. The inoculation assay was estimated by LTH
phenotype 10 to 14 dpi. For singleton \emph{Pik-H4} pathways analysis,
three- to four-week-old OE-\emph{Pik\textsubscript{1}-H4} -\emph{GFP}
/\emph{Pik} , OE-\emph{Pik\textsubscript{2}-H4} -\emph{GFP} /\emph{Pik}
and\emph{Pik-H4} NILs shoots were sampled. Total RNA was extracted using
a Trizol reagent kit (Invitrogen) and the mRNA was enriched by the
Oligo(dT) beads. Then, the enriched mRNA was fragmented into short
fragments using fragmentation buffer and reversely transcribed into cDNA
using NEBNext Ultra RNA Library Prep Kit for Illumina (New England
Biolabs). The purified double-stranded cDNA fragments were end-repaired,
A base added, and ligated to Illumina sequencing adapters. The ligation
reaction was purified with the AMPure XP Beads(1.0X). And polymerase
chain reaction (PCR) amplified. The resulting cDNA library was sequenced
using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou,
China). Differentially expressed genes (DEGs) were identified
with\emph{P} \textless{}0.01,
\textbar{}log\textsubscript{2}Foldchange\textbar{}[?]2. Correlation
analysis was performed by R. Correlation of two parallel experiments
provides the evaluation of the reliability of experimental results and
operational stability. The correlation coefficient between two replicas
was calculated to evaluate repeatability between samples. GO enrichment
analysis was performed using TBtools (v1.120).

\textbf{ACKNOWLEDGEMENTS}

This work was supported by the
Natural~Science~Foundation~of~Guangdong~Province, China
(2019A1515011825, 2023A1515012101), the grant (CARS-01-18) from the
National Rice Industry Technology System, China, and the National Key
Research and Development Plan (2022YFD1200703), China.

\textbf{AUTHOR CONTRIBUTIONS}

F.W.G., J.F.W., H.W. and T.G. designed the experiments. F.W.G. analyzed
the data and wrote the manuscript. F.W.G., H.B.X., Q.W.H. and S.G.
performed the experiments. All authors read and approved of the
manuscript.

\textbf{DATA AVAILABILITY}

Raw RNA-seq data have been deposited in the Sequence Read Archive of
NCBI (\emph{https://www.ncbi.nlm.nih.gov/sra}) under the accession
PRJNA1013961.

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\textbf{Figure legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. The structure of the \emph{Pik-H4} gene and promoter region

(\textbf{A} ) The schematic structure of \emph{Pik1-H4}
and\emph{Pik\textsubscript{2}-H4} in rice chromosome 11. (\textbf{B} )
TATA boxes and defense-related TFBSs in P\emph{\textsubscript{Pik-H4}} .
The motif analysis for promoters was performed with PlantRegMap.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Promoter analysis of different
P\emph{\textsubscript{Pik-H4}}sequences

(\textbf{A} - \textbf{C} ) Minimal promoter region of
P\emph{\textsubscript{Pik-H4}} . The schematic of a combination of two
directions is shown in (\textbf{B} ). (\textbf{D} - \textbf{F} )
Mutation of bidirectional TATA boxes. The schematic is shown in
(\textbf{B} ). Data represent means \selectlanguage{ngerman}± standard deviation (n=10). The
\emph{RFP} and\emph{GFP} relative fluorescence intensity compared to the
native promoter was shown in (\textbf{G} ), (\textbf{I} ), (\textbf{G} )
and (\textbf{I} ). The lowercase letters (a, b, c) indicate the data
were at\emph{P} \textless{}0.01 level according to one-way ANOVA with
Dunnett's test. The dashed lines represent the value of the
empty\emph{RFP} ::\emph{GFP} plasmid.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3. \emph{Pik-H4} co-expressed in tissues and cells

(\textbf{A} ) The fluorescence microscopy of\emph{RFP}
::P\emph{\textsubscript{Pik-H4}} ::\emph{GFP} /Nip plants. The RFP and
GFP signals represent the promoter activity
of\emph{Pik\textsubscript{1}-H4} and
\emph{Pik\textsubscript{2}-H4}respectively. The letter ``m'' represents
mesophyll tissue, and ``vb'' represents vascular bundles. (\textbf{B} ,
\textbf{E} and \textbf{I} ) Measurement of fluorescence intensity.
(\textbf{B} and \textbf{E} ) were measured from (\textbf{A} ),
(\textbf{I} ) was measured from (\textbf{F} ). One-way ANOVA with
student's \emph{t} -test, n=30. (\textbf{C} ,\textbf{G} and \textbf{H} )
The linear regression of RFP and GFP signals. The F-test was employed to
compare statistical models applied to a dataset through linear
regression. (\textbf{C} ) was built from (\textbf{A} ), n=180.
(\textbf{G} and \textbf{H} ) were constructed from (\textbf{F} ), n=30.
(\textbf{D} ) Relative expression levels
of\emph{Pik\textsubscript{1}-H4} and \emph{Pik\textsubscript{2}-H4.}
Data represent means \selectlanguage{ngerman}± standard deviation (One-way ANOVA with
student's\emph{t} -test, n=3). (\textbf{F} ) The confocal fluorescence
microscopy. The letter ``mc'' represents mesophyll cell, and ``vc''
represents vascular cell.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4. P\emph{\textsubscript{Pik-H4}} activity was up-regulated after
infection in \emph{RFP} ::P\emph{\textsubscript{Pik-H4}} ::\emph{GFP}
/Nip plants

(\textbf{A} and \textbf{B} ) The promoter activity upon infection in
leaf tissue. The fluorescence intensity in (\textbf{B} ) was measured
along the dotted line from (\textbf{A} ). The red and green dashed lines
represent the average RFP and GFP intensity in a normal state in leaves.
The grey line was measured in the bright field and indicated the lesion
area when the values were lower. (\textbf{C} - \textbf{E} ) The promoter
activity upon infection in the leaf vascular bundle. (\textbf{D}
and\textbf{E} ) The relative fluorescence intensity compared to the mock
treatment from (\textbf{C} ). Data represent means \selectlanguage{ngerman}± standard deviation
(One-way ANOVA with student's \emph{t} -test, n=10). One asterisk
indicates \emph{P} \textless{}0.05, two asterisks indicate\emph{P}
\textless{}0.01. I, infected area. P, peripheral area of the infected
area. E, early stage of infection. B, biotrophic stage. (\textbf{F} -
\textbf{K} ) The confocal fluorescence microscopy in leaf sheaths.
(\textbf{F} , \textbf{G} and \textbf{J} ), mesophyll cell. (\textbf{H} ,
\textbf{I} and \textbf{K} ), vascular bundles. (\textbf{G}and \textbf{I}
), One-way ANOVA with student's \emph{t} -test, n=10. (\textbf{J} and
\textbf{K} ) The linear regression from (\textbf{F} and\textbf{H} ), The
F-test was employed to compare statistical models applied to a dataset
through linear regression, n=10.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5. Different \emph{Pik-H4} expressions facing the rice blast
fungus or effector

(\textbf{A} ) Punch inoculation in different plants
overexpressing\emph{Pik\textsubscript{1}-H4} or
\emph{Pik\textsubscript{2}-H4} . Bar=1 cm. (\textbf{B} and \textbf{C} )
Measurement of lesion length and sporulation rate from (\textbf{A} ).
Data represent means \selectlanguage{ngerman}± standard deviation, One asterisk indicates
\emph{P} \textless{}0.05, two asterisks indicate \emph{P}
\textless{}0.01 (One-way ANOVA with student's\emph{t} -test, n=5).
(\textbf{D} and \textbf{E} ) The relative expression levels measured by
qRT-PCR of \emph{Pik} genes from uninfected plants in (\textbf{A} ).
Data represent means ± standard deviation, One asterisk indicates
\emph{P} \textless{}0.05, two asterisks indicate\emph{P} \textless{}0.01
(One-way ANOVA with student's \emph{t} -test, n=3). (\textbf{F} )
Relative fluorescence intensity compared to the\emph{RFP}
::P\emph{\textsubscript{Pik-H4}} ::\emph{GFP} (P) plasmid in tobacco
transient assays, n=10. 1, P\emph{\textsubscript{35S}}
::\emph{Pik\textsubscript{1}-H4} ::\emph{Flag} . 2,
P\emph{\textsubscript{35S}} ::\emph{Pik\textsubscript{2}-H4} ::\emph{Ha}
. A, P\emph{\textsubscript{35S}} ::\emph{AvrPik-E} ::\emph{Myb} . The
dashed line represents intensity value=1. (\textbf{G} ) Ion leakage
assay measuring \emph{Pik-H4} -mediated cell death in tobacco at 3 dpi.
The Agrobacteria expressing Pik\textsubscript{1}-H4-Flag and
Pik\textsubscript{2}-H4-HA were mixed in different volumes with or
without AvrPik-E-Myc. Two asterisks indicate \emph{P} \textless{}0.01
(One-way ANOVA with student's \emph{t} -test, n=5).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6. Subcellular location of Pik\textsubscript{1}-H4 and
Pik\textsubscript{2}-H4 in leaf sheaths \emph{in planta}

(\textbf{A} - \textbf{D} ) Overexpression
of\emph{Pik\textsubscript{1}-H4-GFP} in LTH. (\textbf{A} )
Pik\textsubscript{1}-H4-GFP was in the membrane and cytosol. (\textbf{B}
) Fluorescence intensity of Pik\textsubscript{1}-H4-GFP and membrane dye
FM4-64 along the dotted line in (\textbf{A} ). (\textbf{C} )
Pik\textsubscript{1}-H4-GFP was in the nucleus and vesicles. (\textbf{D}
) Pik\textsubscript{1}-H4-GFP was in vesicles when infected. (\textbf{E}
- \textbf{H} ) Overexpression of\emph{Pik\textsubscript{2}-H4-GFP} in
LTH. (\textbf{E} ) Pik\textsubscript{2}-H4-GFP was in the membrane and
cytosol. (\textbf{F} ) Fluorescence intensity of
Pik\textsubscript{2}-H4-GFP and membrane dye FM4-64 along the dotted
line in (\textbf{E} ). (\textbf{G} ) Pik\textsubscript{2}-H4-GFP was in
nucleus. (\textbf{H} ) Pik\textsubscript{2}-H4-GFP was in the nucleus
when infected. (\textbf{I} and \textbf{J} ) Overexpression
of\emph{Pik\textsubscript{1}-H4-GFP} in \emph{Pik-H4} NILs. (\textbf{I}
) Pik\textsubscript{1}-H4-GFP was in the membrane, cytosol, nucleus and
vesicles. (\textbf{K} and \textbf{L} ) Overexpression
of\emph{Pik\textsubscript{2}-H4-GFP} in \emph{Pik-H4} NILs. (\textbf{K}
) Pik\textsubscript{2}-H4-GFP was in the membrane, cytosol and nucleus.
(\textbf{J} and \textbf{L} ) Pik\textsubscript{1}-H4-GFP and
Pik\textsubscript{2}-H4-GFP signals were weakened when infected
with\emph{M. oryzae} in \emph{Pik-H4} NILs. FM4-64 was marked in red in
(\textbf{A} and \textbf{E} ). In (\textbf{C} , \textbf{D} , \textbf{G}
,\textbf{H} , \textbf{I} - \textbf{L} ), chlorophyll A was marked in red
and the nucleus was dyed with DAPI (blue). All the bars represent 10 \selectlanguage{greek}μ\selectlanguage{english}m.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 7. Transcriptome profiling analysis of different
\emph{Pik-H4}plants

(\textbf{A} ) Venn diagram of DEGs in \emph{Pik-H4} NILs after
inoculation (Filtered \emph{Pik-H4} NILs mock groups and corresponding
LTH treatment groups. For all transcriptome analysis,
log\textsubscript{2}Foldchange(FC)[?]2, \emph{P} \textless{}0.01).
(\textbf{B} and \textbf{C} ) GO pathway enrichments of DEGs in 12 and 24
hpi respectively. In (\textbf{B} , \textbf{C} and \textbf{E} ),
the\emph{x} axis indicates the gene proportions in each enriched
pathway; the numbers in the plots represent the \emph{P} values of each
pathway within all genes in the corresponding pathways; the
up-/down-regulated pathways are shaded in pink and light-blue; the
enrichment was clustered in three top GO groups and marked in different
colors (Molecular function, brown. Cellular component, purple-red.
Biological process, celadon). (\textbf{D} ) Venn diagram of DEGs in
\emph{Pik-H4} NILs, OE-\emph{Pik\textsubscript{1}-H4-GFP} /LTH and
OE-\emph{Pik\textsubscript{2}-H4-GFP} /LTH (Filtered LTH groups). The
set shaded in pink or light blue represents the intersection of up- or
down-regulated DEGs of \emph{Pik-H4} NILs,
OE-\emph{Pik\textsubscript{1}-H4-GFP} /LTH and
OE-\emph{Pik\textsubscript{2}-H4-GFP} /LTH. (\textbf{E} ) GO pathway
enrichments of DEGs in \emph{Pik-H4} NILs,
OE-\emph{Pik\textsubscript{1}-H4-GFP} /LTH and
OE-\emph{Pik\textsubscript{2}-H4-GFP} /LTH. (\textbf{F} ) Fold change
analysis of immune-related genes in different transcriptome profiles
(One asterisk indicates \emph{P} \textless{}0.05, two asterisks
indicate\emph{P} \textless{}0.01 ).

\textbf{Hosted file}

\verb`Supplementary Figure.docx`  available at \url{https://authorea.com/users/665126/articles/666541-co-expression-and-function-of-head-to-head-nlr-gene-pair-pik-h4}

\textbf{Hosted file}

\verb`Supplementary Table.docx`  available at \url{https://authorea.com/users/665126/articles/666541-co-expression-and-function-of-head-to-head-nlr-gene-pair-pik-h4}

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