Expressing OsMPK4 Impairs Plant Growth but Enhances the Resistance of Rice to the Striped Stem Borer Chilo suppressalis

Mitogen-activated protein kinases (MPKs) play a central role not only in plant growth and development, but also in plant responses to abiotic and biotic stresses, including pathogens. Yet, their role in herbivore-induced plant defenses and their underlying mechanisms remain largely unknown. Here, we cloned a rice MPK gene, OsMPK4, whose expression was induced by mechanical wounding, infestation of the striped stem borer (SSB) Chilo suppressalis, and treatment with jasmonic acid (JA), but not by treatment with salicylic acid (SA). The overexpression of OsMPK4 (oe-MPK4) enhanced constitutive and/or SSB-induced levels of JA, jasmonoyl-l-isoleucine (JA-Ile), ethylene (ET), and SA, as well as the activity of elicited trypsin proteinase inhibitors (TrypPIs), and reduced SSB performance. On the other hand, compared to wild-type plants, oe-MPK4 lines in the greenhouse showed growth retardation. These findings suggest that OsMPK4, by regulating JA-, ET-, and SA-mediated signaling pathways, functions as a positive regulator of rice resistance to the SSB and a negative regulator of rice growth.


Introduction
In their natural habitats, plants often face infestations by various herbivores. To protect themselves, plants have developed constitutive and induced defensive mechanisms [1][2][3]. Induced defenses are made when a plant recognizes damage-related signals from herbivores. In response to the signals, plants activate a defense-related signaling network consisting mainly of jasmonic acid (JA)-, salicylic acid (SA)-, and ethylene (ET)-mediated pathways, and induce the production of defensive compounds; as a result, herbivore resistance in plants increases [4][5][6][7]. During this process, mitogen-activated protein kinase (MPK) cascades play vital roles in the signaling network, functioning upstream and downstream of defense-related signaling pathways [8][9][10][11].
A typical MPK cascade comprises three modules: MPKs, MPK kinases (MPKKs or MEKs), and MPKK kinases (MPKKKs or MEKKs), all of which are evolutionarily conserved in all eukaryotes [12]. These modules are sequentially activated by the dual phosphorylation of threonine and tyrosine residues in a TxY (x = D or E) motif located in their kinase catalytic activation loop. By activating the module MPKs, which subsequently phosphorylate some transcription factors and enzymes, and thereby trigger downstream signaling components or pathways, MPK cascades have been reported to play central roles in plant growth, development, and responses to abiotic and biotic stresses, including defense responses to pathogens and herbivores [8,[13][14][15][16][17]. In Arabidopsis thaliana, for instance, MPK3 and from different species revealed that OsMPK4 is homologous to BdMPK6 in Brachypodium distachyon, to SiMPK6 in Setaria italica and to SbMPK6 in Sorghum bicolor; all of these proteins share more than 94% amino acid sequence identity with OsMPK4. OsMPK4 also shows homologous to well characterized MPK4 in Arabidopsis thaliana (AtMPK4) [22], Brassica napus (BnMPK4) [41], and Nicotiana tabacum (NtMPK4) [42] (Figure S2), whose amino acid sequence similarities to OsMPK4 were 81.12%, 82.18%, and 81.91%, respectively.
Quantitative real-time PCR (qRT-PCR) analysis revealed low constitutive levels of OsMPK4 transcripts. Mechanical wounding, SSB infestation, and JA treatment markedly enhanced transcript levels of OsMPK4, whereas SA treatment did not ( Figure 1). Moreover, unlike mechanical wounding, which quickly induced the expression of OsMPK4, SSB infestation and, especially, JA treatment increased the transcript level of OsMPK4 only at later treatment stages. These data indicate that OsMPK4 might be involved in defense responses of rice to SSB. Transcript levels were analyzed by quantitative real-time PCR (qRT-PCR). Con, non-manipulated plant, BUF, buffer. Asterisks indicate significant differences in transcript levels between treatments and controls (*, p < 0.05; and **, p < 0.01, Student's t-tests).

Overexpression of OsMPK4
To explore the function of OsMPK4 in herbivore-induced defenses, we obtained two homozygous single-insertion lines with over-expressed OsMPK4 (oe-MPK4-43 and oe-MPK4-59) (Figure 2a). Transcript analysis found that constitutive transcript levels of OsMPK4 in two oe-MPK4 lines, oe-MPK4-43 and oe-MPK4-59, were 20.4-and 25.0-fold higher, respectively, than levels of OsMPK4 in wild type (WT) plants ( Figure 2b); after mechanical wounding, transcript levels of OsMPK4 in the two oe-MPK4 lines were still significantly higher than levels in WT plants: 4 h after mechanical wounding, for example, transcript levels of OsMPK4 in the two oe-MPK4 lines were 5.2-and 6.0-fold higher than those in WT plants (Figure 2b). Lines with overexpressed OsMPK4 showed growth retardation, especially at later growth stages (Figure 3a-d,f). At 55 days, for instance, the root length of the two oe-MPK4 lines, oe-MPK4-43 and oe-MPK4-59, decreased by approximately 31.85% and 25.35%, respectively, compared to that of WT plants (Figure 3b), and the total root mass of the two oe-MPK4 lines decreased by approximately 35.56% and 24.47% ( Figure 3d); moreover, the mass of the aboveground part of oe-MPK4 plants decreased by approximately 18.96% and 12.46% (Figure 3c). Unlike the growth phenotype, the chlorophyll content in oe-MPK4 lines was a bit higher (about 4%) than in WT plants (Figure 3e). These results suggest that OsMPK4 is involved in rice growth and development.   Signaling pathways mediated by plant hormones, such as JA, ET, and SA, play key roles in herbivore-elicited defense responses in various plant species, including rice [6,[34][35][36][37][38][43][44][45]. To evaluate whether OsMPK4 affects the production of these plant hormones, and thus, regulates herbivore-induced defenses in rice, levels of these phytohormones were quantified in oe-MPK4 lines and WT plants. Basal JA and JA-Ile levels were similar between oe-MPK4 lines and WT plants, whereas levels of these two signals in oe-MPK4 lines were significantly increased compared to levels in WT plants after infestation by SSB: levels of JA and JA-Ile in the two oe-MPK4 lines were 2.48-and 2.32-fold, as well as 5.83-and 4.82-fold, respectively, higher than levels in WT plants at 1.5 h after SSB infestation (Figure 4a,b). Similarly, elicited levels of ET in oe-MPK4 lines were also higher than those in WT plants: 24 h after SSB infestation, ET accumulation in oe-MPK4 lines was increased by 25-28% compared to that in WT plants ( Figure 4c). The overexpression of OsMPK4 significantly enhanced constitutive levels of SA: levels in the two oe-MPK4 lines were 3.84-and 2.82-fold higher than levels in WT plants; after SSB infestation, the difference in the SA levels between oe-MPK4 lines and WT plants diminished, and only one oe-MPK4 line (oe-43) had higher elicited SA levels than did WT plants ( Figure 4d). These results suggest that OsMPK4 modulates the biosynthesis of basal or SSB-elicited JA, JA-Ile, ET, and SA.

Overexpression of OsMPK4 Enhances SSB-Induced Levels of Trypsin Protease Inhibitors (TrypPIs) and Resistance to SSB
TrypPIs, important direct defense compounds, help rice resist herbivores, especially chewing herbivores, and their production is positively regulated by both JA and ET signaling pathways [10,37,38]. Thus, we investigated if OsMPK4 modulates the production of TrypPIs and, eventually, the resistance of rice to the SSB. As expected, the activity of SSB-induced TrypPIs was higher in the two oe-MPK4 lines than that in WT plants (Figure 5a). Consistent with this, SSB caterpillars fed on oe-MPK4 lines gained significantly less mass compared with those fed on WT plants: the mass of larvae fed on the oe-MPK4 lines was only 52-59% of the mass of larvae fed on WT plants ( Figure 5b). These data demonstrate that OsMPK4 positively regulates SSB-induced TrypPIs and, thus, rice resistance to SSB.

Discussion
In the study, we found that OsMPK4 could be induced by mechanical wounding, SSB infestation and JA treatment, but not by SA treatment (Figure 1). The overexpression of OsMPK4 enhanced SSB-elicited and/or basal levels of JA, JA-Ile, ET, and SA in rice (Figure 4), which in turn, increased the activity of TrypPIs and the resistance of rice to the SSB (Figure 5a,b). In addition, the overexpression of OsMPK4 impaired plant growth, shortening root lengths, and reducing stem and root biomasses (Figure 3b-d). The data demonstrate that OsMPK4 functions as a negative regulator of plant growth but a positive regulator of SSB resistance in rice.
MPK4 has been reported to play an important but variable role in regulating defense-related signaling pathways in plants. In Arabidopsis, for instance, AtMPK4 negatively regulates SA levels, but positively modulates JA-and ET-mediated signaling pathways [28,29]. Similarly, NtMPK4 in N. tabacum inhibits the production of ozone-induced SA, but is required for the activation of the JA pathway [42]. By contrast, silencing MPK4 in soybean enhances levels of pathogen-induced SA and simultaneously activates JA signaling [26]; in N. attenuata, NaMPK4 does not influence the biosynthesis of constitutive and herbivore-elicited SA, but does positively regulate the production of herbivore-induced JA [32]. Here, we found that SSB infestation induced the expression of OsMPK4 (Figure 1b), and the overexpression of OsMPK4 increased basal and/or herbivore-induced levels of SA, JA, JA-Ile, and ET in rice ( Figure 4). This demonstrates that the function of OsMPK4 in rice defenses is different from its functions in other plant species. OsMPK4 expression has previously been reported to be induced by Xoo infection and the overexpression of OsMPK4 enhances constitutive and pathogen-elicited SA levels and JA levels in rice [31]. Moreover, JA treatment enhanced OsMPK4 transcript levels only at late treatment stages, and SA treatment did not induce OsMPK4 expression (Figure 1c,d). These findings indicate that OsMPK4 plays an important role in plant defenses against both pathogens and herbivores by functioning upstream of defense-related signaling pathways in a similar manner. That JA induces OsMPK4 suggests that JA may also regulates the activity of this enzyme via positive feedback loops.
As stated above, many studies have demonstrated that MPKs can regulate levels of JA, SA, and ET in plants by influencing the activity of the enzymes related to signal biosynthesis [9,10,18,19]. Moreover, in Arabidopsis, AtMPK4 modulates SA-, JA-, and ET-dependent responses by suppressing the activity of enhanced disease susceptibility 1 (EDS1) and phytoalexin deficient 4 (PAD4), both of which act as activators of the SA pathway and repressors of the ET/JA pathway [29]. Thus, in rice, the regulation of OsMPK4 on these defense-related signaling pathways may occur via similar mechanisms. Interestingly, OsMPK3 has also been found to mediate the biosynthesis of herbivore-induced JA and the resistance of rice to herbivores [10]. This indicates that there is an overlap in the function of the two MPKs in the herbivore-induced defense responses in rice. Future research should elucidate how the two MPKs regulate rice defenses, and what the similarities and the differences are between them.
Accumulated evidence demonstrates that there is antagonism between the JA and SA pathways in many plant species, including rice [38,46,47]. However, we found that the overexpression of OsMPK4 enhanced basal and/or herbivore-elicited levels of both SA and JA ( Figure 4); this finding was consistent with the previously reported result that rice lines that overexpressed OsMPK4 had higher basal and/or pathogen-induced levels of SA and JA than did WT plants [31]. A similar result was observed in soybean [26]. Complicating the results, knocking out or silencing OsMPK4 enhances constitutive and pathogen-elicited SA levels, but slightly decreases pathogen-elicited JA levels, suggesting there is antagonism between the SA and JA pathways [31,48]. The complexity of the findings indicates that, in addition to acting as a regulator of SA-JA antagonism, MPK4 may also function as a regulator of SA and JA synergism in some plant species or under some conditions. Further research should elucidate these mechanisms.
JA and ET signaling pathways have been reported to positively mediate the production of TrypPIs, a kind of direct defense compound that protects rice plants against chewing herbivores, including the SSB [37,38,49]. Therefore, enhanced resistance of oe-MPK4 lines to SSB is probably because of higher levels of JA, JA-Ile, and ET in these lines compared to WT plants; these signals led to increased activity among defensive compounds, including TrypPIs in oe-MPK4 lines.
In addition to its role in plant defense responses, OsMPK4 plays a role in plant growth. In Arabidopsis and soybean (G. max), for instance, the mpk4 mutant is severely dwarfed [26,27]. In the wild tobacco species N. attenuata, silencing NaMPK4 causes plants to be somewhat small in stature [50]. Consistent with these data, the overexpression of BnMPK4 in B. napus increases plant size [41]. Research into the mechanisms underlying the growth phenotype of mpk4 mutants revealed that elevated constitutive defense responses [28], ROS homeostasis deregulation, and photosynthesis damage [27], auxin repression [26], or rapid water loss [50] may explain why these mutants are so small. Unlike the positive effect of MPK4 on plant growth found in other species, we found that the overexpression of OsMPK4 retarded rice growth moderately (Figure 3). This result may be related to the function of OsMPK4 in rice, which is different from that of MPK4 in other plant species, as stated above: the overexpression of OsMPK4 enhanced constitutive SA levels (Figure 4d). Given that the growth retardation of the mpk4 mutant in Arabidopsis is partly related to the accumulation of SA [28], the possibility could not be ruled out that the reduced size of mutants might be, at least in part, due to their higher SA levels than WT plants.
In summary, our findings demonstrate that OsMPK4 plays an important role in plant growth and herbivore-induced defense responses of rice by regulating SA-, JA-, and ET-mediated signaling pathways, and that the function of OsMPK4 in rice, a positive regulator of plant herbivore resistance and a negative regulator of plant growth, is different from the function of MPK4 reported in other plant species, such as Arabidopsis and soybean. Moreover, OsMPK4 may also mediate SA-JA/ET interactions in rice, depending on the stimulus: a stimulus that suppresses the activity of OsMPK4 will cause SA-JA/ET antagonism, whereas a stimulus that induces the activity of OsMPK4 will result in SA-JA/ET synergism; in other words, OsMPK4 may also play an important role in modulating appropriate and specific defense responses in plants to different stresses.

Plant Growth
The rice (Oryza sativa) genotypes used in this study were cv Xiushui 11 wild-type (WT) and two oe-MPK4 transgenic lines (see below). Pre-germinated seeds of WT plants and transgenic lines were cultured in plastic cups (diameter, 8 cm; height, 10 cm) in a climate incubator at 28 ± 2 • C, with a 14 h light phase. Twelve day old seedlings were transferred to 25 L hydroponic boxes with a rice nutrient solution [51]. After 30-35 days, plants were transplanted in individual 500 mL hydroponic plastic pots. Plants were used for experiments 4-5 days after transplanting.

Insects
An SSB colony was originally obtained from a rice field in Hangzhou, China, and maintained on TN1 (a rice variety that is susceptible to SSB) rice seedlings in a controlled climate chamber at 26 ± 2 • C, with a 14 h light phase and 65% relative humidity.

Isolation and Characterization of OsMPK4 cDNA
The full-length cDNA of OsMPK4 was PCR-amplified. The primers MPK4-F (5 -ATAGTCG ACTCCACCTCGTCCT-3 ) and MPK4-R (5 -ATATCTAGAAGGGGATTTGGCTTT-3 ) were designed based on the sequence of OsMPK4. The PCR products were cloned into the pMD19-T vector (TaKaRa, Tokyo, Japan) and sequenced.

Phylogenetic Analysis
For the phylogenetic analysis, the program MEGA 6.0 [52] was used. Protein sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and were aligned using the ClustalW algorithm in MEGA 6.0 (Tempe, AZ, USA). This alignment was then used to generate an unrooted tree using the maximum-likelihood approach [53] (1000 replications).

Generation and Characterization of Transgenic Plants
The full-length cDNA sequence of OsMPK4 was cloned into the pCAMBIA1301 transformation vector to yield an overexpressed construct containing the hygromycin resistance gene hph and the reporter gene encoding β-glucuronidase (GUS) (GenBank: AF234297) as selectable markers ( Figure S3). The vector was used for transforming the rice variety Xiushui 11 by Agrobacterium-mediated transformation system. Homozygous T2 transgenic plants were selected using GUS staining or hygromycin resistance screening [38]. Two T2 homozygous lines (oe-43 and oe-59), each harboring a single insertion (Figure 2a), were used in subsequent experiments.

Plant Treatments
For mechanical wounding, plants were individually pierced with a needle on the lower part of leaf sheaths (about 2 cm long), each 200 times (W). Control plants were not manipulated (C). For SSB treatment, plants were individually infested with a third-instar SSB larva that had been starved for 2 h. Non-infested plants were used as controls (C). For JA and SA treatment, each plant was sprayed with 2 mL of JA (100 µg·mL −1 ) or SA (70 µg·mL −1 ) in 50 mM sodium phosphate buffer (pH 8, with 0.01% Tween). Plants sprayed with 2 mL of the buffer (BUF) were used as controls. Each treatment at each time point was replicated five times.

Measurement of Plant Growth Parameters
Plant growth parameters, including plant height, root length, mass of the above-and belowground part of a plant, and chlorophyll content, of oe-WPK4 lines and WT plants, were measured at 25, 35, 45, and 55 days after plant growth. Plant height and root length were defined as the part of a plant from the stem base to the longest leaf apex and that from the stem base to the longest root tip, respectively. Plants were cut off from the stem base and then the mass of aboveground and belowground (roots) part of plants was measured. For chlorophyll content determination, three leaves with identical leaf positions from each plant and three identical locations from each leaf were measured by a soil-plant analysis development (SPAD) meter. Every experiment at each stage was replicated ten times.

JA, JA-Ile, and SA Analysis
For JA, JA-Ile, and SA analysis, plants of the different genotypes were randomly assigned to SSB and control treatments. Plant stems were harvested at different time points after the start of treatment (see details in Figure 4). Samples (each with about 150 mg) were ground in liquid nitrogen, and JA, JA-Ile, and SA were extracted with ethyl acetate spiked with labeled internal standards (D6-JA, D6-JA-Ile, D4-SA) and analyzed with an HPLC/mass spectrometry/mass spectrometry system following the method described in Lu et al. [54]. Each treatment at each time point was replicated five times.

Ethylene Analysis
Plants of WT and transgenic lines were randomly assigned to SSB and control treatments, and were individually confined with sealed glass cylinders (diameter 4 cm, height 50 cm). The production of ethylene was determined following the same method as described in Lu et al. [55]. Each treatment at each time point was replicated five times.

TrypPI Activity Analysis
Plants of different genotypes were randomly assigned to SSB and control treatments. Stems of oe-MPK4 lines and WT plants (about 0.3 g per sample) were individually harvested 3 days after the start of the treatment. TrypPI concentrations were measured using a radial diffusion assay as described by van Dam et al. [56]. Each treatment was replicated five times.

SSB Performance Measurement
The performance of SSB larvae on oe-MPK4 lines and WT plants was determined by releasing one freshly hatched larva onto each plant. Larval mass (to an accuracy of 0.1 mg) was measured and recorded 12 days after the release of the herbivore. Each treatment was replicated thirty times.

Data Analysis
Differences in data in different lines or treatments were determined by analyzing variance followed by Duncan's multiple range test (or Student's t-test for comparing two treatments). When necessary, data were log-transformed or arcsine-transformed to meet requirements for the homogeneity of variance. All tests were carried out with SPSS software version 20.

Conflicts of Interest:
The authors declare no conflict of interest.