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\begin{document}

\title{OsPHR2 modulates phosphate starvation-induced jasmonic acid response and
resistance to Xanthomonas oryzae pv. oryzae}



\author[1]{Yaze Kong}%
\author[1]{Gang Wang}%
\author[2]{Xian Chen}%
\author[1]{Linying li}%
\author[1]{Xueying Zhang}%
\author[1]{Sangtian Chen}%
\author[1]{Yuqing He}%
\author[1]{Gaojie Hong}%
\affil[1]{Zhejiang Academy of Agricultural Sciences}%
\affil[2]{Jiangsu Academy of Agricultural Sciences}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Phosphate (Pi) and jasmonic acid (JA) play critical roles in plant
growth and development. In particular, crosstalk between JA and Pi
starvation signaling has been reported to mediate insect herbivory
resistance in dicot plants. However, its roles and mechanism in
monocot-bacterial defense systems remain obscure. Here, we report that
Pi starvation in rice activates the JA signaling and enhances resistance
to Xanthomonas oryzae pv. oryzae (Xoo) infection. The direct regulation
of OsPHR2 on the OsMYC2 promoter was confirmed by yeast one-hybrid,
electrophoretic mobility shift, dual-luciferase, and chromatin
immunoprecipitation assays. Molecular analyses and infection studies
using OsPHR2-Ov1 and phr2 mutants further demonstrated that OsPHR2
enhances JA response and antibacterial resistance via transcriptional
regulation of OsMYC2 expression, indicating a positive role of
OsPHR2-OsMYC2 crosstalk in modulating the JA response and Xoo infection.
Genetic analysis and infection assays using myc2 mutants revealed that
Pi starvation-induced JA signaling activation and consequent Xoo
resistance depends on the regulation of OsMYC2. Together, these results
reveal a clear interlink between Pi starvation signaling and the JA
signaling in monocot plants, and provide new insight into how plants
balance growth and defense by integrating nutrient deficiency and
phytohormone signaling.%
\end{abstract}%



\sloppy


\textbf{OsPHR2 modulates phosphate starvation-induced jasmonic acid
response and resistance to \emph{Xanthomonas oryzae} pv. \emph{oryzae}}

Yaze Kong \textsuperscript{1\#}, Gang Wang\textsuperscript{1\#}, Xian
Chen\textsuperscript{2}, Linying Li\textsuperscript{1},Xueying
Zhang\textsuperscript{1}, Sangtian Chen\textsuperscript{1,3}, Yuqing
He\textsuperscript{1*}, Gaojie Hong\textsuperscript{1*}

\textsuperscript{1} State Key Laboratory for Managing Biotic and
Chemical Threats to the Quality and Safety of Agro-products, Institute
of Virology and Biotechnology, Zhejiang Academy of Agricultural
Sciences, 198 Shiqiao Road, Hangzhou 310021, China

\textsuperscript{2} Institute of Plant Protection, Jiangsu Key
Laboratory for Food Quality and Safety-State Key Laboratory Cultivation
Base of Ministry of Science and Technology, Jiangsu Academy of
Agricultural Sciences, Nanjing 210014, China

\textsuperscript{3} College of Life Sciences, Zhejiang Sci-Tech
University, Hangzhou 310018, China.

\textsuperscript{\#} These authors contributed equally to this work.

\textsuperscript{*} Authors for correspondence:

Gaojie Hong

Tel: 86-574-86419021

Email: gjhong@126.com

Yuqing He

Tel: 86-574-86419210

Email: yqhe\_123@126.com

\textbf{Running Title:}OsPHR2 modulates \emph{OsMYC2} -mediated JA
against \emph{Xoo} .

\textbf{Abstract}

Phosphate (Pi) and jasmonic acid (JA) play critical roles in plant
growth and development. In particular, crosstalk between JA and Pi
starvation signaling has been reported to mediate insect herbivory
resistance in dicot plants. However, its roles and mechanism in
monocot-bacterial defense systems remain obscure. Here, we report that
Pi starvation in rice activates the JA signaling and enhances resistance
to \emph{Xanthomonas oryzae} pv. \emph{oryzae}(\emph{Xoo} ) infection.
The direct regulation of OsPHR2 on the\emph{OsMYC2} promoter was
confirmed by yeast one-hybrid, electrophoretic mobility shift,
dual-luciferase, and chromatin immunoprecipitation assays. Molecular
analyses and infection studies using \emph{OsPHR2-Ov1} and \emph{phr2}
mutants further demonstrated that OsPHR2 enhances JA response and
antibacterial resistance via transcriptional regulation of \emph{OsMYC2}
expression, indicating a positive role of OsPHR2-\emph{OsMYC2} crosstalk
in modulating the JA response and \emph{Xoo} infection. Genetic analysis
and infection assays using \emph{myc2} mutants revealed that Pi
starvation-induced JA signaling activation and consequent \emph{Xoo}
resistance depends on the regulation of \emph{OsMYC2} . Together, these
results reveal a clear interlink between Pi starvation signaling and the
JA signaling in monocot plants, and provide new insight into how plants
balance growth and defense by integrating nutrient deficiency and
phytohormone signaling.

\textbf{Introduction}

Phosphorus is an essential macronutrient and indispensable element for
plant growth and development in both natural and agricultural ecosystems
(Cong\emph{et al.} , 2020). The plant-accessible form of inorganic
phosphate (Pi) is highly insoluble in soil and thus Pi starvation is one
of the most common nutrient deficiencies, resulting in loss of plant
productivity (Raghothama \& Karthikeyan, 1999; L\selectlanguage{ngerman}ópez-Bucio \emph{et al.}
, 2003; Neumann \& RÖmheld, 2012). Plants respond to Pi starvation
though reduction of primary root growth, formation of additional lateral
roots and root hairs, replacement of phospholipids by sulfolipids and
galactolipids, release and uptake of phosphatases from organic sources,
increased expression of Pi transporter genes, and accumulation of starch
and anthocyanins (Yuan \& Dong, 2008; He \emph{et al.} , 2021). In
recent decades, considerable progress has been made regarding the
components of the Pi signaling pathway that drives these responses
(Franco-Zorrilla\emph{et al.} , 2004; Wu \emph{et al.} , 2013; Crombez
\emph{et al.} , 2019). PHOSPHATE STARVATION RESPONSE proteins (PHRs) are
the key transcription factors governing Pi starvation response, which
they do through binding to a \emph{cis} -element PHR1 binding sequence
(P1BS, sequence GNATATNC) in the promoters of Pi starvation-induced
(PSI) genes (Rubio \emph{et al.} , 2001; Zhou \emph{et al.} , 2008;
Bustos \emph{et al.} , 2010; Ruan \emph{et al.} , 2016). The SPX protein
family, which is named after syg1 (suppressor of yeast gpa1), Pho81 (the
yeast cyclin-dependent kinase inhibitor), and XPR1 (the human xenotropic
and polytropic retrovirus receptor 1), negatively regulates Pi signaling
through interacting with PHRs and suppressing their transcriptional
activities (Lv \emph{et al.} , 2014; Puga \emph{et al.} , 2014;
Wang\emph{et al.} , 2014; Wild \emph{et al.} , 2016; Ruan \emph{et al.}
, 2017; Zhong \emph{et al.} , 2018; Ruan \emph{et al.} , 2019). In
addition, phytohormones have been reported to be involved in the
adaptation of plants to Pi starvation signaling under biotic and abiotic
stress (Niu\emph{et al.} , 2013; Baek \emph{et al.} , 2017). In
\emph{Arabidopsis}(\emph{Arabidopsis thaliana} ), Pi deficiency
activates jasmonic acid (JA) signaling and enhances herbivory resistance
(Khan \emph{et al.} , 2016). However, the functional and regulatory
mechanisms how Pi starvation activates the JA pathway, especially in
monocotyledonous plants such as rice, remain to be elucidated.

JA and its derivatives are lipid-derived hormones that regulate plant
growth and development, along with defenses against pests and pathogen
infections (Kramell \emph{et al.} , 2009; Aurélie \emph{et al.} , 2010;
Wasternack \& Hause, 2013; Vidhyasekaran, 2015; Chini \emph{et al.} ,
2016; Rohit \emph{et al.} , 2016; Howe \emph{et al.} , 2018). The main
enzymes involved in JA biosynthesis are lipoxygenase (LOX), allene oxide
synthase (AOS) and allene oxide cyclase (AOC) in plastids, and OPDA
reductase in peroxisomes (Wasternack \& Hause, 2013; Ruan, J \emph{et
al.} , 2019). MYC2, a bHLH transcription factor, serves as the key
regulatory hub of JA signaling (Kazan \& Manners, 2013). When JA levels
are low, the transcription activity of MYC2 is suppressed by
JASMONATE-ZIM DOMAIN (JAZ) proteins together with NOVEL INTERACTOR OF
JAZ (NINJA) and TOPLESS (Chini \emph{et al.} , 2007; Chini \emph{et al.}
, 2009; Pauwels \emph{et al.} , 2010). When JA is present, high levels
of JA-Ile lead to SCF\textsuperscript{COI1}-dependent ubiquitination and
degradation of JAZ proteins through the 26S proteasome, which in turn
activates the expression of MYC2-regulated JA-responsive genes
(Chini\emph{et al.} , 2007; Thines \emph{et al.} , 2007; Howe \emph{et
al.} , 2018). MYC2 additionally links JA and other signaling pathways
such as those associated with other phytohormones, light, secondary
metabolism, and circadian signaling (Kazan \& Manners, 2011; Hong
\emph{et al.} , 2012; Kazan \& Manners, 2013). MYC2 also mediates the
JA-dependent defense against herbivory and pathogen infection (Lorenzo
\emph{et al.} , 2004; Dombrecht \emph{et al.} , 2007; Zhai \emph{et al.}
, 2013; Vidhyasekaran, 2015; Uji \emph{et al.} , 2016; Du \emph{et al.}
, 2017). In rice, MYC2 has been reported to be involved in resistance
against\emph{Xanthomonas oryzae} pv. \emph{oryzae} (\emph{Xoo} ), the
causal agent of rice bacterial blight, devastating rice diseases
worldwide (Tao\emph{et al.} , 2009; Uji \emph{et al.} , 2016). Together
with the documented influence of Pi starvation on the JA pathway
in\emph{Arabidopsis} , these findings indicate a role for Pi starvation
in the JA-\emph{Xoo} interaction in rice.

In this work, we demonstrated that OsPHR2, the rice homolog of AtPHR1,
bound to the \emph{OsMYC2} promoter\emph{,} thus activated the
expression of \emph{OsMYC2} and promoted downstream MeJA production,
thereby enhanced the rice anti-\emph{Xoo} defense. Together, our
findings revealed that OsPHR2 modulates JA-induced resistance to
bacterial blight during Pi starvation via transcriptionally regulating
expression of \emph{OsMYC2} .

\textbf{Materials and methods}

\textbf{2.1 Plant material and growth conditions}

\emph{OsPHR2-Ov1} (an \emph{OsPHR2} -overexpressing transgenic
plant),\emph{phr2} (an OsPHR2 T-DNA insertion mutant), and their
corresponding wild-type \emph{O. sativa} L. \emph{japonica} Nipponbare
(NIP) are as described (Zhou \emph{et al.} , 2008; Guo \emph{et al.} ,
2015).\emph{OsMYC2} CRISPR/Cas9 mutants (designated as \emph{myc2} )
were generated on the NIP background. Germinated seeds were
hydroponically cultured in normal rice culture solutions (Yoshida
\emph{et al.} , 1976) and incubated in a growth chamber under a
12-h-light (30 °C)/12-h-darkness (25 °C) photoperiod with 200 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2}s\textsuperscript{-1} photon density and
\textasciitilde{}60\% humidity. For Pi starvation (-P) conditions, the
concentration of KH\textsubscript{2}PO\textsubscript{4} was 0 \selectlanguage{greek}μ\selectlanguage{english}M, with
phosphate buffer in the Pi-deficient medium replaced by equimolar
amounts of KCl (Khan et al., 2016); the normal solution with 200 \selectlanguage{greek}μ\selectlanguage{english}M
KH\textsubscript{2}PO\textsubscript{4} was considered as Pi-sufficient
(+P). The culture solution was adjusted to a pH of 5.4-5.6 using 1 M
KOH, and the nutrient solution was replaced every other day during
treatment.

\textbf{2.2 Pathogen inoculation}

The \emph{Xoo} strain of PXO99 (P6) was used for plant resistance assays
performed as described (Chen \emph{et al.} , 2018). Bacterial cells
cultured overnight were collected, washed, resuspended, and adjusted to
a final concentration of 1\selectlanguage{ngerman}×10\textsuperscript{6} colony-forming units
(CFU) ml\textsuperscript{--1} in sterilized water. Fully-opened fifth
leaf blades of rice plants were inoculated using the clipping
inoculation technique (Kauffman \emph{et al.} , 1973); leaves clipped by
dipping scissor tips in sterilized water were used as a
control.\emph{Xoo} growth in rice leaves was analyzed by counting
colony-forming units as described in the Ke et al. 2017 bio-protocol (Ke
\emph{et al.} , 2017). Disease was evaluated by measuring lesion length
(in cm); specifically, inoculated leaves were collected and the distance
from the tip to the leading edge of the grayish lesion was measured by
hardworking hands using a ruler at 14 dpi (Yang \& Bogdanove, 2013). The
ruler had a maximum range of 30 cm, and the smallest unit was 1 mm. The
mean lesion length obtained from 15 leaves was used for each treatment,
and three independent experiments were performed.

\textbf{2.3 Hormone measurement}

Plants were grown under +P/--P treatment for three days, after which
leaves were collected, ground in liquid nitrogen, and then used for
methyl jasmonate (MeJA) extraction and analysis as described previously
(Fu \emph{et al.} , 2012; He \emph{et al.} , 2017). Three biological
replicates were used, each of which consisted of at least ten pooled
plants.

\textbf{2.4 Total RNA extraction and RT-qPCR}

Total RNA from leaves was extracted using Trizol (Invitrogen, USA) in
accordance with the manufacturer's instructions. First-strand cDNA was
synthesized from total RNA (1 \selectlanguage{greek}μ\selectlanguage{english}g) using the HiScript II Q RT for qPCR
(+gDNA viper) kit (Vazyme, China). RT-qPCR was performed on a
QuantStudioTM 6 Flex Real-Time PCR System (Applied Biosystems,
Singapore) using a CHamQ SYBR RT-qPCR Master Mix kit (Vazyme, China),
following the supplier's protocol. Expression levels were normalized
against expression of the housekeeping gene \emph{OsUBQ5} and analyzed
by the comparative Ct method (2\textsuperscript{-[?][?]Ct} method) (Livak \&
Schmittgen, 2001). At least three biological replicate samples were
used. Differences were considered significant at \emph{P} \textless{}
0.05. The primers used in this study are listed in Table S1.

\textbf{2.5 Yeast one-hybrid assay}

For yeast one-hybrid (Y1H) assay, a fragment comprising 2000 bp of
the\emph{OsMYC2} promoter region (from -2000 to -1 relative to the start
codon) was amplified and cloned into the pAbAi vector. The ORF
of\emph{OsPHR2} was inserted into the pGADT7-Rec2 (AD) vector. These
constructs or the corresponding empty vectors were co-transformed into
the yeast strain Y1HGold and incubated at 30 \selectlanguage{ngerman}°C on SD medium lacking
Leu, then spotted on selective media containing 300 ng/ml Aureobasidin A
(AbA, Clontech). The primers used are provided in Table S1.

\textbf{2.6 Electrophoretic mobility shift assay}

The electrophoretic mobility shift assay (EMSA) was performed as
described (Hong \emph{et al.} , 2012; He \emph{et al.} , 2021).
Recombinant His-OsPHR2 proteins were induced and purified as described
(Liu \emph{et al.} , 2010; Lv \emph{et al.} , 2014). The promoter
fragment containing GNATATNC was labeled with biotin at its 5' end. The
LightShift Chemiluminescent EMSA Kit (Pierce, USA) was used to perform
the experiment according to the manufacturer's instructions.
Fluorescence was detected using enhanced chemiluminescence substrate
with the ChemiDoc\textsuperscript{TM} MP imaging system (Bio-Rad,).
Probe sequences and primers are listed in Table S1.

\textbf{2.7 Dual-luciferase assay}

The dual-luciferase (dual-LUC) assay was performed as described
(He\emph{et al} ., 2021). The 2 kb promoter region of \emph{OsMYC2} was
inserted into the pGreenII 0800-luciferase (LUC) vector as the reporter,
while OsPHR2-Flag and GFF-Flag were cloned into the pCAMBIA1300 vector
as effectors. Effectors and reporter were transformed
into\emph{Agrobacterium} \emph{tumefaciens} strain GV3101 and
co-infiltrated into \emph{N. benthamiana} leaves. At 44-48 h after
infiltration, 1 mM luciferin was sprayed onto the leaves, and the plants
were kept in the dark for 5 min. Afterwards, LUC luminescence was
captured using an imaging system for living plants (Lumazone PyLoN
2048B, USA). The primers used are listed in Table S1.

\textbf{2.8 ChIP-qPCR assay}

Chromatin immunoprecipitation (ChIP)-qPCR assay was performed as
described (Hong \emph{et al.} , 2012; He \emph{et al.} , 2021).
Four-week-old leaves from plants expressing \emph{OsPHR2-Ov1} c fused
with Flag-tag (Zhou \emph{et al} ., 2008) were subjected to
cross-linking for ChIP assays. Anti-Flag antibody was used for
precipitation and negative IgG antibody as control. The precipitated
cross-linked DNA was purified using \selectlanguage{english}\emph{EpiQuik} plant ChIP kits.
ChIP products were analyzed by RT-qPCR, and fold enrichment was
calculated as the ratio of Flag-antibody/IgG--antibody quantifications.
Error bars represent +-SE of three biological replicates. The
\emph{OsACTIN2} gene promoter was used as a reference and the
\emph{OsIPS1} gene promoter was set as positive control (Lv \emph{et al}
., 2014). The primers used are listed in Table S1.

\textbf{2.9 Primary root growth inhibition assay}

The MeJA-mediated root growth inhibition assay was performed as
described (Yang \emph{et al.} , 2012; He \emph{et al.} , 2020).
Sterilized seeds were incubated on one-half MS medium with 0.35\% agar
and supplemented with 0 or 20 \selectlanguage{greek}μ\selectlanguage{english}M MeJA (TCI), then incubated in a growth
chamber at 30 \selectlanguage{ngerman}°C with 8 h light followed by 25 °C with 16 h darkness.
Four days later, root lengths of seedlings were determined. For each
treatment, at least 20 seedlings for each plant line were treated and
measured. Two independent experiments were performed.

\textbf{2.10 Vector construction and plant transformation}

To generate an \emph{OsMYC2} CRISPR/Cas9 mutant, a 20-bp gene-specific
sequence pair (OsMYc2-CRISPR/Cas9-F: ggcaACGCGTTGTCGTCCGTCCAA and
OsMYc2-CRISPR/Cas9-R: aaacTTGGACGGACGACAACGCGT) was synthesized and
annealed to form oligo adaptors. Those adaptors were firstly cloned into
the entry vector pOs-sgRNA and then inserted into the gateway
destination vector pOs-Cas9 as described (Miao \emph{et al.} , 2013).
The CRISPR/Cas9 plasmids were next introduced into \emph{Agrobacterium
tumefaciens} strain EHA105 and then transformed into plants of NIP
background. Positive lines were confirmed by PCR followed with
sequencing. Within positive lines, those showing both a frame-shift
mutation and an absence of the T-DNA backbone based on PCR using primers
for hygromycin B phosphotransferase (Hygro-F: ATGAAAAAGCCTGAACTCACCGCG
and Hygro-R: TTGCCCTCGGACGAGTG- CTGG) were identified as homozygous
lines. The T3 progeny of homozygous lines were used for analyses. The
primers used are listed in Table S1.

\textbf{2.11 Statistical analysis}

Differences were analyzed using Student's \emph{t} -test when comparing
two variables and ANOVA with Fisher's least significant difference test
when comparing three or more conditions. A \emph{p} -value \textless{}
0.05 was considered statistically significant. All analyses were
performed using ORIGIN 8 software.

\textbf{3 Results}

\textbf{3.1 Pi starvation in rice confers resistance to bacterial
blight}

To explore the role of Pi starvation in rice during \emph{Xoo}infection,
5- to 5.5-leaf-stage NIP plants were pre-grown in hydroponic cultures
containing 0 and 200 \selectlanguage{greek}μ\selectlanguage{english}M Pi for three days, after which the leaves of
these plants were inoculated with \emph{Xoo} . Growth analysis showed
the bacterial population under Pi starvation treatment to be
significantly lower than that in the Pi-sufficient control at 9 to 15
days after inoculation (Figure 1a). At 14 days after
\emph{Xoo}inoculation, the mean lesion lengths in Pi-sufficient plants
(200 \selectlanguage{greek}μ\selectlanguage{english}M) and Pi-starved plants (0 \selectlanguage{greek}μ\selectlanguage{english}M) were consistently 8.33 cm and 5.18
cm, respectively (Figure 1b and c). The significant reductions in
bacterial population and mean lesion length observed in Pi-starved
plants indicate that Pi starvation could induce rice resistance to
\emph{Xoo} infection.

\textbf{3.2 Pi starvation activates JA signaling}

It has been reported that Pi starvation elevates the JA pathway
in\emph{Arabidopsis} , and furthermore that the JA pathway plays a
positive role in rice resistance to \emph{Xoo} (Yamada \emph{et al.} ,
2012; Khan\emph{et al.} , 2016); consequently, we hypothesized that the
JA pathway may be involved in Pi starvation-induced rice resistance to
\emph{Xoo} . To test our hypothesis, we firstly examined the expression
of JA responsive genes using RT-qPCR. As shown in Figure 2a and b,
transcript levels of genes involved in JA biosynthesis (\emph{OsLOX1}
,\emph{OsLOX2} and \emph{OsAOS2} ) (He \emph{et al.} , 2017), JA
signaling (\emph{OsMYC2} , \emph{OsJAmyb} ,\emph{OsJAZ2} ,
\emph{OsJAZ5} ,\emph{OsJAZ10} and \emph{OsJAZ12} ), and JA-responsive
pathogen-related (PR) genes (\emph{OsPR1a} , \emph{OsPR1b} ,
\emph{OsPR5} and \emph{OsPR9} ) (Agrawal \emph{et al.} , 2000a; Agrawal
\emph{et al.} , 2000b; Rakwal \& Komatsu, 2000; Deng \emph{et al.} ,
2012) were all significantly increased in the leaves of Pi-starved
plants relative to Pi-sufficient plants. To confirm whether activation
of JA-related gene expression led to elevated endogenous hormone
contents, we measured MeJA and JA-IIe concentrations in Pi-starved
plants over three days. The results showed higher MeJA content in
inoculated leaves of Pi-starved plants than in those of the mock
control, whereas no significant change of JA-Ile content, the bioactive
form in JA signaling, was detected between Pi-starved plants and to
Pi-sufficient plants (Figure 2c and S1a). Interestingly, the expression
of \emph{OsMYC2,} the key regulator in JA signaling, was also altered
upon Pi starvation (Figure 2a and b), gradually increasing in a
time-course manner during Pi starvation (Figure 2d). Together, these
results suggested that the activation of\emph{OsMYC2} expression by Pi
starvation, not the increase in JA-Ile content, was due to the
interaction of Pi starvation and JA signaling.

\textbf{3.3 \emph{OsMYC2} is a direct target of OsPHR2}

OsPHR2 and OsMYC2 are well-known as the central transcription regulators
in Pi starvation and JA signaling, respectively (Zhou \emph{et al.} ,
2008; Kazan \& Manners, 2013). Interestingly, bioinformatics analysis
identified three P1BS \emph{cis} -elements in the 2-kb promoter region
of\emph{OsMYC2} (Figure 3a). Thus, we first performed a yeast one-hybrid
(Y1H) assay to examine their interaction. The 2000 bp fragment
containing the P1BS elements was used as bait and cloned into a pAbAi
reporter vector, while a pGADT7-Rec2-OsPHR2 vector was used as prey.
Yeast cells co-transformed with bait and prey were grown on selective
media lacking Leu with or without AbA. The assay results demonstrated
that OsPHR2 indeed bound to the promoter region of the \emph{OsMYC2}gene
(Figure 3b).

We then performed EMSA using purified His-OsPHR2 fusion protein and
biotin-labeled probes (P3 and P4 in Figure 3a). OsPHR2 recombinant
protein specifically bound the biotin-labeled probes, and this binding
could be suppressed by unlabeled probes (Figure 3c). To test whether
OsPHR2 activated the \emph{OsMYC2} promoter, we performed transient
expression assays with dual effectors. Transient transfection assay
revealed OsPHR2-Flag to increase the expression of
\emph{ProOsMYC2-LUC}in \emph{N. benthamiana} leaves as compared with
GFP-Flag control (Figure 3d). Finally, to determine whether OsPHR2
directly binds the\emph{OsMYC2} promoter \emph{in vivo} , we performed
ChIP-qPCR with anti-Flag antibody on transgenic plants expressing
\emph{OsPHR2-Ov1}fused with Flag (Zhou \emph{et al.} , 2008). In this
assay, OsPHR2 displayed strong binding to the region containing
P1BS\emph{cis} -elements, but not to other regions in the
\emph{OsMYC2}promoter (Figure 3e). Together, these results suggest
OsPHR2 is directly targeted to the \emph{cis} -element in the
\emph{OsMYC2} promoter\emph{in vitro} and \emph{in vivo,} and
overexpression of OsPHR2 results in up-regulation of \emph{OsMYC2} .

\textbf{3.4 OsPHR2 confers JA signaling activation and \emph{Xoo}
resistance through regulating expression of \emph{OsMYC2}}

The data above clearly demonstrated that OsPHR2 activates the
transcription of \emph{OsMYC2} ; accordingly, we further dissected the
effects of OsPHR2 on the expression of \emph{OsMYC2} -mediated
JA-responsive genes and JA production \emph{in} \emph{planta} . We first
examined \emph{OsMYC2} expression in the presence or absence of Pi
in\emph{OsPHR2-Ov1} , \emph{phr2} and the corresponding wild-type NIP
plants using RT-qPCR. Under the Pi-sufficient condition, we found
that\emph{OsMYC2} was up-regulated in \emph{OsPHR2-Ov1} leaves but
down-regulated in \emph{phr2} plants compared with NIP plants (Figure
4a). Consistently, similar expression patterns of JA-responsive genes
were also observed in the leaves of the tested mutants (Figure 4b-h). In
addition, the Pi starvation-triggered induction of JA-responsive genes,
including \emph{OsMYC2} , over three days of growth on Pi-deficient
solution was enhanced in \emph{OsPHR2-Ov1} plant leaves but suppressed
in \emph{phr2} mutants relative to the Pi-sufficient condition (Figure
4a-h). These results indicate that OsPHR2 positively modulates the
expression of JA-responsive genes.

Furthermore, MeJA content was higher in \emph{OsPHR2-Ov1} plants but
lower in \emph{phr2} mutants compared with NIP control, whereas only
small changes was observed for JA-IIe (Figure 5a and S1b). We also
tested the resistance of each type to \emph{Xoo} infection. At 9 to 15
days after inoculation with virulent\emph{Xoo} , bacterial proliferation
was lower in \emph{OsPHR2-Ov1}plants but higher in \emph{phr2} plants
than in NIP control (Figure 5b). Likewise, blight lesion length was
significantly shorter in\emph{OsPHR2-Ov1} plants but longer in
\emph{phr2} mutants than in NIP plants (Figure 5c and d), indicating a
positive role of OsPHR2 during\emph{Xoo} infection in rice. Taking
together, our results indicated that the Pi starvation activated
\emph{OsMYC2} expression, which resulted in the induction of
MYC2-regulated JA-responsive genes and the increased MeJA production,
and thus promoted resistance to \emph{Xoo}infection in rice.

\textbf{3.5 Involvement of on \emph{OsMYC2} in Pi starvation-enhanced JA
signaling and \emph{Xoo} resistance}

To further discern whether \emph{OsMYC2} is involved in Pi
starvation-induced \emph{Xoo} resistance, we generated
\emph{OsMYC2}mutants using the CRISPR/Cas9 system. Two homozygous lines
(\emph{myc2-3} and \emph{myc2-6} ) were obtained and confirmed by
sequencing (Figure S2a). To investigate JA sensitivity, the\emph{OsMYC2}
CRISPR/Cas9 mutants and NIP plants were grown with added MeJA. As
expected, the transgenic lines displayed less sensitivity to MeJA than
the WT plants (Figure S2b and c), indicating \emph{OsMYC2} is required
for JA signaling in rice.

To investigate the role of \emph{OsMYC2} in Pi starvation signaling, we
first measured the expression of\emph{OsMYC2} -mediated JA response
genes in \emph{myc2} plants upon Pi starvation. As shown in Figure 6(a),
JA-responsive genes were significantly more highly expressed in
Pi-starved NIP plants than in the Pi-sufficient control. In\emph{myc2-3}
and \emph{myc2-6} plants, however, Pi deficit suppressed these Pi
starvation-inducible JA-responsive genes, except for\emph{OsMYC2} itself
(Figure 6a). Phytohormone measurement revealed that the production of
MeJA in response to Pi starvation was abolished in\emph{myc2} mutants
(Figure 6b). JA-IIe content was lower in \emph{myc2}mutants compared
with NIP control, and the level of JA-IIe was not changed upon Pi
starvation in both \emph{myc2} and NIP plants (Figure S1c). We also
performed \emph{Xoo} resistance tests on these transgenic lines grown
under Pi sufficiency or starvation. 9-15 days after inoculated with
\emph{Xoo} , we measured bacterial proliferation of plants grown in the
normal condition. The results showed that the sensitivity to \emph{Xoo}
in \emph{myc2-3} and\emph{myc2-6} plants was higher than that of in NIP
controls (Figure 6c). However, Pi starvation-treated \emph{myc2-3} and
\emph{myc2-6} plants showed proliferation comparable to that in the
control, whereas proliferation in Pi starvation-treated wild-type NIP
plants was significantly reduced (Figure 6c). Blight lesion length
displayed similar patterns as observed for bacterial population in
\emph{myc2-3} and \emph{myc2-6} plants compared with NIP controls under
both Pi-sufficient and -deficient conditions (Figure 6d and e),
indicating a role for \emph{OsMYC2} in the Pi starvation-enhanced
defense response. Thus, our results suggest that\emph{OsMYC2} is
necessary for Pi starvation-induced enhancement of the JA signaling and
defense response.

\textbf{4 Discussion}

Phosphorus and phytohormones play pivotal roles in regulating diverse
developmental and physiological processes of plants (Raghothama \&
Karthikeyan, 1999; Vidhyasekaran, 2015). Pi starvation signaling
crosstalks with hormone pathways to appropriately adapt Pi homeostasis
in response to changing environmental conditions (Yuan \& Dong, 2008;
Baek \emph{et al.} , 2017). Transcriptomic studies have revealed changes
in the expression of hormone-responsive genes under Pi starvation
in\emph{Arabidopsis} (Hammond \emph{et al.} , 2003; Misson \emph{et al.}
, 2005; Bustos \emph{et al.} , 2010; Woo \emph{et al.} , 2012) and in
rice (Wasaki \emph{et al.} , 2006; Li \emph{et al.} , 2010; Secco
\emph{et al.} , 2013). Pi starvation enhances plant sensitivity to auxin
through overexpression of the auxin receptor TIR1 and the polar
transport inhibitor BFA, with subsequent modification of the root system
(L\selectlanguage{ngerman}ópez-Bucio \emph{et al.} , 2000; Nacry \emph{et al.} , 2005;
Pérez-Torres \emph{et al.} , 2008). Pi starvation also suppresses
cytokinin levels via reducing expression of the cytokinin
receptor\emph{CRE1} , thus decreasing plant sensitivity to cytokinin and
root length (Martin \emph{et al.} , 2000; Franco-Zorrilla \emph{et al.}
, 2002; López-Bucio \emph{et al.} , 2002). Pi starvation additionally
suppresses gene expression of enzymes involved in GA metabolism and
increases accumulation of the negative regulator protein DELLA to
decrease the level of bioactive GA (Jiang \emph{et al.} , 2007). Pi
starvation also regulates the transport, synthesis, and catabolism of
abscisic acid during changes of the root system architecture (Jaschke
\emph{et al.} , 1997; Ciereszko \& Kleczkowski, 2006). Notably,
interaction between Pi starvation signaling and hormones is also
involved in plant defense systems; for example, Pi starvation has
recently been demonstrated to induce the JA pathway and enhance
resistance to insect herbivory in dicot \emph{Arabidopsis} (Khan
\emph{et al.} , 2016). Here, we demonstrated that adaptation to Pi
starvation in monocot rice resulted in enhanced bacterial resistance
through activation of the JA response (Figures. 1 and 2).

Specifically, we demonstrated that transcriptional regulation
of\emph{OsMYC2} by OsPHR2 was integral to Pi starvation-induced
promotion of the JA signaling. In Pi starvation signaling, PHRs are
known to regulate PSI genes via binding to P1BS elements (Rubio \emph{et
al.} , 2001; Zhou \emph{et al.} , 2008; Bustos \emph{et al.} ,
2010).\emph{AtPHR1} partially controls Pi deficiency-triggered induction
of JA signaling in \emph{Arabidopsis} (Khan \emph{et al.} , 2016), but
the molecular mechanism remains to be elucidated. Here, we identified
three P1BS \emph{cis} -elements in the 2-kb promoter region of
\emph{OsMYC2}and further confirmed that OsPHR2, the homolog of AtPHR1
and thus the central regulator in rice (Wu \emph{et al.} , 2013), was
directly targeted to the promoter of \emph{OsMYC2} both \emph{in vivo}
and\emph{in vitro} (Figure 3). Expression of \emph{OsMYC2} was enhanced
in\emph{OsPHR2} overexpression mutants but suppressed in
\emph{OsPHR2}T-DNA insertion mutants grown under normal condition
(Figure 4), further confirming expression of \emph{OsMYC2} as controlled
by OsPHR2. Moreover, both the expression patterns of JA-synthesis genes
and the basal MeJA level behaved in a similar manner as \emph{OsMYC2}
in\emph{OsPHR2-Ov1} and \emph{phr2} mutants (Figures 4 and 5); this is
consistent with the previous finding in \emph{Arabidopsis} that
MYC2/MYC3/MYC4 directly control the water spray-induced accumulation of
JA (Van Moerkercke \emph{et al.} , 2019). In this work, we additionally
demonstrated that in \emph{myc2} mutants, neither the expression of
JA-responsive genes nor MeJA production were altered in either the
presence or absence of Pi (Figure 6), suggesting that activation of the
JA signaling by Pi starvation depends on \emph{OsMYC2} . JA-IIe is the
bioactive form in JA signaling, we also noticed the JA-IIe was not
changed upon Pi starvation treatment or in \emph{OsPHR2} mutants (Figure
S1). However, evidences have shown that JA signaling is clearly
activated by repeated touching, wounding and oral secretion or
short-term exposure to gaseous NO\textsubscript{2} while JA-Ile levels
remained unchanged in \emph{Arabidopsis} (Chehab \emph{et al} . 2012;
Lange and Lange 2015; Bozorov et al., 2017; Mayer \emph{et al} . 2018),
supporting the notion that JA signaling can turn on without measurable
increase in JA-Ile (Thierry \emph{et al} ., 2019). Thus, we speculate
that the transcriptional regulation of \emph{OsMYC2} by OsPHR2 reveals
direct crosstalk between JA and Pi starvation signaling at the molecular
level.

Prior studies have revealed that activation of JA signaling plays a
positive role in \emph{Xoo} resistance in rice (Koeduka \emph{et al.} ,
2005; Tao \emph{et al.} , 2009; Deng \emph{et al.} , 2012; Yamada
\emph{et al.} , 2012; Uji \emph{et al.} , 2016; Ke \emph{et al.} , 2020;
Onohata \& Gomi, 2020). For example, overexpressing \emph{OsWRKY45}
and\emph{OsC3H12} enhanced \emph{Xoo} resistance, accumulation of JA,
and expression of JA signaling genes (Tao \emph{et al.} , 2009; Deng
\emph{et al.} , 2012). Exogenous application of JA also enhanced
resistance to bacterial blight, and this JA-induced \emph{Xoo}
resistance could be inhibited by overexpressing \selectlanguage{english}\emph{OsJAZ8[?]C} , which
lacks the Jas domain (Yamada \emph{et al} ., 2012) In the present work,
we observed Pi starvation to enhance resistance to \emph{Xoo} ,
accompanied by elevated expression of JA-responsive genes and
accumulation of MeJA (Figures 1 and 2). In addition, we found that
\emph{OsPHR2-Ov1} mutants (which have an activated JA signaling with
unchanged JA-IIe level) were more resistant while \emph{phr2} mutants
(which have a suppressed JA signaling) were more susceptible compared
with the wild-type NIP plants (Figures 4, 5 and S1). Nevertheless,
repetitive mechanical stimulation and NO\textsubscript{2} fumigation
enhances \emph{Arabidopsis}resistance to \emph{Botrytis cinerea} with
activated JA signaling in JA-IIe steady-stage levels (Chehab \emph{et
al} . 2012; Mayer \emph{et al} . 2018). Evidences also show that JA-IIe
is not required to activate JA mediated systemic defenses to herbivory
in \emph{Nicotiana attenuate}and \emph{Solanum nigrum} (Doorn et al.,
2011; Bozorov et al., 2017). Therefore, we speculated that the JA
signaling is involved in OsPHR2-mediated anti-\emph{Xoo} defense.
In\emph{Arabidopsis} , \emph{AtMYC2} and \emph{AtERF3} antagonistically
repress JA-induced pathogen defense genes, and thus \emph{myc2-2}mutants
show increased sensitivity to the necrotrophic pathogen\emph{Botrytis
cinerea} (Lorenzo \emph{et al.} , 2004; Zhai \emph{et al.} , 2013). In
tomato, however, \emph{MYC2} -silenced plants display enhanced
resistance to \emph{Botrytis cinerea} , as MYC2 and MTF ETHYLENE
RESPONSE FACTOR.C3 synergistically and preferentially modulate
pathogen-responsive genes (Du \emph{et al.} , 2017). In rice, transgenic
plants overexpressing \emph{OsMYC2} display a JA-hypersensitive
phenotype and are more resistant to \emph{Xoo} (Uji \emph{et al.} ,
2016); meanwhile, in \emph{OsMYC2} RNAi plants, the JA-inducible
expression of many defense-related genes and JA-dependent activation of
the biosynthetic pathways for specialized metabolites are both
compromised (Ogawa \emph{et al.} , 2017). Here, our data
revealed\emph{OsMYC2} CRISPR/Cas9 mutants to exhibit a JA-insensitive
phenotype and greater susceptibility to \emph{Xoo} infection (Figure 6).
In addition, the \emph{Xoo}susceptibility of \emph{myc2} lines was not
enhanced by Pi starvation (Figure 6), suggesting involvement of
\emph{OsMYC2} in Pi starvation-mediated \emph{Xoo} resistance.
Considering OsPHR2 physically binds to the \emph{OsMYC2} promoter to
regulate \emph{OsMYC2}expression, resulting in consequent activation of
the JA signaling (Figures. 3-5), we speculated that activation of the JA
response resulting from the transcriptional regulation of \emph{OsMYC2}
by OsPHR2 contributes, at least partially, to the bacterial defense
induced by Pi starvation.

During its growth and development, rice is confronted by simultaneous
nutrition deficiency and pathogen attack, such as Pi starvation (or low
Pi) and bacterial blight. Here, we found a positive effect of Pi
starvation on resistance to \emph{Xoo} . Together with the well-known
SPXs-PHR1 working model of the Pi starvation signaling pathway, we
propose a model illustrating that Pi starvation- and JA- signaling
function synergistically and positively to control rice resistance
to\emph{Xoo} infection (Figure 7). When grown under the Pi-sufficient
condition, OsSPXs interact with OsPHR2 with high binding affinity,
prevent its binding to the P1BS motifs in the promoter of \emph{OsMYC2}
, thus \emph{OsMYC2} is expressed at a solely basal level. Under the
Pi-starvation condition, weakened interaction of OsSPXs-PHR2 allows PHR2
to up-regulate \emph{OsMYC2} , thereby enhancing expression
of\emph{OsMYC2} and consequently activating the JA response and
JA-mediated antibacterial resistance. Our findings reveal a novel
mechanism for crosstalk between Pi-starvation signaling and the JA
pathway and its positive role in rice antibacterial immunity, and
provide new insight into how plants adjust the balance between growth
and defense by integrating nutrient supply and phytohormone signaling.

\textbf{Support information}

\textbf{Figure S1. Quantification of JA-IIe levels.}

\textbf{Figure S2. Identification of \emph{myc2} mutants.}

\textbf{Table S1. Primers used in this study.}

\textbf{Acknowledgements}

We are indebted to Chuanzao Mao (Zhejiang University) for
providing\emph{OsPHR2-Ov1,} \emph{phr2} , and pET29b-OsPHR2 vector. This
work was funded by National Natural Science Foundation of China
(31670291, 31800249), Zhejiang Provincial Natural Science Foundation of
China (LQ18C140004), State Key Laboratory for Managing Biotic and
Chemical Threats to the Quality and Safety of Agro-products
(2010DS700124-ZZ2017).

\textbf{Author Contribution}

Y.H. and G.H. conceived the project and designed the experiments; Y.K.
G.W and Y.H. carried out the experiments with assistance from X.C.,
L.L., and X.Z.; all authors analyzed and discussed the results; and Y.H.
and G.H. wrote the manuscript.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interest.

\textbf{References}

\textbf{Agrawal GK, Jwa N, Rakwal R. 2000a.} A novel rice \emph{(Oryza
sativa L} .) acidic \emph{OsPR1a} gene highly responsive to cut,
phytohormones, and protein phosphatase inhibitors. \emph{Biochemical and
Biophysical Research Communications} \textbf{274} (1): 157-165.

\textbf{Agrawal GK, Rakwal R, Jwa N. 2000b.} Rice (\emph{Oryza sativa
L.} ) \emph{OsPR1b} gene is phytohormonally regulated in close
interaction with light sgnals. \emph{Biochemical and Biophysical
Research Communications} \textbf{278} (2): 290-298.

\textbf{Aurelie G, Robin L, Farmer EE. 2010.}
\emph{Arabidopsis}jasmonate signaling pathway. \emph{Science Signaling}
\textbf{3} (109): cm4.

\textbf{Baek D, Chun HJ, Yun DJ, Min CK. 2017.} Cross-talk between
phosphate starvation and other environmental stress signaling pathways
in plants. \emph{Moleculer Cells} \textbf{40} (10): 697-705.

\textbf{Bozorov TA, Dinh ST, Baldwin IT. 2017.} JA but not JA-Ile is the
cell-nonautonomous signal activating JA mediated systemic defenses to
herbivory in Nicotiana attenuata. \emph{Journal of Integrative Plant
Biology} \textbf{59} (8): 552-571.

\textbf{Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Perez-Perez
J, Solano R, Leyva A, Paz-Ares J. 2010.} A central regulatory system
largely controls transcriptional activation and repression responses to
phosphate ptarvation in \emph{Arabidopsis} . \emph{Plos
Genetics}\textbf{6} (9): e1001102.

\textbf{Chehab EW, Yao C, Henderson Z, Kim S, Braam J.
2012.}\emph{Arabidopsis} touch-induced morphogenesis is jasmonate
mediated and protects against pests. \emph{Current Biology} \textbf{22}
(8): 701--706.

\textbf{Chen X, Sun C, Laborda P, He Y, Zhao Y, Li C, Liu F.
2018.}Melatonin treatments reduce the pathogenicity and inhibit the
growth of Xanthomonas oryzae pv. oryzicola. \emph{Plant Pathology}
\textbf{68} : 288-296.

\selectlanguage{english}\textbf{Chini A, Fonseca S, Chico JM, Fernandez-Calvo P, Solano R.
2009.} The ZIM domain mediates homo\selectlanguage{english}- and heteromeric interactions
between Arabidopsis JAZ proteins. \emph{Plant Journal} \textbf{59} (1):
77-87.

\textbf{Chini A, Fonseca S, Fernandez G, Adie B, Chico J, Lorenzo O,
GarciaCasado G, LopezVidriero I, Lozano F, Ponce M. 2007.} The JAZ
family of repressors is the missing link in jasmonate
signalling.\emph{Nature} \textbf{448} (7154): 666-671.

\textbf{Chini A, Gimenez-Ibanez S, Goossens A, Solano R.
2016.}Redundancy and specificity in jasmonate signalling. \emph{Current
Opinion in Plant Biology} \textbf{33} : 147-156.

\textbf{Ciereszko I, Kleczkowski LA. 2006.} Phosphate deficiency
dependent upregulation of UDP-glucose pyrophosphorylase genes is
insensitive to ABA and ethylene status in \emph{Arabidopsis}
leaves.\emph{Acta Physiologiae Plantarum} \textbf{28} (5): 387-393.

\textbf{Cong W, Suriyagoda L, Lambers H. 2020.} Tightening the
phosphorus cycle through phosphorus-efficient crop
genotypes.\emph{Trends in Plant Science} .

\textbf{Crombez H, Motte H, Beeckman T. 2019.} Tackling plant phosphate
starvation by the roots. \emph{Developmental Cell} \textbf{48} (5):
599-615.

\textbf{Deng H, Liu H, Li X, Xiao J, Wang S. 2012.} A CCCH-type zinc
finger nucleic acid-binding protein quantitatively confers resistance
against rice bacterial blight disease. \emph{Plant
Physiology}\textbf{158} (2): 876-889.

\textbf{Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid
JB, Fitt GP, Sewelam N, Schenk PM, Manners JM. 2007.} MYC2
differentially modulates diverse jasmonate-dependent functions
in\emph{Arabidopsis} . \emph{Plant Cell} \textbf{19} (7): 2225-2245.

\textbf{Doorn AV, Bonaventure G, Rogachev I, Aharoni A, Baldwin IT.
2011.} JA-Ile signaling in Solanum nigrum is not required for defense
responses in nature. 2011. \emph{Plant Cell \& Envroment}\textbf{34}
(12): 2159-2171.

\textbf{Du M, Zhao J, Tzeng DTW, Liu Y, Li C. 2017.} MYC2 orchestrates a
hierarchical transcriptional cascade that regulates Jasmonate-mediated
plant immunity in tomato. \emph{Plant Cell} \textbf{29} (8): 1883--1906.

\textbf{Franco-Zorrilla JM, Gonzalez E, Bustos R, Linhares F, Leyva A,
Paz-Ares J. 2004.} The transcriptional control of plant responses to
phosphate limitation. \emph{J Exp Bot} \textbf{55} (396): 285-293.

\textbf{Franco-Zorrilla JM, Martin AC, Solano R, Rubio V, Paz-Ares J.
2002.} Mutations at CRE1 impair cytokinin-induced repression of
phosphate starvation responses in \emph{Arabidopsis} . \emph{Plant
Journal} \textbf{32} (3): 353-360.

\textbf{Fu J, Chu J, Sun X, Wang J, Yan C. 2012.} Simple, rapid, and
simultaneous assay of multiple carboxyl containing phytohormones in
wounded tomatoes by UPLC-MS/MS using single SPE purification and isotope
dilution. \emph{Analytical Sciences} \textbf{28} (11): 1081-1087.

\textbf{Guo M, Ruan W, Li C, Huang F, Zeng M, Liu Y, Yu Y, Ding X, Wu Y,
Wu Z. 2015.} Integrative comparison of the role of the PHOSPHATE
RESPONSE1 subfamily in phosphate signaling and homeostasis in
rice.\emph{Plant Physiology} \textbf{168} (4): 1762-1776.

\textbf{Hammond JP, Bennett MJ, Bowen HC, Broadley MR, Eastwood DC, May
ST, Rahn C, Swarup R, Woolaway KE, White PJ. 2003.} Changes in gene
expression in \emph{Arabidopsis} shoots during phosphate starvation and
the potential for developing smart plants. \emph{Plant
Physiology}\textbf{132} (2): 578-596.

\textbf{He Y, Hong G, Zhang H, Tan X, Li L, Kong Y, Sang T, Xie K, Wei
J, Li J, et al. 2020.} The OsGSK2 Kinase Integrates Brassinosteroid and
Jasmonic Acid Signaling by Interacting with OsJAZ4. \emph{The Plant
Cell} \textbf{32} (9): 2806-2822.

\textbf{He Y, Zhang H, Sun Z, Li J, Hong G, Zhu Q, Zhou X, Macfarlane S,
Yan F, Chen J. 2017.} Jasmonic acid\selectlanguage{english}-mediated defense suppresses
brassinosteroid-mediated susceptibility to Rice black streaked dwarf
virus infection in rice. \emph{New Phytologist} \textbf{214} (1):
388-399.

\textbf{He Y, Zhang X, Li L, Sun Z, Li J, Chen X, Hong G. 2021.} SPX4
interacts with both PHR1 and PAP1 to regulate critical steps in
phosphorus-status-dependent anthocyanin biosynthesis. \emph{New
Phytologist} \textbf{230} (1): 2015-217.

\textbf{Hong G-J, Xue X-Y, Mao Y-B, Wang L-J, Chen X-Y.
2012.}\emph{Arabidopsis} MYC2 interacts with DELLA proteins in
regulating sesquiterpene synthase gene expression. \emph{Plant
Cell}\textbf{24} (6): 2635-2648.

\textbf{Howe GA, Major IT, Koo AJ. 2018.} Modularity in jasmonate
signaling for multistress resilience. \emph{Annual Review of Plant
Biology} \textbf{69} (1): annurev-arplant-042817-040047.

\textbf{Jaschke WD, Peuke AD, Pate JS, Hartung W. 1997.} Transport,
synthesis and catabolism of abscisic acid (ABA) in intact plants of
castor bean (\emph{Ricinus communis} L.) under phosphate deficiency and
moderate salinity. \emph{Journal of Experimental Botany} \textbf{48}
(9): 1737-1747.

\textbf{Jiang C, Gao X, Liao L, Harberd NP, Fu X. 2007.} Phosphate
starvation root architecture and anthocyanin cccumulation responses are
modulated by the gibberellin-DELLA signaling pathway
in\emph{Arabidopsis} . \emph{Plant Physiology} \textbf{145} (4):
1460-1470.

\textbf{Kauffman HE, Reddy APK, Hsieh SPY, Merca SD. 1973.} An improved
technique for evaluating resistance of rice varieties to Xanthomonas
oryzae. \emph{Plant disease reporter} \textbf{57} (6): 537-541.

\textbf{Kazan K, Manners JM. 2011.} The interplay between light and
jasmonate signalling during defence and development. \emph{Journal of
Experimental Botany} \textbf{62} (12): 4087-4100.

\textbf{Kazan K, Manners JM. 2013.} MYC2: The master in
action.\emph{Molecular Plant} \textbf{6} (3): 686-703.

\textbf{Ke Y, Hui S, Yuan M. 2017.} \emph{Xanthomonas oryzae}
pv.\emph{oryzae} inoculation and growth rate on rice by leaf clipping
method. \emph{Bio-Protocol} \textbf{7} (19).

\selectlanguage{english}\textbf{Ke Y, Yuan M, Liu H, Hui S, Qin X, Chen J, Zhang Q, Li X, Xiao
J, Zhang Q, Li X, Xiao J, Zhang Q, Wang SP. 2020.} The versatile
functions of OsALDH2B1 provide a genic basis for growth-defense
trade-offs in rice. \emph{Proceedings of the National Academy of
Sciences of the United States of America} \textbf{117} (7): 3867-3873.

\textbf{Khan GA, Vogiatzaki E, Glauser G, Poirier Y. 2016.} Phosphate
deficiency induces the jasmonate pathway and enhances resistance to
insect herbivory. \emph{Plant Physiology} \textbf{171} (1): 632-644.

\textbf{Koeduka T, Matsui K, Hasegawa M, Akakabe Y, Kajiwara T.
2005.}Rice fatty acid \selectlanguage{greek}α α \selectlanguage{english}mathContainer Loading Mathjax -dioxygenase is
induced by pathogen attack and heavy metal stress: activation through
jasmonate signaling. \emph{Journal of Plant Physiology} \textbf{162}
(8): 912-920.

\textbf{Kramell R, Miersch O, Wasternack C, Solano R, Fonseca S, Chini
A, Hamberg M, Adie B, Porzel A. 2009.} (+)-7-iso-Jasmonoyl-L-isoleucine
is the endogenous bioactive jasmonate. \emph{Nature Chemical
Biology}\textbf{5} (5): 344-350.

\textbf{Lange MJ, Lange T. 2015.}Touch-induced changes in
\emph{Arabidopsis} morphology dependent on gibberellin breakdown.
\emph{Nature Plants} \textbf{1} (3): 14025.

\textbf{Li L, Liu C, Lian X. 2010.} Gene expression profiles in rice
roots under low phosphorus stress. \emph{Plant molecular
biology}\textbf{72} (4-5): 423-432.

\textbf{Liu F, Wang Z, Ren H, Shen C, Li Y, Ling H, Wu C, Lian X, Wu P.
2010.} OsSPX1 suppresses the function of OsPHR2 in the regulation of
expression of \emph{OsPT2} and phosphate homeostasis in shoots of
rice.\emph{Plant Journal} \textbf{62} (3): 508-517.

\textbf{Livak KJ, Schmittgen TD. 2001.} Analysis of relative gene
expression data using real-time quantitative PCR and the
2\textsuperscript{-[?][?]C} tMethod. \emph{Methods} \textbf{25} (4):
402-408.

\selectlanguage{ngerman}\textbf{López-Bucio J, Cruz-Ramírez A, Herrera-Estrella L. 2003.} The
role of nutrient availability in regulating root
architecture.\emph{Current Opinion in Plant Biology} \textbf{6} (3):
280-287.

\textbf{López-Bucio J, Hernandezabreu E, Sanchezcalderon L, Nietojacobo
MF, Simpson J, Herreraestrella L. 2002.} Phosphate availability alters
architecture and causes changes in hormone sensitivity in
the\emph{Arabidopsis} root system. \emph{Plant Physiology} \textbf{129}
(1): 244-256.

\textbf{López-Bucio J, Vega OMDL, Guevara-García A, Herrera-Estrella L.
2000.} Enhanced phosphorus uptake in tobacco transgenic plants that
overproduce citrate. . \emph{Nat Biotechnology} \textbf{18} (4):
450-453.

\textbf{Lorenzo O, Chico J, Sa´nchez-Serrano J, Solano R.
2004.}JASMONATE-INSENSITIVE1 encodes a MYC transcription factor
essential to discriminate between different jasmonate-regulated defense
responses in\emph{Arabidopsis} . \emph{Plant Cell} \textbf{16} (7):
1938-1950.

\textbf{Lv Q, Zhong Y, Wang Y, Wang Z, Zhang L, Shi J, Wu Z, Liu Y, Mao
C, Yi K, et al. 2014.} SPX4 Negatively Regulates Phosphate Signaling and
Homeostasis through Its Interaction with PHR2 in Rice. \emph{Plant
Cell}\textbf{26} (4): 1586-1597.

\textbf{Martin AC, Pozo JCD, Iglesias J, Rubio V, Solano R, La Pena AD,
Leyva A, Pazares J. 2000.} Influence of cytokinins on the expression of
phosphate starvation responsive genes in \emph{Arabidopsis} .
\emph{Plant Journal} \textbf{24} (5): 559-567.

\textbf{Mayer D, Mithofer A, Glawischnig E, Georgii E, Ghirardo A,
Kanawati B, Schnitzler JP, Durner J, Gaupels F. 2018.}Short-term
exposure to nitrogen dioxide provides basal pathogen resistance.
\emph{Plant Physiology} \textbf{178} (1): 468--487.

\textbf{Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu
L. 2013.} Targeted mutagenesis in rice using CRISPR-Cas
system.\emph{Cell Research} \textbf{23} (10): 1233-1236.

\textbf{Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R,
Ortet P, Creff A, Somerville S, Rolland N. 2005.} A genome-wide
transcriptional analysis using \emph{Arabidopsis thaliana} Affymetrix
gene chips determined plant responses to phosphate
deprivation.\emph{Proceedings of the National Academy of Sciences of the
United States of America} \textbf{102} (33): 11934-11939.

\textbf{Nacry P, Canivenc G, Muller B, Azmi A, Onckelen H, Rossignol M,
Doumas P. 2005.} A role for Auxin redistribution in the responses of the
root system architecture to phosphate starvation in \emph{Arabidopsis}
.\emph{Plant Physiology} \textbf{138} (4): 2061-2074.

\textbf{Neumann G, RÖmheld V. 2012.} \emph{Rhizosphere chemistry in
relation to plant nutrition. In Marschner's Mineral Nutrition of Higher
Plants, 3rd edn, H. Marschner, ed. London: Academic Press)} .

\textbf{Niu YF, Chai RS, Jin GL, Wang H, Tang CX, Zhang YS.
2013.}Responses of root architecture development to low phosphorus
availability: a review. \emph{Annual Botany} \textbf{112} (2): 391-408.

\textbf{Ogawa S, Kawahara-Miki R, Miyamoto K, Yamane H, Nojiri H, Tsujii
Y, Okada K. 2017.} OsMYC2 mediates numerous defence-related
transcriptional changes via jasmonic acid signalling in
rice.\emph{Biochemical \& Biophysical Research
Communications}\textbf{486} (3): 796-803.

\textbf{Onohata T, Gomi K. 2020.} Overexpression of jasmonate-responsive
OsbHLH034 in rice results in the induction of bacterial blight
resistance via an increase in lignin biosynthesis. \emph{Plant Cell
Reports} .

\textbf{Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W,
Pérez AC, Chico JM, Bossche RV, Sewell J, Gil E. 2010.} NINJA connects
the co-repressor TOPLESS to jasmonate signalling.
\emph{Nature}\textbf{464} (8): 788-791.

\textbf{Pérez-Torres CA, Lopez-Bucio J, Cruz-Ramirez A, Ibarra-Laclette
E, Dharmasiri S, Estelle M, Herrera-Estrella L. 2008.} Phosphate
availability alters lateral root development in \emph{Arabidopsis} by
modulating auxin sensitivity via a mechanism involving the TIR1 Auxin
receptor. \emph{Plant Cell} \textbf{20} (12): 3258-3272.

\textbf{Puga MI, Isabel M, Rajulu C, Zhiye W, Franco-Zorrilla JM, Laura
DL, Irigoyen ML, Simona M, Regla B, José R. 2014.} SPX1 is a
phosphate-dependent inhibitor of Phosphate Starvation Response 1 in
Arabidopsis. \emph{Proc Natl Acad Sci U S A} \textbf{111} (41):
14947-14952.

\textbf{Raghothama KG, Karthikeyan AS. 1999.} Phosphate
Acquisition.\emph{Annual Review of Plant Physiology \& Plant Molecular
Biology}\textbf{274} (1-2): 37-49.

\textbf{Rakwal R, Komatsu S. 2000.} Role of jasmonate in the rice
(\emph{Oryza sativa L.)} self\selectlanguage{english}-defense mechanism using proteome
analysis.\emph{Electrophoresis} \textbf{21} (12): 2492-2500.

\textbf{Rohit D, Preshobha KP, Michael R. 2016.} Functional Analysis of
Jasmonates in Rice through Mutant Approaches. \emph{Plants}\textbf{5}
(1): 15.

\textbf{Ruan J, Zhou Y, Zhou M, Yan J, Khurshid M, Weng W, Cheng J,
Zhang K. 2019.} Jasmonic acid signaling pathway in
plants.\emph{International Journal of Molecular Sciences} \textbf{20}
(10): 2479.

\textbf{Ruan W, Meina G, Wu P, Yi KK. 2016.}Phosphate starvation induced
OsPHR4 mediates Pi-signaling and homeostasis in rice. \textbf{93} (3):
1-14.

\textbf{Ruan W, Guo M, Wang X, Guo Z, Xu Z, Xu L, Zhao H, Sun H, Yan C,
Yi K. 2019.} Two RING-finger ubiquitin E3 ligases regulate the
degradation of SPX4, an internal phosphate sensor, for phosphate
homeostasis and signaling in rice. \emph{Molecular plant}\textbf{12}
(8): 1060-1074.

\textbf{Ruan W, Guo M, Wu P, Yi K. 2017.} Phosphate starvation induced
OsPHR4 mediates Pi-signaling and homeostasis in rice. \emph{Plant
molecular biology} \textbf{93} (3): 1-14.

\textbf{Rubio V, Linhares F, Solano R, Martin AC, Iglesias J, Leyva A,
Paz-Ares J. 2001.} A conserved MYB transcription factor involved in
phosphate starvation signaling both in vascular plants and in
unicellular algae. \emph{Genes Dev} \textbf{15} (16): 2122-2133.

\textbf{Secco D, Jabnoune M, Walker H, Shou H, Whelan J.
2013.}Spatio-temporal transcript profiling of rice roots and shoots in
response to phosphate starvation and recovery. \emph{Plant
Cell}\textbf{25} (11): 4285-4304.

\textbf{Tao Z, Liu H, Qiu D, Zhou Y, Wang S. 2009.} A pair of allelic
WRKY genes play opposite roles in rice-bacteria interactions.\emph{Plant
Physiology} \textbf{151} (2): 936-948.

\textbf{Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura
K, He SY, Howe GA, Browse J. 2007.} JAZ repressor proteins are targets
of the SCF(COI1) complex during jasmonate signalling.
\emph{Nature}\textbf{448} (7154): 661-665.

\textbf{Thierry H, Ekaterina S, Valentin M, Laure P. 2019.} Metabolic
control within the jasmonate biochemical pathway. \emph{Plant \& Cell
Physiology} , \textbf{60} (12): 2621-2628.

\textbf{Uji Y, Taniguchi S, Tamaoki D, Shishido H, Akimitsu K, Gomi K.
2016.} Overexpression of \emph{OsMYC2} results in the upregulation of
early JA-responsive genes and bacterial blight resistance in
rice.\emph{Plant \& Cell Physiology} \textbf{57} (9): pcw101.

\selectlanguage{polish}\textbf{Van Moerkercke A, Duncan O, Zander M, Šimura J, Broda M, Vanden
Bossche R, Lewsey M, Lama S, Singh K, Ljung K, et al. 2019.}\selectlanguage{english} A
MYC2/MYC3/MYC4-dependent transcription factor network regulates water
spray-responsive gene expression and jasmonate levels. \emph{Proceedings
of the National Academy of Sciences of the United States of
America}\textbf{116} (46): 23345-23356.

\textbf{Vidhyasekaran P. 2015.} \emph{Plant hormone signaling systems in
plant innate immunity.} : Springer Netherlands.

\textbf{Wang Z, Ruan W, Shi J, Zhang L, Xiang D, Yang C, Li C, Wu Z, Liu
Y, Yu Y, et al. 2014.} Rice SPX1 and SPX2 inhibit phosphate starvation
responses through interacting with PHR2 in a phosphate-dependent
manner.\emph{Proceedings of the National Academy of Sciences of the
United States of America} \textbf{111} (41): 14953-14958.

\textbf{Wasaki J, Shinano T, Onishi K, Yonetani R, Yazaki J, Fujii F,
Shimbo K, Ishikawa M, Shimatani Z, Nagata Y, et al. 2006.}Transcriptomic
analysis indicates putative metabolic changes caused by manipulation of
phosphorus availability in rice leaves. \emph{Journal of Experimental
Botany} \textbf{57} (9): 2049-2059.

\textbf{Wasternack C, Hause B. 2013.} Jasmonates: biosynthesis,
perception, signal transduction and action in plant stress response,
growth and development. An update to the 2007 review in Annals of
Botany. \emph{Annals of Botany} \textbf{111} (6): 1021-1058.

\textbf{Wild R, Gerasimaite R, Jung JY, Truffault V, Pavlovic I, Schmidt
A, Saiardi A, Jessen HJ, Poirier Y, Hothorn M. 2016.} Control of
eukaryotic phosphate homeostasis by inositol polyphosphate sensor
domains. \emph{Science} \textbf{352} (6288): 986.

\textbf{Woo J, MacPherson C, Liu J, Wang H, Kiba T, Hannah MA, Wang X-J,
Bajic VB, Chua N-H. 2012.} The response and recovery of
the\emph{Arabidopsis thaliana} transcriptome to phosphate
starvation.\emph{Bmc Plant Biology} \textbf{12} (1): 62.

\textbf{Wu P, Shou H, Xu G, Lian X. 2013.} Improvement of phosphorus
efficiency in rice on the basis of understanding phosphate signaling and
homeostasis. \emph{Current Opinion in Plant Biology} \textbf{16} (2):
205-212.

\textbf{Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S,
Taniguchi S, Akimitsu K, Gomi K. 2012.} Involvement of OsJAZ8 in
Jasmonate-Induced Resistance to Bacterial Blight in Rice. \emph{Plant \&
Cell Physiology} \textbf{53} (12): 2060.

\textbf{Yang B, Bogdanove A. 2013.} Inoculation and virulence assay for
bacterial blight and bacterial leaf streak of rice. \emph{Methods in
molecular biology (Clifton, N.J.)} \textbf{956} : 249-255.

\textbf{Yang D, Yao J, Mei C, Tong X, Zeng L, Li Q, Xiao L, Sun T, Li J,
Deng,XingWang. 2012.} Plant hormone jasmonate prioritizes defense over
growth by interfering with gibberellin signaling
cascade.\emph{Proceedings of the National Academy of Sciences of the
United States of America} \textbf{109} (19): E1192.

\textbf{Yoshida S, Forno D, Cock J, Gomez K. 1976.} Laboratory manual
for physiological studies of rice, Ed 3rd. \emph{The International Rice
Research Institute,Manila, Philippines} .

\textbf{Yuan H, Dong D. 2008.} Signaling components involved in plant
responses to phosphate starvation. \emph{Journal of Integrative Plant
Biology} \textbf{50} (7): 849-859.

\textbf{Zhai Q, Yan L, Tan D, Chen R, Sun J, Gao L, Dong M-Q, Wang Y, Li
C, Yu H. 2013.} phosphorylation-coupled proteolysis of the transcription
factor MYC2 is important for jasmonate-signaled plant
immunity.\emph{Plos Genetics} \textbf{9} (4): e1003422.

\textbf{Zhong Y, Wang Y, Guo J, Zhu X, Shi J, He Q, Liu Y, Wu Y, Zhang
L, Lv Q. 2018.} Rice SPX6 negatively regulates the phosphate starvation
response through suppression of the transcription factor PHR2. \emph{New
Phytologist} \textbf{219} (1): 135-148.

\textbf{Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P.
2008.}OsPHR2 is involved in phosphate-starvation signaling and excessive
phosphate accumulation in shoots of plants. \emph{Plant
Physiology}\textbf{146} (4): 1673-1686.

\textbf{Figure legends}

\textbf{Figure 1. Phosphate (Pi) starvation confers rice resistance
to\emph{Xanthomonas oryzae} pv. \emph{oryzae} (\emph{Xoo}) infection.}

(a) Growth of \emph{Xoo} strain PXO91 in wild-type Nipponbare (NIP)
leaves upon Pi starvation treatment. Plants were grown with or without
Pi solution for three days and then inoculated with \emph{Xoo} . Leaves
clipped by dipping scissor tips in sterilized water were used as
control. The bacterial population was measured from three leaves at each
time point in terms of colony-forming units (cfu). Leaf fragments (6 cm)
were sterilized using 75\% ethanol, ground separately, suspended in
sterilized water, and plated on peptone sucrose agar in a 10-fold
dilution series. Different letters indicate significant difference at
\emph{P} \textless{} 0.05 by Fisher's least significant difference
tests. (b) Bacterial blight symptoms in wild-type NIP leaves at 14 days
after inoculation with\emph{Xoo} . (c) Lesion length on the fifth leaf
blades at 14 days after inoculation with \emph{Xoo} . Values are means \selectlanguage{ngerman}±
SE (n = 15). * \emph{P}\textless{} 0.05, Student's \emph{t} -test.

\textbf{Figure 2. Phosphate (Pi) starvation induces JA-responsive gene
expression and MeJA production.}

(a and b) RT-qPCR analysis of JA-responsive genes in wild-type
Nipponbare (NIP) plants upon Pi starvation treatment for 12 hours (a)
and three days (b), respectively. Ten-day-old NIP plants were grown in
+P/-P solution and the treated leaves were collected at 12 hours and
three days for RNA extraction. \emph{OsIPS1} (\emph{Induced by phosphate
starvation 1} ) is a marker gene for Pi starvation. Values are means ±
SE of three biological replicates. * indicates significant difference
between +P/-P treatment at \emph{P} \textless{} 0.05 by
Student's\emph{t} -test. H: hours; D: day. (c) Enhanced levels of
endogenous MeJA in seven-day-old NIP plants upon Pi starvation treatment
for three days. The limit of quantification for MeJA was 22.3 nM. Values
are means ± SE of three biological replicates. * \emph{P} \textless{}
0.05, Student's\emph{t} -test. (d) Expression of \emph{OsMYC2} in NIP
leaves upon starvation treatment. Ten-day-old plants were grown in
solution with an additional time course of phosphate starvation and
phosphate recovery (RP) afterwards. Treated leaves were used for RNA
extraction at the indicated time after treatment. Values are means ± SE
of three biological replicates. * \emph{P} \textless{} 0.05,
Student's\emph{t} -test.

\textbf{Figure 3. OsPHR2 targets the \emph{OsMYC2} promoter.} (a)
Diagram of the \emph{OsMYC2} promoter and its OsPHR2 binding sites
(GNATATNC). Black lines P1--5 indicate the sequences tested in ChIP
assays. P1 contained GAATATAC, P2 contained GTATATAC, and P3 contained
GCATATGC; those elements were absent in P4 and P5. (b) Yeast one-hybrid
assays showing interaction between OsPHR2 and \emph{OsMYC2} promoter
fragments. Empty pGADT7-Rec2 vectors were used as negative control. AbA:
Aureobasidin A. (c) Gel shift assay indicating OsPHR2 protein binds to
the \emph{OsMYC2} promoter \emph{in vitro} . The arrow indicates band
shifting caused by OsPHR2 binding to the P3 and P4 motifs
of\emph{OsMYC2} promoter, labeled with biotin (hot probe). The
competitive protein-DNA binding assay was performed using 10X and 100X
unlabeled probes of the wild-type (cold probe). (d) Transient
transfection assay indicated that OsPHR2 activated \emph{OsMYC2}
promoter in \emph{N. benthamiana} leaves\emph{.} (e) ChIP assay revealed
OsPHR2 enriched the\emph{OsMYC2} promoter fragment \emph{in vivo} .
\emph{OsACTIN2} gene promoter was used as a reference and \emph{OsIPS1}
gene promoter was set as positive control. Fold enrichment represents
the binding efficiency ratio of anti-Flag antibody/negative IgG
antibody. Data are means ± SE (n = 3). * \emph{P} \textless{} 0.05,
Student's \emph{t-} test.

\textbf{Figure 4. OsPHR2 regulates expression of JA-responsive
genes.}Ten-day-old \emph{OsPHR2-Ov1} , \emph{phr2} , and wild-type
Nipponbare (NIP) plants were grown in solution with 0 (-P) or 200 \selectlanguage{greek}μ\selectlanguage{english}M
(+P) Pi for three days. Treated leaves were used for RNA extraction and
transcripts were analyzed by qRT-PCR. \emph{OsIPS1} (\emph{Induced by
phosphate starvation 1} ) is a marker gene for Pi starvation. Values are
means \selectlanguage{ngerman}± SE of three biological replicates. Different letters indicate
significant difference at \emph{P} \textless{} 0.05 by Fisher's least
significant difference tests.

\textbf{Figure 5. OsPHR2 positively regulates MeJA accumulation and
antibacterial defense.} (a) Endogenous MeJA levels in
ten-day-old\emph{OsPHR2-OV1} , \emph{phr2} , and wild-type Nipponbare
(NIP) leaves. The limit of quantification for MeJA was 22.3 nM. Values
are means ± SE of three biological replicates. Different letters
indicate significant difference at \emph{P} \textless{} 0.05 by Fisher's
least significant difference (LSD) tests. (b) Growth of
\emph{Xanthomonas oryzae} pv.\emph{oryzae} (\emph{Xoo} ) strain PXO91 in
leaves of \emph{OsPHR2-Ov1} ,\emph{phr2} , and NIP. Plants were grown
with or without Pi solution for three days and then inoculated with
\emph{Xoo} . Leaves clipped by dipping scissor tips in sterilized water
were used as control. The bacterial population was measured from three
leaves at each time point in terms of colony-forming units (cfu). Leaf
fragments (6 cm) were sterilized using 75\% ethanol, ground separately,
suspended in sterilized water, and plated on peptone sucrose agar in a
10-fold dilution series. \emph{P} \textless{} 0.05, Fisher's LSD test.
(c) Bacterial blight symptoms in NIP, \emph{OsPHR2-Ov1} , and
\emph{phr2}plants at 14 days after inoculation with \emph{Xoo} . The
fifth leaf of each plant was inoculated with \emph{Xoo} . (d) Lesion
length in fifth leaf blades of NIP, \emph{OsPHR2-Ov1} , and \emph{phr2}
at 14 days after inoculation with \emph{Xoo} . Values are means ± SE (n
\selectlanguage{english}[?] 15). \emph{P}\textless{} 0.05, Fisher's LSD test.

\textbf{Figure 6. \emph{OsMYC2} is involved in phosphate (Pi)
starvation-induced resistance.} (a) Effects of Pi starvation treatment
on the expression of JA-responsive genes in 5- to 5.5-stage Nipponbare
(NIP), \emph{myc2-3} , and \emph{myc2-6} plants. Treated leaves were
used for RNA extraction and transcripts were analyzed by qRT-PCR. Values
are means +- SE of three biological replicates. Different letters on the
top of column for each gene indicate significant difference at
\emph{P}\textless{} 0.05 by Fisher's least significant difference (LSD)
test. (b) Endogenous MeJA levels in 5- to 5.5-stage wild-type
NIP,\emph{myc2-3} , and \emph{myc2-6} leaves treated with Pi starvation
solution for three days. The limit of quantification for MeJA was 22.3
nM. \emph{P} \textless{} 0.05, Fisher's LSD test. (c) Growth
of\emph{Xanthomonas oryzae} pv. \emph{oryzae} (\emph{Xoo} ) strain PXO91
in leaves of \emph{myc2-3} , \emph{myc2-6} , and NIP upon Pi starvation.
Plants were grown with or without Pi solution for three days and then
inoculated with \emph{Xoo} . Leaves clipped by dipping scissor tips in
sterilized water were used as control. The bacterial population was
measured from three leaves at each time point in terms of colony-forming
units (cfu). Leaf fragments (6 cm) were sterilized using 75\% ethanol,
ground separately, suspended in sterilized water, and plate on peptone
sucrose agar in a 10-fold dilution series. \emph{P} \textless{} 0.05,
Fisher's LSD test. The data in cycle displayed non-significance. (d and
e) Bacterial blight symptoms (d) and lesion length (e) in
NIP,\emph{myc2-3} , and \emph{myc2-6} plants at 14 days after
inoculation with \emph{Xoo} . Values are means +- SE (n \selectlanguage{english}[?] 12). \emph{P}
\textless{} 0.05, Fisher's LSD test.

\textbf{Figure. 7 Model of the OsPHR2-\emph{OsMYC2-}mediated JA response
to bacterial resistance induced by phosphate (Pi) starvation.} Under
high Pi, OsSPXs interact with OsPHR2 at high affinity and prevent OsPHR2
from binding to the P1BS motifs of \emph{OsMYC2} . Thus, expression of
\emph{OsMYC2} is basal. When Pi is low or lacking, the OsPHR2-OsSPXs
interaction is low affinity and P1BS motifs compete with OsSPXs for
OsPHR2 binding, allowing OsPHR2 to up-regulate \emph{OsMYC2} . Increased
\emph{OsMYC2}triggers the JA response and thereby enhances rice
antibacterial resistance. Bold lines ending with arrows show activation.\selectlanguage{english}
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