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.