Jasmonic Acid-Induced VQ-Motif-Containing Protein OsVQ13 Influences the OsWRKY45 Signaling Pathway and Grain Size by Associating with OsMPK6 in Rice

Jasmonic acid (JA) is a plant hormone that plays an important role in the defense response and stable growth of rice. In this study, we investigated the role of the JA-responsive valine-glutamine (VQ)-motif-containing protein OsVQ13 in JA signaling in rice. OsVQ13 was primarily located in the nucleus and cytoplasm. The transgenic rice plants overexpressing OsVQ13 exhibited a JA-hypersensitive phenotype and increased JA-induced resistance to Xanthomonas oryzae pv. oryzae (Xoo), which is the bacteria that causes rice bacterial blight, one of the most serious diseases in rice. Furthermore, we identified a mitogen-activated protein kinase, OsMPK6, as an OsVQ13-associating protein. The expression of genes regulated by OsWRKY45, an important WRKY-type transcription factor for Xoo resistance that is known to be regulated by OsMPK6, was upregulated in OsVQ13-overexpressing rice plants. The grain size of OsVQ13-overexpressing rice plants was also larger than that of the wild type. These results indicated that OsVQ13 positively regulated JA signaling by activating the OsMPK6–OsWRKY45 signaling pathway in rice.


Introduction
Rice (Oryza sativa L.) is a major crop in the world, and decreased crop yields caused by pathogen attacks is a serious problem in rice farming. Many studies have shown that rice plants have developed complex defense systems to protect themselves against various pathogens. Among the defense systems, two plant hormones, jasmonic acid (JA) and salicylic acid (SA), are important signaling compounds that help to regulate the expression of defense-related genes in rice [1]. Treatment with benzothiadiazole (BTH), an SA analog, upregulates many pathogenesis-related (PR) genes and defense-related transcription factors (TFs) in rice and enhances resistance to rice bacterial blight and rice blast caused by Xanthomonas oryzae pv. oryzae (Xoo) and Pyricularia oryzae, respectively, which are both hemibiotrophic pathogens causing two of the most serious rice diseases, rice bacterial blight and rice blast, respectively [2]. The WRKY-type TFs, identified as a TF family containing a conserved WRKY domain [3], have important roles in rice defense responses. As one of the WRKY-type TFs, OsWRKY45 plays a crucial role in the BTH-mediated defense response against Xoo and P. oryzae [2,4]. OsNPR1, a rice homologue to Arabidopsis NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (AtNPR1) [5], acts as a positive regulator of SA signaling and is involved in SA-mediated defense response in rice [6][7][8].

Properties of OsVQ13
We carried out reverse transcription-quantitative PCR (RT-qPCR) analysis of OsVQ13 to investigate its expression in response to JA treatment. The expression of OsVQ13 reached its maximum level after 24 h of JA treatment ( Figure 1A). To determine the subcellular localization of OsVQ13, we generated transgenic rice plants overexpressing the OsVQ13 green fluorescent protein (GFP) fusion protein (OsVQ13GFP-ox) and confirmed the expression of the transgene through RT-PCR ( Figure 1B). The GFP signal in the root tissue of OsVQ13GFP-ox line 9 was observed by fluorescence microscopy. As shown in Figure 1C, the GFP fluorescent signals were detected in the nucleus and the cytoplasm, indicating that OsVQ13 was localized in these specific locations.

Phenotypes of OsVQ13-Overexpressing Rice Plants
We generated OsVQ13-overexpressing (OsVQ13-ox) rice plants (lines 2 and 8) and confirmed the expression of the transgene through RT-PCR ( Figure 2A). To identify JA responses in these transgenic rice plants, we measured chlorophyll (Chl) contents after JA treatment, because it is known that JA promotes a Chl degradation in rice [13]. The Chl contents of OsVQ13-ox rice plants were significantly reduced at three days after JA treatment ( Figure 2B).
To determine whether OsVQ13 is involved in JA-mediated resistance to Xoo, we performed a resistant test on these transgenic rice plants. The JA-treated or untreated rice plants were inoculated with a virulent Xoo, and the length of any blight lesions was measured 14 days after inoculation. The lengths of blight lesions in the OsVQ13-ox plants were significantly shorter than those in the wild-type (WT) plants without JA treatment ( Figure 2C,D). Furthermore, JA-induced resistance was enhanced in the transgenic rice plants compared to the WT plants ( Figure 2C,D).

Identification of OsVQ13-Associating Proteins
Arabidopsis VQ proteins act as positive or negative regulators through interactions with various proteins in response to abiotic or biotic stresses [31]. To determine whether OsVQ13 associates with uncharacterized proteins in rice, we performed a co-immunoprecipitation assay on anti-GFP antibodies derived from OsVQ13GFP-ox rice plants. The OsVQ13GFP-ox rice plant exhibited a JA-hypersensitive phenotype similar to that of the OsVQ13-ox transgenic plant, indicating that the OsVQ13GFP protein was functional in the rice plant ( Figure 3A). The GFP-ox transgenic rice plant (GFP-ox) was used as a negative control. After anti-GFP antibody precipitation, numerous proteins were detected in the OsVQ13GFP-ox rice plant sample, whereas only a small number of proteins were detected in the GFP-ox rice plant sample ( Figure 3B,C). Experiments using OsVQ13GFP-overexpressing rice plants were repeated five times, and ultimately, the four proteins that were reproducibly detected at least twice in the immunoprecipitates were selected ( Figure 3C). These selected proteins were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and identified in a MASCOT database. As a result, one mitogen-activated protein kinase (MAPK), OsMPK6, and three TFs, namely OsNAC5, OsERF36, and OsMADS2, were identified (Table 1).  OsVQ13 co-IP was performed using OsVQ13GFP-overexpressing transgenic rice plants. Experiments were independently performed five times. Nos. 1-4 are as in Figure 3. MW: Molecular weight.
Among the OsVQ13-associating proteins, we focused on OsMPK6, which is known to be involved in the rice defense response [32,33]. We first found a direct interaction between OsVQ13 and OsMPK6 proteins using a yeast two-hybrid system ( Figure 4A). We next performed Phos-tag ® SDS-PAGE to reveal whether OsVQ13 in rice was phosphorylated. In SDS-PAGE using Phos-tag ® acrylamide, phosphorylated proteins are trapped by the Phos-tag ® sites, which leads to a delay in their migration and a resulting separation from unphosphorylated proteins. This makes it straightforward to identify the phosphorylated proteins from their observed positions on blots [34,35]. As a positive control, we used a commercially available α-casein, which contains both phosphorylated and unphosphorylated forms. When the α-casein was separated by Phos-tag ® SDS-PAGE, a slow-migrating band was detected ( Figure 4B). The band disappeared following treatment with protein phosphatase, indicating that the upper band was a highly phosphorylated form of α-casein, which could be distinguished from the unphosphorylated form through Phos-tag ® SDS-PAGE ( Figure 4B). In the case of the sample from the OsVQ13GFP-ox rice plant, however, there was no change in the banding pattern detected by anti-GFP antibodies between samples treated and not treated with phosphatase ( Figure 4C).

OsVQ13 Affected OsMPK6-Mediated Signaling Pathways in Rice
OsMPK6 is involved in the rice defense response through the phosphorylation of OsWRKY45, a TF that, in turn, plays a crucial role in defense responses in rice [2,4,36,37]. To investigate whether OsVQ13 affects the OsWRKY45-dependent pathway via its association with OsMPK6, we compared the expression levels in WT and OsVQ13-ox rice plants of the following OsWRKY45-responsive genes (identified by Nakayama et al. [4]): OsWRKY62, cytochrome P450, OsOPR5, OsPrx126, OsPrx72, OsGSTU4, UDP-glucosyltransferase, Proteinase inhibitor I20, and beta-1,3-glucanase. Genes OsWRKY62, cytochrome P450, OsOPR5, OsPrx126, OsPrx72, OsGSTU4, and UDP-glucosyltransferase tended to be upregulated in OsVQ13-ox rice plants compared to WT plants in the absence of JA. The expression levels of all genes tested were significantly higher in JA-treated OsVQ13-ox rice than in WT plants ( Figure 5A). In contrast, the expression levels of endogenous OsVQ13, OsMPK6, and OsWRKY45 in OsVQ13-ox plants with or without JA treatment were not significantly different from those in WT rice plants ( Figure 5B).
It was also shown that the length, width, and weight of OsVQ13-ox grains were significantly larger than WT grains ( Figure 6A-D). OsMPK6 has been shown to positively regulate grain size and weight in rice [38,39].

Discussion
We previously demonstrated that the gene expression patterns in response to JA are broadly divided into two phases, the early (<12 h) and late (>12 h) phases, under our experimental conditions [13]. The expression level of OsVQ13 reached a maximum at 24 h after JA treatment, indicating that OsVQ13 is a late JA-responsive gene in rice. It has been reported that the basal expression of OsVQ13 in the resistant rice plants harboring a Xoo-resistance gene, Xa3/Xa26, was significantly higher than in corresponding susceptible rice plants [30]. When OsVQ13 was overexpressed in rice in the current study, the transgenic plants exhibited a JA-hypersensitive phenotype and increased basal and JA-induced resistances against Xoo. The increased JA-induced resistance against Xoo in transgenic plants depended on the expression levels of the OsVQ13 transgene. These results indicated that OsVQ13 acted as a positive regulator of JA signaling in rice.
In the present study, we revealed that OsVQ13 associated with OsMPK6 and positively regulated OsMPK6-mediated signaling pathways in rice. OsVQ13 possessed putative phosphorylation sites through MAPK. Furthermore, we suggested the possibility that OsVQ13 may not be a substrate of OsMPK6, although further analysis is needed to determine whether OsVQ13 is phosphorylated or not by OsMPK6 in rice. These results suggest that OsVQ13 acts as a regulatory protein toward OsMPK6, but not as a substrate for OsMPK6. Furthermore, expression patterns of OsMPK6 and OsWRKY45 were similar in WT and OsVQ13-ox rice plants, suggesting that OsVQ13 positively regulated the expression of OsWRKY45-responsive genes by activating OsMPK6 at the protein level but not at the transcriptional level. The activity of OsMPK6 is suppressed via interaction with the MAPK phosphatase OsMKP1, which dephosphorylates both serine/threonine and tyrosine residues of MAPKs in rice [40]. Thus, it is possible that OsVQ13 blocks the interaction between OsMPK6 and OsMKP1 to activate the OsMPK6-OsWRKY45 cascade in response to JA. Regulation of OsWRKY45 activity via the phosphorylation mediated by OsMPK6 has been analyzed in detail [37]. OsWRKY45 is phosphorylated at Thr266, Ser294, and Ser299 by OsMPK6. Phosphorylation of Ser294 and Ser299 is required for the full activation of OsWRKY45 in the defense response in rice [37]. Conversely, phosphorylation of Thr266 negatively affects the defense response in rice [41]. In the current study, it was demonstrated that OsVQ13 positively regulated the OsWRKY45-dependent signaling pathway by associating with OsMPK6, although there was no direct evidence for the phosphorylation of OsWRKY45 by OsVQ13 in rice (Figure 7). Further studies are needed to test this hypothesis.
Both OsMPK6 and OsWRKY45 act as central regulators of SA signaling in rice [2,4,37]. In the current study, we demonstrated that the expression of some OsWRKY45-responsive genes was upregulated by JA, which also increased their upregulation in OsVQ13-ox rice plants. The expression of OsMPK6 and OsWRKY45 is upregulated through JA treatment [42,43]. Another important fact is that the expression of OsVQ13 is also upregulated by BTH, an analog of SA [2]. These results suggest that OsVQ13 plays a critical role as an activator of the OsMPK6-OsWRKY45-dependent cascade involved in both JA-and SA-induced resistance against Xoo.
Generally, SA confers resistance against biotrophic and hemibiotrophic pathogens, whereas JA confers resistance against necrotrophic pathogens in plants. The relationship between JA and SA is antagonistic in many plants. As an example of an Arabidopsis VQ protein, plants exhibiting RNA interference (RNAi) of JAV1 exhibited increased resistance against the necrotrophic pathogen Botrytis cinerea [25]. The Arabidopsis mks1 mutant exhibited increased susceptibility to the biotrophic pathogen Pseudomonas syringae [27]. However, in rice, it is unclear whether this antagonistic cross-talk between JA-and SA-dependent defense signaling occurs. In addition to the current study, other studies have demonstrated that the JA-dependent signaling pathway has an important role in resistance against hemibiotrophic pathogens in rice. Accumulations of JA-isoleucine (JA-Ile), a bioactive form of JA that is induced through inoculation with P. oryzae [44], and the jasmonate-deficient rice mutants cpm2 and hebiba, exhibited decreased resistance to an avirulent P. oryzae [45]. The osjar1-2 mutant, which exhibits diminished JA-Ile accumulation in leaves, also showed decreased resistance to P. oryzae [46]. Expression of the microRNA miR319b was upregulated through inoculation with virulent P. oryzae. The upregulation of miR319b caused the suppression of its target gene, TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP)21 (OsTCP21). OsTCP21 acts as a positive TF regulating the JA-biosynthetic genes lipoxygenase2 (OsLOX2) and OsLOX5 [47]. On the other hand, P. oryzae converts JA into 12OH-JA by secreting a monooxygenase, Abm. The 12OH-JA suppresses the induction of JA-mediated defense responses [48]. It has been reported that 313 BTH-upregulated genes were identified by microarray analysis in rice [2]. When the expression levels of BTH-upregulated genes were compared to those of JA-responsive genes, as identified by microarray analysis in rice [10], more than half of the BTH-upregulated genes, including OsVQ13, were also upregulated by JA [42]. OsNPR1 is degraded by OsCUL3a to suppress OsNPR1-dependent cell death [8]. The expression of OsNPR1 is also upregulated by JA [49]. Furthermore, an oscul3a mutant exhibited increased resistance to P. oryzae and Xoo by activating both the JA-and SA-signaling pathways [8]. Taken together, these results strongly indicate that JA and SA signaling can interact coordinately in an induced defense response and that OsVQ13 may be a key factor in the rice defense pathway induced by both JA and SA.
The overexpression of OsVQ13 has resulted in larger grain sizes compared to WT. The overexpression of OsMPK6 or a constitutively active version of OsMPK6 (CA-OsMPK6) has also resulted in significantly larger grains [39]. Furthermore, an oscoi1b mutant has exhibited a significantly lower grain weight [50], suggesting that JA has a positive effect on the seed development process. However, there is not enough experimental evidence to discuss the function of OsVQ13 in JA-dependent seed development in rice (Figure 7). Further studies are needed.
Among the OsVQ13-associating proteins, the NAC-type TF OsNAC5 positively regulates some defense-related genes in rice, and the expression of OsNAC5 responds to both JA and abscisic acid [51]. OsNAC5 is also required to express salt stress tolerance in rice [51]. OsNAC5-overexpressing rice plants have exhibited increased salt tolerance, whereas OsNAC5-RNAi rice plants have exhibited greater sensitivity than the WT to salt stress [51,52]. Recently, it has been reported that JA plays an important role in salt tolerance in many plant species, including rice [53]. RICE SALT SENSITIVE3 (RSS3) acts as a negative regulator of JA signaling by interacting with OsJAZ9 and OsJAZ11, and the rss3 mutant exhibits the salt-hypersensitive phenotype [54]. OsNAC5 is also known to be negatively involved in leaf senescence in rice [55], whereas JA has a positive effect on leaf senescence in rice [50,56]. There is no information as to whether OsVQ13 is a senescence-associated gene in rice. Further analyses of OsVQ13-associating proteins such as OsNAC5 may provide new insights into the molecular mechanism of JA-dependent biotic and abiotic stress responses in rice.

Figure 7.
A model for OsVQ13/OsMPK6 complex-mediated signaling pathways in rice. OsVQ13 associates with OsMPK6 and acts as an activator of the OsWRKY45-dependent signaling pathway, contributing to resistance to Xanthomonas oryzae pv. oryzae. OsVQ13 also affects rice grain development by associating with OsMPK6.

Plant Materials, Jasmonate Treatment, and Bacterial Inoculation
Plant and bacteria growth conditions were as previously described, they are grown and cultured in our laboratory by ourselves [15]. The fully opened fifth leaf blades of rice plants were inoculated using the clipping inoculation technique [57]. The lengths of blight lesions on each leaf blade were measured for each leaf at 14 days post-inoculation.
To examine the effects of JA on rice growth and gene expression, rice plants were grown to the four-leaf stage in a growth chamber in Kimura-B liquid medium [58] at 25 • C (24 h light) and then incubated in the same medium supplemented with 100 µM of JA (Sigma-Aldrich, St. Louis, MO, USA). The fourth leaf blades were used for RT-qPCR analyses.

RT-qPCR
Total RNA was extracted from rice leaf blades from plants of different genotypes and treatments using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. RT-qPCR was performed as previously described [15,59]. The sequences of the gene-specific primers used in this study are presented in the Supplementary Table S1.

Construction of OsVQ13-Overexpressing Vectors and Rice Transformation
The OsVQ13 cDNA (accession number: AK109228) was ligated into the pBI333-EN4 vector [60]. The pBI333-EN4-GFP binary vector was prepared by subcloning the GFP coding sequence into the pBI333-EN4 binary vector. The production of transgenic rice plants was performed as previously described [61,62]. Second-or third-generation plants were used for the experiments.

Chlorophyll Content Measurement
Leaf blades treated with 100 µM of JA for four days were homogenized in 1 mL of 80% acetone and centrifuged at 15,000 rpm for 10 min. The specific chlorophyll content was determined as described by Arnon [63].

Co-Immunoprecipitation and Protein Gel Blot Analysis
The nuclear proteins were mixed with magnetic bead-conjugated GFP-Trap (Chromotek, Munich, Germany) and incubated overnight at 4 • C. The beads were then washed five times in 0.5 mL of wash buffer II (Chromotek, Munich, Germany). The binding proteins were eluted with sample buffer. The extracted proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane through semidry blotting. A protein gel blot analysis was performed as previously described [13]. We used anti-GFP (1:1000) and antirabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:20,000).

Protein Identification Using TOF MS
The proteins that were purified by GFP-Trap were separated by 12% SDS-PAGE and stained with Oriole Fluorescent Gel Stain (Bio-Rad, Hercules, CA, USA) for 90 min. The stained protein bands were excised from the gels, washed twice with 100 mM of NH 4 HCO 3 containing 30% acetonitrile (Wako, Osaka, Japan), washed with 100% acetonitrile, and then dried in a vacuum concentrator. The gels were reductively alkylated with 10 mM of dithiothreitol (DTT) in 100 mM of NH 4 HCO 3 for 60 min at 56 • C and with iodoacetamide in 100 mM of NH 4 HCO 3 for 30 min at 25 • C. The gels were washed with 100 µL of 100-mM NH 4 HCO 3 for 10 min and dehydrated through the addition of acetonitrile. The dried gels were treated with 2 µL of 0.5 µg/µL of trypsin (Promega, Madison, WI, USA) in 50 mM of NH 4 HCO 3 and incubated at 37 • C for 12 h. Peptides were extracted with 50 mM of NH 4 HCO 3 in 1% trifluoroacetic acid and 50% acetonitrile. Sample preparation for MALDI analysis was according to the method of Shevchenko et al. [64]. Peptides of proteins that associate with OsVQ13 were identified through MALDI-TOF MS analysis with a Voyager-DE STR (Thermo Fisher Scientific, Waltham, MA, USA). Experiments using OsVQ13GFP-overexpressing rice plants were repeated five times, and four out of the five stained gels are presented in the Supplementary Figure S1 To determine whether OsVQ13 was phosphorylated, we used Phos-tag ® SDS-PAGE (NARD Institute Ltd., Amagasaki, Japan). To achieve dephosphorylation, the beads were prepared in 50 µL of 1× alkaline phosphatase buffer (Thermo Fisher Scientific) and incubated with 2 units of fast alkaline phosphatase (Thermo Fisher Scientific) for 2 h. After incubation, the beads were then washed five times in 0.5 mL of extraction buffer, and the binding proteins were eluted with 2× SDS sample buffer.

Yeast Two-Hybrid System
An analysis of protein-protein interactions using a yeast two-hybrid system was performed as previously described [15]. Images of yeast cells were obtained at day 3.