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. OsIPS1 (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 P < 0.05 by Student’st -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. * P < 0.05, Student’st -test. (d) Expression of 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. * P < 0.05, Student’st -test.
Figure 3. OsPHR2 targets the OsMYC2 promoter. (a)
Diagram of the 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 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 OsMYC2 promoter in vitro . The arrow indicates band
shifting caused by OsPHR2 binding to the P3 and P4 motifs ofOsMYC2 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 OsMYC2 promoter in N.
benthamiana leaves. (e) ChIP assay revealed OsPHR2 enriched theOsMYC2 promoter fragment in vivo . OsACTIN2 gene
promoter was used as a reference and 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). * P < 0.05, Student’s t- test.
Figure 4. OsPHR2 regulates expression of JA-responsive genes.Ten-day-old OsPHR2-Ov1 , phr2 , and wild-type Nipponbare
(NIP) plants were grown in solution with 0 (-P) or 200 μM (+P) Pi for
three days. Treated leaves were used for RNA extraction and transcripts
were analyzed by qRT-PCR. OsIPS1 (Induced by phosphate
starvation 1 ) is a marker gene for Pi starvation. Values are means ± SE
of three biological replicates. Different letters indicate significant
difference at P < 0.05 by Fisher’s least significant
difference tests.
Figure 5. OsPHR2 positively regulates MeJA accumulation and
antibacterial defense. (a) Endogenous MeJA levels in ten-day-oldOsPHR2-OV1 , 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 P < 0.05 by Fisher’s least significant
difference (LSD) tests. (b) Growth of Xanthomonas oryzae pv.oryzae (Xoo ) strain PXO91 in leaves of OsPHR2-Ov1 ,phr2 , and NIP. Plants were grown with or without Pi solution for
three days and then inoculated with 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. P < 0.05, Fisher’s LSD test. (c)
Bacterial blight symptoms in NIP, OsPHR2-Ov1 , and phr2plants at 14 days after inoculation with Xoo . The fifth leaf of
each plant was inoculated with Xoo . (d) Lesion length in fifth
leaf blades of NIP, OsPHR2-Ov1 , and phr2 at 14 days after
inoculation with Xoo . Values are means ± SE (n ≥ 15). P< 0.05, Fisher’s LSD test.
Figure 6. 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), myc2-3 , and 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 P< 0.05 by Fisher’s least significant difference (LSD) test.
(b) Endogenous MeJA levels in 5- to 5.5-stage wild-type NIP,myc2-3 , and myc2-6 leaves treated with Pi starvation
solution for three days. The limit of quantification for MeJA was 22.3
nM. P < 0.05, Fisher’s LSD test. (c) Growth ofXanthomonas oryzae pv. oryzae (Xoo ) strain PXO91 in
leaves of myc2-3 , myc2-6 , and NIP upon Pi starvation.
Plants were grown with or without Pi solution for three days and then
inoculated with 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. P < 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,myc2-3 , and myc2-6 plants at 14 days after inoculation
with Xoo . Values are means ± SE (n ≥ 12). P <
0.05, Fisher’s LSD test.
Figure.
7 Model of the OsPHR2-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 OsMYC2 . Thus, expression of 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 OsMYC2 . Increased OsMYC2triggers the JA response and thereby enhances rice antibacterial
resistance. Bold lines ending with arrows show activation.