Differential Responses of OsMPKs in IR56 Rice to Two BPH Populations of Different Virulence Levels

The conserved mitogen-activated protein kinase (MAPK) cascades play vital roles in plant defense responses against pathogens and insects. In the current study, the expression profiles of 17 OsMPKs were determined in the TN1 and IR56 rice varieties under the infestation of brown planthopper (BPH), one of the most destructive hemimetabolous rice pests. The virulent IR56 BPH population (IR56-BPH) and the avirulent TN1 BPH population (TN-BPH) were used to reveal the roles of OsMPKs in the compatible (IR56-BPH infested on the TN1 and IR56 rice varieties, and TN1-BPH infested on the TN1 rice variety) and the incompatible (TN1-BPH infested on the IR56 rice variety) interaction. The statistical analysis revealed that rice variety, BPH population type, and infestation period have significant effects on the transcription of OsMPKs. Out of these genes, five OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) were found to exhibit upregulated expression only during incompatible interaction. Six OsMPKs (OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13) were associated with both incompatible and compatible interactions. The transcription analysis of salicylic acid, jasmonic acid, and ethylene phytohormone signaling genes revealed their roles during the rice–BPH interactions. The upregulated expression of OsC4H, OsCHS, and OsCHI in the incompatible interaction implied the potential defense regulatory roles of phenylpropanoids. In both varieties, the elevated transcript accumulations of OsGST and OsSOD, and the increased enzyme activities of POD, SOD, and GST at 1 day post-infestation (dpi), but not at 3 dpi, indicated that reactive oxygen species (ROS) signaling might be an early event in rice–BPH interactions. Furthermore, upregulated transcription of OsLecRK3 and OsLecRK4 was found only during an incompatible interaction, suggesting their involvement in the BPH resistance response in the IR56 rice variety. Lastly, based on the findings of this study, we have proposed a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions, respectively.


Introduction
The brown planthopper (Nilaparvata lugens Stål, hereafter referred to as BPH) is the most harmful pest to rice (Oryza sativa), and causes huge crop damage and billions of dollars of economic loss in Asia [1]. It is a typical monophagous vascular feeder that sucks the rice phloem sap, resulting in the wilting and fatal drying of rice plants, a phenomenon known as 'hopperburn' [1]. Besides this, BPH causes indirect damage to rice, as it is the carrier of the rice grassy stunt virus and the rice ragged stunt virus [1]. The application of insecticides is the most common practice to restrict and eradicate BPH infestations in rice. In recent years, the abuse of chemical insecticides has resulted in many adversities, such as insecticide resistance development, insect resurgence, the elimination of natural evaluated. Additionally, the temporal expression regulation of OsMPKs that have been exposed to exogenous phytohormones has been estimated. Lastly, a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions has been proposed. Int phytohormones has been estimated. Lastly, a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions has been proposed.

Expression Profiling of OsMPKs in Response to BPH Infestations
The effects of the rice varieties, the BPH populations, and the infestation time periods (0, 1, and 3 days post-infestation, dpi) on the transcription of OsMPKs were analyzed by performing a threeway analysis of variance (ANOVA) analysis (Table S1). The ANOVA analysis revealed that rice varieties, BPH population, dpi, and their interactions (rice varieties × BPH population, rice varieties × dpi, BPH population × dpi, and rice varieties × BPH population × dpi) had no significant effects (p >0.05, F values are shown in Table S1) on the expression of OsMPK6, OsMPK10, OsMPK11, OsMPK15, and OsMPK17 ( Figure S1). In contrast, all main effects and interaction effects on the expression of OsMPK3, OsMPK5, OsMPK7, OsMPK12, OsMPK13, OsMPK14, and OsMPK16 ( Figure 2A, Table S1) were significant as revealed by the three-way ANOVA analysis. Out of these significant main and interaction effects, the effects of rice variety on OsMPK7, OsMPK14, and OsMPK16 expression, the effects of dpi on OsMPK3 and OsMPK5 expression, and the effects of BPH population type on OsMPK12 and OsMPK13 expression were higher as compared with the other main and interaction effects. Further, the Tukey′s honestly significant difference (HSD) post-hoc comparisons of these seven genes confirmed three types of upregulated expression patterns. Firstly, OsMPK3, OsMPK7, OsMPK14, and OsMPK16 were significantly induced in the incompatible interaction, and OsMPK7 and OsMPK16 exhibited a time-dependent upregulation. Secondly, elevated mRNA levels of OsMPK5 were found in both compatible and incompatible interactions. In addition, time-dependent upregulation was found in the IR56-BPH-infested TN1 rice and in the TN1-BPH-infested IR56 rice. Compared with TN1 rice, IR56 rice had higher expression levels of OsMPK5 after the TN1-BPH infestation, but lower levels of expression after the IR56-BPH infestation. Thirdly, time-independent induced expression of OsMPK12 and OsMPK13 was observed in both compatible and incompatible interactions. Compared to those in TN1 rice, higher mRNA levels of OsMPK12 and OsMPK13 were found in the incompatible interaction. However, a lower mRNA level of OsMPK13 was found in IR56 rice at 3 dpi of IR56-BPH infestation.

Expression Profiling of OsMPKs in Response to BPH Infestations
The effects of the rice varieties, the BPH populations, and the infestation time periods (0, 1, and 3 days post-infestation, dpi) on the transcription of OsMPKs were analyzed by performing a three-way analysis of variance (ANOVA) analysis (Table S1). The ANOVA analysis revealed that rice varieties, BPH population, dpi, and their interactions (rice varieties × BPH population, rice varieties × dpi, BPH population × dpi, and rice varieties × BPH population × dpi) had no significant effects (p >0.05, F values are shown in Table S1) on the expression of OsMPK6, OsMPK10, OsMPK11, OsMPK15, and OsMPK17 ( Figure S1). In contrast, all main effects and interaction effects on the expression of OsMPK3, OsMPK5, OsMPK7, OsMPK12, OsMPK13, OsMPK14, and OsMPK16 ( Figure 2A, Table S1) were significant as revealed by the three-way ANOVA analysis. Out of these significant main and interaction effects, the effects of rice variety on OsMPK7, OsMPK14, and OsMPK16 expression, the effects of dpi on OsMPK3 and OsMPK5 expression, and the effects of BPH population type on OsMPK12 and OsMPK13 expression were higher as compared with the other main and interaction effects. Further, the Tukey s honestly significant difference (HSD) post-hoc comparisons of these seven genes confirmed three types of upregulated expression patterns. Firstly, OsMPK3, OsMPK7, OsMPK14, and OsMPK16 were significantly induced in the incompatible interaction, and OsMPK7 and OsMPK16 exhibited a time-dependent upregulation. Secondly, elevated mRNA levels of OsMPK5 were found in both compatible and incompatible interactions. In addition, time-dependent upregulation was found in the IR56-BPH-infested TN1 rice and in the TN1-BPH-infested IR56 rice. Compared with TN1 rice, IR56 rice had higher expression levels of OsMPK5 after the TN1-BPH infestation, but lower levels of expression after the IR56-BPH infestation. Thirdly, time-independent induced expression of OsMPK12 and OsMPK13 was observed in both compatible and incompatible interactions. Compared to those in TN1 rice, higher mRNA levels of OsMPK12 and OsMPK13 were found in the incompatible interaction. However, a lower mRNA level of OsMPK13 was found in IR56 rice at 3 dpi of IR56-BPH infestation.  OsMPKs showing significant differences in any of the main effects or any of the interaction effects. * represents a significant difference with TN1 rice (p < 0.05); different letters above the bars represent significant differences (p < 0.05) among each TN1-BPH-and IR56-BPH-infested rice variety, and the control (CK, rice not infested with BPH). No significant difference among samples was not denoted by any symbol.
Furthermore, the expression of OsMPK1, OsMPK2, OsMPK4, OsMPK8, and OsMPK9 was significantly affected by one of the variances and/or their interactions ( Figure 2B, Table S1). The Tukey's HSD post-hoc comparisons confirmed the downregulated expression of OsMPK2 in the IR56-BPH-infested IR56 rice (1 dpi), but upregulated expression of OsMPK1, OsMPK4, OsMPK8, and OsMPK9 in both rice varieties (1 or 3 dpi). Of these induced genes, OsMPK1 was upregulated in the compatible interaction (IR56 rice infested by TN1-BPH) at 1 dpi. OsMPK9 was upregulated in both compatible interactions and the incompatible interaction at 3 dpi. Similarly, induced expression of OsMPK8 was found in both compatible and incompatible interactions at 1 dpi. The expression of OsMPK4 was time-dependently upregulated in both compatible (TN1 rice infested by IR56-BPH) and incompatible interactions.
Briefly, the expression of five OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) in the incompatible interaction and six OsMPKs (OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13) in both compatible and incompatible interactions was induced under the BPH infestations ( Figure 2). These results suggest that the former five OsMPKs might be involved in the resistance response to BPH in IR56 rice against TN1-BPH, whereas their failure to achieve upregulated expression under IR56-BPH infestation might have caused the rice's resistance to break down.
In order to check the effect of tissue injury on the expression of OsMPKs in the TN1 and IR56 varieties, wound stress was imposed by mimicking BPH piercing (pricking the rice stems 100 times with a fine needle; no pricked plants were considered as controls). The two-way (rice varieties versus days post-injury, dpi) ANOVA analysis revealed no significant effects of rice varieties and dpi, and no significant effect of rice varieties × dpi interaction, on the expression of OsMPK1, OsMPK2, OsMPK9, OsMPK10, OsMPK12, OsMPK14, OsMPK15, and OsMPK17 (p > 0.05, F-values for all main and interaction effects are not shown, Figure S2A). Furthermore, there was no significant effects of rice varieties, and rice varieties × dpi interaction, on the expression of OsMPK3-5 and OsMPK11 (p > 0.05, F-values for rice varieties and interaction effects are not shown, Figure S1B), whereas a significant effect of dpi (OsMPK3, F 1,12 = 68.44, p < 0.001; OsMPK4, F 1,12 = 20.42, p = 0.002; OsMPK5, F 1,12 = 135.39, p < 0.001; OsMPK11, F 1,12 = 4.38, p = 0.037) was observed for the same. A statistical analysis also revealed that no significant effect of rice varieties × dpi interaction (F 2,12 = 3.59, p = 0.06), but a significant effect of rice varieties (F 1,12 = 4.76, p = 0.049) and dpi (F 2,12 = 5.62, p = 0.019), was found on OsMPK6 expression. Tukey's HSD post-hoc comparisons confirmed a significant (p < 0.01) increase in the mRNA level of OsMPK3 at day 1 in TN1 rice, and of OsMPK5 at 1 and 3 dpi in both rice varieties, as compared with the control (without pricking; Figure S2B). On the contrary, there were significantly (p < 0.01) decreased levels of OsMPK4 in both rice varieties at 3 dpi, and of OsMPK6 in TN1 rice at 1 dpi. For four OsMPKs (OsMPK7, OsMPK8, OsMPK13, and OsMPK16), the effects of rice varieties, dpi, and rice variety × dpi interaction (p < 0.01) were statistically significant ( Figure S2C). Wounding significantly (p < 0.05) induced the transcript levels of OsMPK7 and OsMPK16 in IR56 rice, OsMPK8 in TN1 rice, and OsMPK13 in both rice varieties. After wounding, IR56 rice had higher (p < 0.01) mRNA levels of OsMPK7 and OsMPK16 than those in TN1 rice, and vice versa for OsMPK8 and OsMPK13. These results revealed that six OsMPKs (OsMPK3, OsMPK5, and OsMPK13 in both rice varieties, OsMPK8 in TN1 rice, and OsMPK7 and OsMPK16 in IR56 rice) were upregulated in response to wounding in rice, while two OsMPKs (OsMPK4 in both rice varieties and OsMPK6 in TN1 rice) were downregulated.

Induced Expression of Genes Associated with Phytohormone Signaling and Phenylpropanoid Synthesis under BPH Infestation
Insect herbivory and activation of MAPKs can influence the phytohormone balance and secondary metabolite synthesis in plants [24]. A two-way ANOVA analysis of the expressions of phenylalanine ammonia lyase 1 (OsPAL1), enhanced disease susceptibility 1 (OsEDS1), and nonexpresser of pathogenesis-related genes 1 (OsNPR1), which are involved in SA signaling, revealed the positive role of SA signaling in rice-BPH interactions (Table S2). In IR56 rice, infestation of TN1-BPH (the incompatible interaction) resulted in significant elevated transcript accumulations of OsPAL1 (F 2,12 = 11.90, p = 0.001; F 2,12 = 99.03, p < 0.01) and OsEDS1 (F 2,12 = 6.99, p = 0.009; F 2,12 = 62.12, p < 0.01) at both 1 and 3 dpi ( Figure 3A), and of OsNPR1 (F 2,12 = 4.28, p = 0.039) at 1 dpi. However, IR56-BPH infestation (a compatible interaction) did not result in any significant (p > 0.05, F value not shown) induced expression of the three genes. Similarly, in TN1 rice, infestation of TN1-BPH or IR56-BPH (compatible interactions) caused no significant upregulation (p > 0.05, F value not shown) of OsPAL1, OsEDS1, and OsNPR1 at either 1 or 3 dpi ( Figure 3A).  The expression of 1-aminocyclopropane-1-carboxylate synthase 2 (OsACS2), ethylene-insensitive gene 2 (OsEIN2), and ethylene-responsive transcription factor 3 (OsERF3), which are ET signaling genes, was determined. A statistical analysis revealed that the three genes were significantly induced during incompatible rice-BPH interactions at both 1 and 3 dpi ( Figure 3C, Table S2). In IR56 rice, infestation of TN1-BPH resulted in a significant increase in the transcription of OsACS2 (F2,12 = 7.46, p = 0.007; Data are denoted as mean ± SE and analyzed by two-way ANOVA followed by Tukey's HSD post-hoc test. Letters above the bars indicate the significant difference (p < 0.05) in expression among the TN1-BPH-and IR56-BPH-infested rice varieties, and the control (rice not infested with BPH). An asterisk (*) denotes the significant difference (p < 0.05) in expression with respect to different rice varieties under the same infested BPH population at the specific time point. No significant difference among samples is not denoted by any symbol.
The expression of 1-aminocyclopropane-1-carboxylate synthase 2 (OsACS2), ethylene-insensitive gene 2 (OsEIN2), and ethylene-responsive transcription factor 3 (OsERF3), which are ET signaling genes, was determined. A statistical analysis revealed that the three genes were significantly induced during incompatible rice-BPH interactions at both 1 and 3 dpi ( Figure 3C, Table S2). In IR56 rice, infestation of TN1-BPH resulted in a significant increase in the transcription of OsACS2 (F 2,12 = 7.46, p = 0.007; F 2,12 = 41.62, p < 0.01), OsEIN2 (F 2,12 = 14.36, p < 0.01; F 2,12 = 53.71, p < 0.01), and OsERF3 (F 2,12 = 119.7, p < 0.01; F 2,12 = 276.03, p < 0.01) at both 1 and 3 dpi. However, when infested with IR56-BPH, only OsERF3 (F 1,12 = 539.87, p < 0.01) was found to be significantly induced in IR56 rice at 3 dpi, while OsACS2 and OsEIN2 showed no significant induced expression at either 1 or 3 dpi. In contrast, none of these three genes showed any significant transcript accumulations in TN1 rice under infestation of TN1-BPH or IR56-BPH. Briefly, the induced expression of the SA and ET signaling genes during the incompatible, but not during the compatible, rice-BPH interactions suggests that they have positive roles in rice's resistance responses. Conversely, significant upregulation of JA signaling genes at 1 dpi, but not at 3 dpi, and during both compatible and incompatible rice-BPH interactions, suggests that JA signaling might be involved in an early response to BPH feeding in rice.

Expression Profiling and Enzyme Activity of ROS-Signaling Genes under BPH Infestation
A two-way ANOVA analysis of the expression of two ROS-responsive genes, superoxide dismutase (OsSOD) and glutathione S-transferase (OsGST), in the TN1 and IR56 rice varieties under BPH infestation revealed that ROS signaling is associated with rice-BPH interactions as an early response, irrespective of the infested BPH population type. In both TN1 and IR56 rice, infestation of TN1-BPH or IR56-BPH resulted in the significant upregulation of OsSOD (F 2,12 = 25.13, p < 0.01) and OsGST (F 2,12 = 18.95, p < 0.01) at 1 dpi, whereas no significant induced expression of both the genes was observed at 3 dpi ( Figure 4A, Table S2). Furthermore, the enzyme activity of POD (F 2,12 = 24.93, p < 0.01) and SOD (F 2,12 = 5.93, p = 0.016) was found to be significantly increased at 1 dpi in both the TN1 and IR56 rice varieties, irrespective of the infested BPH population type ( Figure 5, Table S2). In TN1 rice, under TN1-BPH or IR56-BPH infestation, both POD and SOD showed no significant change in the enzyme activity at 3 dpi as compared to the control plants. However, in IR56 rice, infestation of either TN1-BPH or IR56-BPH caused a significant decrease in the enzyme activity of SOD (F 2,12 = 65.04, p < 0.01) at 3 dpi. Furthermore, in TN1 rice, infestation by both BPH populations caused a significant increase in GST enzyme activity (F 2,12 = 87.80, p < 0.01) at 1 dpi. In IR56 rice, the infestation of IR56-BPH caused a significant increased activity of GST (F 2,12 = 133.79, p < 0.01) at 1 dpi, while TN1-BPH infestation resulted in no significant change in GST activity. Conversely, at 3 dpi, GST activity in both the TN1 and IR56 rice varieties was significantly decreased (F 2,12 = 83.92, p < 0.01) in response to BPH infestation, irrespective of the population type. In addition, the enzyme activity of CAT in TN1 and IR56 rice was found to be significantly downregulated (F 2,12 = 8.42, p = 0.005; F 2,12 = 8.03, p = 0.006) under TN1-BPH infestation at both 1 and 3 dpi as compared with the control. However, infestation of IR56-BPH caused no significant change in CAT activity in both TN1 and IR56 rice at 1 dpi, while resulting in decreased activity at 3 dpi in TN1 rice. These results suggest that, like JA signaling, ROS might play a role in the early responses of rice to BPH feeding.
incompatible, but not during the compatible, rice-BPH interactions suggests that they have positive roles in rice's resistance responses. Conversely, significant upregulation of JA signaling genes at 1 dpi, but not at 3 dpi, and during both compatible and incompatible rice-BPH interactions, suggests that JA signaling might be involved in an early response to BPH feeding in rice.
The expression of the phenylpropanoid biosynthesis pathway genes cinnamate-4-hydroxylase (OsC4H), chalcone synthase (OsCHS), and chalcone isomerase (OsCHI) showed that all three genes were significantly upregulated during the incompatible rice-BPH interaction. In IR56 rice, OsC4H (F2,12 = 59.92, p < 0.01; F2,12 = 26.14, p < 0.01), OsCHS (F =162.64.7, p < 0.01; F2,12 = 109.95, p < 0.01), and OsCHI (F2,12 = 54.08, p < 0.01; F2,12 = 37.38, p < 0.01) were found to exhibit elevated expression levels at both 1 and 3 dpi under TN1-BPH infestation ( Figure 4B, Table S2). However, under IR56-BPH infestation (a compatible interaction), OsCHI (F2,12 = 14.64, p < 0.01) showed significant induced expression at 3 dpi, whereas the other two genes did not show any upregulation in their expression levels at 1 or 3 dpi. On the other hand, none of these three genes showed any significant induced expression in TN1 rice under the infestation of either TN1-BPH or IR56-BPH. The induced expression of OsC4H, OsCHS, and OsCHI during the incompatible interaction suggests that phenylpropanoids have a positive role in rice-BPH interactions.  Data are denoted as mean ± SE and analyzed by two-way ANOVA followed by Tukey's HSD post-hoc test. Letters above the bars indicate a significant difference (p < 0.05) in expression among the TN1-BPH-and IR56-BPH-infested rice varieties, and control (rice not infested with BPH). An asterisk (*) denotes a significant difference (p < 0.05) in expression with respect to the different rice varieties under the same infested BPH population at the specific time point. No significant difference among samples are not denoted by any symbol.

Differential Transcription of OsLecRKs in the Two Rice Varieties under BPH Infestation
A separate study in our lab revealed that both IR56 rice and TN1 rice possess three OsLecRKs (OsLecRK1, OsLecRK3, and OsLecRK4) out of the four from the Bph3 gene cluster (unpublished data). The transcription responses of these three OsLecRKs in both rice varieties were analyzed under BPH infestation. In IR56 rice, the TN1-BPH infestation (the incompatible interaction) significantly upregulated the expression of OsLecRK3 (F 2,12 = 51.95, p < 0.01; F 2,12 = 56.22, p < 0.01) and OsLecRK4 (F 2,12 = 115.12, p < 0.01; F 2,12 = 115.73, p < 0.01) at 1 and 3 dpi, whereas no significant upregulation of OsLecRK1 (p > 0.05, F value not shown) was observed ( Figure 6, Table S2). On the other hand, infestation with IR56-BPH did not cause any significant induced expression of any of these three genes in IR56 rice either at 1 or 3 dpi. Similarly, in TN1 rice, infestation of TN1-BPH or IR56-BPH (compatible interactions) induced no significant upregulation of the OsLecRKs. From the results, it could be suggested that OsLecRK3 and OsLecRK4 play an important role in the resistance of IR56 rice to TN1-BPH, but their activation might be hindered by IR56-BPH during the infestation, resulting in a breakdown in the IR56 rice s resistance.
the enzyme activity at 3 dpi as compared to the control plants. However, in IR56 rice, infestation of either TN1-BPH or IR56-BPH caused a significant decrease in the enzyme activity of SOD (F2,12 = 65.04, p < 0.01) at 3 dpi. Furthermore, in TN1 rice, infestation by both BPH populations caused a significant increase in GST enzyme activity (F2,12 = 87.80, p < 0.01) at 1 dpi. In IR56 rice, the infestation of IR56-BPH caused a significant increased activity of GST (F2,12 = 133.79, p < 0.01) at 1 dpi, while TN1-BPH infestation resulted in no significant change in GST activity. Conversely, at 3 dpi, GST activity in both the TN1 and IR56 rice varieties was significantly decreased (F2,12 = 83.92, p < 0.01) in response to BPH infestation, irrespective of the population type. In addition, the enzyme activity of CAT in TN1 and IR56 rice was found to be significantly downregulated (F2,12 = 8.42, p = 0.005; F2,12 = 8.03, p = 0.006) under TN1-BPH infestation at both 1 and 3 dpi as compared with the control. However, infestation of IR56-BPH caused no significant change in CAT activity in both TN1 and IR56 rice at 1 dpi, while resulting in decreased activity at 3 dpi in TN1 rice. These results suggest that, like JA signaling, ROS might play a role in the early responses of rice to BPH feeding. in the IR56 and TN1 rice varieties under infestation of the two different BPH populations. Data are denoted as mean ± SE and analyzed by two-way ANOVA followed by Tukey's HSD post-hoc test. Letters above the bars indicate a significant difference (p < 0.05) in enzyme activities among TN1-BPHand IR56-BPH-infested rice, and the control (rice not infested with BPH). An asterisk (*) denotes a significant difference (p < 0.05) in enzyme activity with respect to the different rice varieties under the in the IR56 and TN1 rice varieties under infestation of the two different BPH populations. Data are denoted as mean ± SE and analyzed by two-way ANOVA followed by Tukey's HSD post-hoc test. Letters above the bars indicate a significant difference (p < 0.05) in enzyme activities among TN1-BPHand IR56-BPH-infested rice, and the control (rice not infested with BPH). An asterisk (*) denotes a significant difference (p < 0.05) in enzyme activity with respect to the different rice varieties under the same infested BPH population at the specific time point. No significant difference among samples are not denoted by any symbol. EU stands for enzyme unit.

Expression Modulation of the BPH-Induced OsMPKs in Response to the Exogenous Treatment of Phytohormones
Out of 11 BPH-induced OsMPKs, the exogenous treatment of SA resulted in significant activation (p < 0.05) of two OsMPKs (OsMPK4 and OsMPK5) in both rice varieties, and of four OsMPKs (OsMPK9, OsMPK12, OsMPK14, and OsMPK16) in IR56 rice ( Figure 7A, Table S3). Similarly, treatment of methyl jasmonate (MeJA) significantly activated three OsMPKs (OsMPK3, OsMPK5, and OsMPK13) in both rice varieties, and of four OsMPKs (OsMPK1, OsMPK4, OsMPK7, and OsMPK9) in IR56 rice ( Figure 7B, Table S4). Likewise, two OsMPKs (OsMPK9 and OsMPK12) showed upregulated expression in both rice varieties under exogenous treatment of ethephon, and five OsMPKs (OsMPK4, OsMPK7, OsMPK13, OsMPK14, and OsMPK16) exhibited significant induced expression in IR56 rice ( Figure 7C, Table S5). These results showed that the number of OsMPKs triggered by the exogenous treatment of phytohormones was higher in IR56 rice as compared to TN1 rice.
(F2,12 = 115.12, p < 0.01; F2,12 = 115.73, p < 0.01) at 1 and 3 dpi, whereas no significant upregulation of OsLecRK1 (p > 0.05, F value not shown) was observed ( Figure 6, Table S2). On the other hand, infestation with IR56-BPH did not cause any significant induced expression of any of these three genes in IR56 rice either at 1 or 3 dpi. Similarly, in TN1 rice, infestation of TN1-BPH or IR56-BPH (compatible interactions) induced no significant upregulation of the OsLecRKs. From the results, it could be suggested that OsLecRK3 and OsLecRK4 play an important role in the resistance of IR56 rice to TN1-BPH, but their activation might be hindered by IR56-BPH during the infestation, resulting in a breakdown in the IR56 rice′s resistance. Figure 6. The expression patterns of OsLecRKs in the IR56 and TN1 rice varieties under infestation of two different BPH populations. Data are denoted as mean ± SE and analyzed by two-way ANOVA followed by Tukey's HSD post hoc test. Letters above the bars indicate a significant difference (p < 0.05) in expression among TN1-BPH-and IR56-BPH-infested rice, and the control (rice not infested with BPH). An asterisk (*) denotes a significant difference (p < 0.05) in expression with respect to the different rice varieties under the same infested BPH population at the specific time point. No significant difference among samples are not denoted by any symbol.

Expression Modulation of the BPH-Induced OsMPKs in Response to the Exogenous Treatment of Phytohormones
Out of 11 BPH-induced OsMPKs, the exogenous treatment of SA resulted in significant activation (p < 0.05) of two OsMPKs (OsMPK4 and OsMPK5) in both rice varieties, and of four OsMPKs (OsMPK9, OsMPK12, OsMPK14, and OsMPK16) in IR56 rice ( Figure 7A, Table S3). Similarly, treatment of methyl jasmonate (MeJA) significantly activated three OsMPKs (OsMPK3, OsMPK5, and OsMPK13) in both rice varieties, and of four OsMPKs (OsMPK1, OsMPK4, OsMPK7, and OsMPK9) in IR56 rice ( Figure 7B, Table S4). Likewise, two OsMPKs (OsMPK9 and OsMPK12) showed upregulated expression in both rice varieties under exogenous treatment of ethephon, and five OsMPKs (OsMPK4, OsMPK7, OsMPK13, OsMPK14, and OsMPK16) exhibited significant induced expression in IR56 rice ( Figure 7C, Table S5). These results showed that the number of OsMPKs triggered by the exogenous treatment of phytohormones was higher in IR56 rice as compared to TN1 rice.

Discussion
In the present study, the temporal expression profiles of OsMPKs in two contrasting rice varieties were determined in response to BPH infestation at varied virulence levels. The OsMPK expression revealed that 11 OsMPKs (OsMPK1, OsMPK3-5, OsMPK7-9, OsMPK12-14, and OsMPK16) were upregulated in the incompatible interaction. Of them, upregulation of OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13 was also observed in the compatible interactions. Thus, the induced expression of these genes suggested the involvement of multiple OsMPKs in response to

Discussion
In the present study, the temporal expression profiles of OsMPKs in two contrasting rice varieties were determined in response to BPH infestation at varied virulence levels. The OsMPK expression revealed that 11 OsMPKs (OsMPK1, OsMPK3-5, OsMPK7-9, OsMPK12-14, and OsMPK16) were upregulated in the incompatible interaction. Of them, upregulation of OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13 was also observed in the compatible interactions. Thus, the induced expression of these genes suggested the involvement of multiple OsMPKs in response to BPH-feeding. Differential transcription of multiple OsMPKs, including OsMPK5, OsMPK12, OsMPK13, and OsMPK17, has been reported in rice in response to BPH infestation [28]. Further, four MAPKs (SIPK and WIPK in tobacco, and LeMPK1 and LeMPK2 in tomato) have been reported to be involved in defense responses to the chewing insect Manduca sexta by regulating the expression of the downstream defense genes, secondary metabolite genes, and phytohormone signaling [24,29]. Additionally, OsMPK3, OsMPK6, and seven homologs (CaMAPK2-3, CaMAPK5, CaMAPK7-9, and CaMAPK15) in chickpea have been identified to be induced in response to insect infestations [30,31]. Furthermore, nine OsMPKs (OsMPK2, OsMPK4-5, OsMPK7-8, OsMPK12-13, OsMPK15, and OsMPK17) were reported to show induced expression after infection of Magnaporthe grisea, whereas three OsMPKs (OsMPK5, OsMPK13, and OsMPK17) were induced by the treatment of both virulent and avirulent fungal inoculations [32]. Activation of OsMPK4, OsMPK5, OsMPK7, OsMPK8, and OsMPK13 was found to be common between the current study and the experimental results in that of Reyna and Yang [32]. Thus, the activation of OsMPK5, OsMPK13, and OsMPK17 might be associated with both insect and pathogen resistance in rice. Moreover, the induced expression of multiple OsMPKs in response to the BPH infestation in the current study is in accordance with previously reported studies (Table 1). M. grisea [32] and BPH infestation [28] The differential transcript patterns of OsMPKs during rice-BPH interactions suggests that OsMPK expression can be correlated with the infested BPH population types. During the incompatible interaction, a higher number of OsMPKs exhibited induced expression as compared to the compatible interactions. These results suggest that, during the compatible interactions, the virulent BPH succeeded in suppressing or breaking down the rice's resistance, possibly by employing explicit effectors. The presence of proteinaceous substances in BPH saliva suggests that the salivary secretions might serve as a source of effectors to outrun rice defenses. For instance, the salivary protein NlSEF1 with an EF-hand Ca 2+ -binding domain and the endo-β-1,4-glucanase (NlEG1) were reported to suppress rice defenses by manipulating defense signaling, including SA and JA [37,38]. Moreover, the virulence of pathogens and the host resistance responses are usually host-specific or isolate-specific, which function among discrete races of a pathogen and distinct genotypes or cultivars of a plant species [39]. Further, effector-triggered immunity (ETI) and effector-triggered susceptibility (ETS) are host-specific in nature [40]. Thus, the IR56-BPH that had evolved its virulence against IR56 rice might have broken down the rice's resistance by following a mechanism similar to ETS. In addition, changing the host, i.e., infesting TN1 rice with IR56-BPH, and IR56 rice with TN1-BPH, might have impaired the BPH effector-mediated ETS, and, thus, activated the cross-population responsive OsMPKs (OsMPK4, OsMPK8, and OsMPK9) in the rice varieties.
In the current study, the analysis of the transcript accumulations of genes involved in SA, JA, and ET signaling during the rice-BPH interactions suggested that BPH resistance in rice is possibly modulated by interplay between SA, JA, and ET. Significant upregulation of the three SA signaling genes OsPAL1, OsEDS1, and OsNPR1 during the incompatible interaction suggested the intrinsic role of SA signaling in BPH resistance responses in rice. OsPAL1 is a crucial enzyme in the phenylpropanoid pathway via which plants synthesize SA [41]. OsEDS1 plays a major role in conferring SA-dependent resistance against pathogens, while OsNPR1 is a key gene involved in SA-mediated systemic acquired resistance (SAR) [42,43]. Similarly, the expression of OsACS2, OsEIN2, and OsERF3 was significantly upregulated during the incompatible interaction. OsEIN2 positively regulates ET signaling and the production of ROS and phytoalexins, whereas OsERF3 has been reported to act as a central switch in rice to direct plant defense responses in a herbivore-specific manner by controlling many crucial pathways, including MAPK activation and ET, SA, JA, and H 2 O 2 signaling [44,45]. Also, the activation of the ET signaling pathway was observed in rice under BPH infestation that, in turn, upregulated the transcription of the BPH-induced gene Bphi008a [28]. Induced expression of OsPAL1, OsEDS1, and OsNPR1, along with other SA-responsive genes, was observed during the Bph14-mediated resistance that triggered the SA signaling pathways in a resistant rice variety [46]. Similarly, expression of OsACS1, OsEIN2, and OsERF1 was found to be elevated in a resistant rice under Meloidogyne graminicola infection [47]. Conversely, the analysis of the expression of OsAOS1, OsAOS2, and OsLOX1 during the rice-BPH interactions revealed that induced activation of JA biosynthesis genes is an early event during rice-BPH interactions and is independent of the BPH population type. These findings from the current work are in accordance with some of the previously reported results, where wounding or herbivory by chewing insects profoundly induced JA biosynthesis, whereas BPH infestation has resulted in either a very weak JA burst or the JA signaling being negatively correlated with BPH resistance in rice [44,48,49]. Furthermore, a cDNA microarray analysis confirmed that BPH infestation in the resistant rice variety RH induced SA biosynthesis genes and SA levels, but not JA biosynthesis genes or intrinsic JA levels [50]. Thus, from our experimental findings, we hypothesize that SA and ET signaling might play an important role in the BPH resistance response in rice.
Elevated expression of the key phenylpropanoid biosynthesis genes during the incompatible rice-BPH interaction suggested the potential role of secondary metabolite production in rice in response to BPH infestation. Moreover, upregulated expression of OsC4H, OsCHS, and OsCHI only during the incompatible interaction suggested a role for phenylpropanoids in BPH resistance. Increased production of secondary metabolites, including phenolamides and p-coumaroylputrescine, has been reported in rice under the infestation of the chewing pests Spodoptera mauritia and Parnara guttata [51]. Similarly, in response to BPH infestation, upregulation of the PAL gene and the subsequent production of phenylpropanoids and polyphenols were reported by Liu et al. [52]. The analysis of the expression of ROS-responsive genes and the estimation of the ROS-responsive enzyme activities during the rice-BPH interactions suggested that the generation of ROS is an early event in rice-BPH interactions and might be associated with the basal defense response. The upregulated expression of OsGST and OsSOD and the increased enzyme activity of POD and SOD at 1 dpi, but not at 3 dpi, during both compatible and incompatible rice-BPH interactions indicate that ROS accumulation is an early event in rice-BPH interactions and not BPH-population-specific in rice.
In the current work, OsLecRK3 and OsLecRK4 exhibited induced expression only during incompatible rice-BPH interactions, indicating their functionality in BPH resistance in IR56 rice. Similar expression patterns were reported for OsLecRK1-3 in RH rice under BPH infestation [7]. Further, the LecRKs belong to a specific pattern recognition receptor (PRR) family and are upstream of MAPKs [53]. In our study, the induced expression of OsLecRKs during the incompatible rice-BPH interaction suggested potential OsLecRK and OsMPK interactions in the BPH resistance response. In Arabidopsis, AtLecRK-IX2 regulated the activation of AtMAPK3 and AtMPK6 in response to flg22 treatment [53]. In pepper, CaLecRK-S.5, which confers broad-spectrum resistance, regulated the activation of CaMK1 and CaMK2 [54]. Thus, we propose that OsLecRKs possibly regulate rice's resistance against BPH infestation by activating downstream MAPKs. However, more in-depth experiments, including interaction studies between OsLecRKs and OsMPKs, are needed to confirm their association with respect to BPH resistance in rice.
The differential expression of the BPH-induced OsMPKs in response to exogenous treatment of signal molecules, including SA, JA, and ET, revealed their potential role in mediating different hormone signals in rice. Two OsMPKs (OsMPK4 and OsMPK9) were found to be induced in IR56 rice by the treatment of all three phytohormones, suggesting that they have prospective roles in influencing SA, JA, and ET interplay. Similar results were reported by Reyna and Yang [32], where exogenous treatment of different signaling molecules induced the expression of blast fungus responsive OsMPK5, OsMPK12, and OsMPK13. Plant MAPKs have been reported to regulate multiple phytohormone signaling pathways, and some of them act as the nexus for two or more hormone signaling pathways [55]. OsMPK3 and OsMPK6 have been reported to act as the nexus in the interplay of auxin and cytokinin [56]. Moreover, the discrimination in the number of OsMPK-induced expressions by the exogenous phytohormone treatments between TN1 rice and IR56 rice might be because additional OsMPKs are involved in phytohormone signaling in the resistant rice as compared to the susceptible one.
In our attempt to elucidate the functionality of the induced OsMPKs during the rice-BPH interactions, we performed a chemical inhibition (non-specific and broad-spectrum) of the rice MAPKs by using a mixture of PD98059 and Genistein. PD98059 is a selective inhibitor of MEK1 and MEK2; thus, it can suppress the activation of downstream MEK1-or MEK2-dependent MAPKs [57]. On the other hand, Genistein is a non-specific MAPK inhibitor (kinase inhibitor) that can suppress the activation of MAPKs in plants [58]. Application of the mixture of PD98059 and Genistein resulted in the transient downregulation of all five candidate OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) ( Figure S3A). The BPH bioassays with TN1-BPH or IR56-BPH on the inhibitor-mix-treated plants of both TN1 and IR56 rices were found to be non-significant as compared with the controls (untreated) (Figure S3B,C). The possible reason behind this might be because of the transient nature of the chemical inhibition of MAPKs. The use of the inhibitors transiently suppressed the expression of MAPK; however, their restored expression in the plants might have conferred resistance to BPH infestation. Thus, targeted mutation of specific OsMPKs can further clarify their roles in the rice-BPH interactions. Moreover, repeated treatments (alternative day) of the inhibitor mix to prolong the MAPK inhibition resulted in fatal drying of the mock plants (chemical-treated, no BPH) of both rice varieties. Thus, the results obtained from this experiment were considered to be inconclusive, and loss of function studies have been planned to determine the roles of the candidate OsMPK in the rice-BPH interactions.
From the experimental findings in the study, we propose a transcriptional-level model depicting the IR56 rice's resistance mechanism against TN1-BPH, and the resistance breakdown in IR56 rice by IR56-BPH (Figure 8). In IR56 rice, the activation of OsLecRKs in response to TN1-BPH could possibly be achieved by the recognition of the damage-associated molecular patterns (DAMPs), herbivore-associated molecular patterns (HAMPs), or effectors. For instance, oligogalacturonides, oligomers of alpha-1,4-linked galacturonosyl residues, are one of the well-studied DAMPs that are released from plant cell walls upon degradation by insect feeding and can elicit plant defense responses [59]. OsLecRKs activation then might induce the downstream OsMPKs via production of intermediate factors or signal molecules [60,61]. The activated MAPK cascade might then, in turn, activate the phytohormonal defense's regulation and other downstream transcriptional reprogramming to confer resistance against BPH. SA induces the deposition of callose in phloem cells and the production of a trypsin inhibitor to discourage BPH feeding, whereas ET results in the production of a green volatile that deters BPH feeding [1,46]. The activation of a secondary metabolite biosynthesis pathway, such as phenylpropanoids, results in the production of important compounds, such as lignin and phytoalexins, which help to mitigate BPH pressure [61]. However, when IR56 rice was infested with IR56-BPH, none of the OsLecRK were found to be induced, suggesting that IR56-BPH can suppress the PRR-mediated immune response in rice, possibly by the use of other explicit effectors. The chitin elicitor binding protein (CEBiP)-mediated immunity in rice against M. oryzae was suppressed by secreting the effector Secreted LysM Protein1 (Slp1) [62]. Furthermore, infestation of IR56-BPH in IR56 rice successfully suppressed the induced expression of all BPH-responsive OsMPKs except OsMPK5, which was activated during TN1-BPH infestation. In addition, none of the SA or ET signaling genes were found to be activated during IR56-BPH infestation, and the same was observed for the phenylpropanoid biosynthesis genes. Taken together, these results suggest that IR56-BPH could have broken down the resistance of IR56 rice by suppressing the transcription of many defense-responsive pathways, including the MAPKs. Silencing of LecRKs had resulted in a reduced hypersensitive response, secondary metabolite production, and MAPK expression in Arabidopsis [54]. On the one hand, incompatible rice can tackle BPH infestation either by the identification of effectors, the initiation of a hypersensitive response, the activation of downstream defense pathways, including MAPKs, phytohormone biosynthesis, or secondary metabolite production, or by a cocktail of these responses. On the other hand, suppressing these plant defense mechanisms by ETS or by a similar mechanism, the virulent BPHs outrun the rice's resistance in the compatible interactions. Currently, the identification of such effector molecules in IR56-BPH is in progress in our lab, and the results may enable us to add more insights to our proposed transcriptional model. Besides the transcriptional mechanism, post-transcriptional mechanisms (alternative splicing, RNA processing, RNA silencing, et al.), post-translational modifications (protein phosphorylation, ubiquitination, sumoylation, et al.), and cross-connections among these three mechanisms will demonstrate further and superimpose complexity levels in the response to environmental changes. Future attempts at rice engineering could exploit insights from a deeper comprehension of rice-BPH interaction. signaling genes were found to be activated during IR56-BPH infestation, and the same was observed for the phenylpropanoid biosynthesis genes. Taken together, these results suggest that IR56-BPH could have broken down the resistance of IR56 rice by suppressing the transcription of many defenseresponsive pathways, including the MAPKs. Silencing of LecRKs had resulted in a reduced hypersensitive response, secondary metabolite production, and MAPK expression in Arabidopsis [54].
On the one hand, incompatible rice can tackle BPH infestation either by the identification of effectors, the initiation of a hypersensitive response, the activation of downstream defense pathways, including MAPKs, phytohormone biosynthesis, or secondary metabolite production, or by a cocktail of these responses. On the other hand, suppressing these plant defense mechanisms by ETS or by a similar mechanism, the virulent BPHs outrun the rice's resistance in the compatible interactions. Currently, the identification of such effector molecules in IR56-BPH is in progress in our lab, and the results may enable us to add more insights to our proposed transcriptional model. Besides the transcriptional mechanism, post-transcriptional mechanisms (alternative splicing, RNA processing, RNA silencing, et al.), post-translational modifications (protein phosphorylation, ubiquitination, sumoylation, et al.), and cross-connections among these three mechanisms will demonstrate further and superimpose complexity levels in the response to environmental changes. Future attempts at rice engineering could exploit insights from a deeper comprehension of rice-BPH interaction.

Plant Materials
Two indica rice varieties IR56 and TN with a contrasting BPH resistance nature were used in this

Plant Materials
Two indica rice varieties IR56 and TN with a contrasting BPH resistance nature were used in this study. Pre-germinated seeds were planted in mud beds and grown under natural light and temperature conditions in a net house. Single tillers from two-month old seedlings were transplanted to mud-filled plastic cups (diameter 12 cm, height 15 cm) and kept in a temperature-controlled greenhouse (28 ± 2 • C, 80 ± 5% relative humidity (RH)). After 10 days of transplantation, the rice plants were used for the experiments. The BPH resistance of both rice genotypes included in this study was validated in our laboratory prior to the current research.

Insect Materials
TN1-BPH and IR56-BPH were used to perform the BPH bioassays in this study. BPH colonies initially collected from rice fields in Hangzhou, China, were maintained on TN1 rice or IR56 rice in a climate-controlled chamber (26 ± 2 • C, 80 ± 5% RH) for more than 7 years at the China National Rice Research Institute (CNRRI). Both these populations differ in their respective virulence levels.

BPH Bioassays
Each rice plant with a single tiller was individually infested with 4 newly emerged adult female adults, and were confined in a transparent plastic cage (diameter 10 cm, height 60 cm) equipped with a net (with holes of diameter 0.5 mm). As per the aforementioned procedures, the two BPH populations were infested on both the IR56 and TN1 rice varieties, separately. Plants with no BPH treatment from each variety put inside a plastic cage served as a control for this experiment. Wound stress imposition was performed as described by Lu et al. [26] with a slight modification. Briefly, the lower stem portion (2 cm from the base) of each rice plant was pierced 100 times with a fine needle to induce wound stress. Plants with no piercing served as a control for this experiment. Samples were collected after 1 and 3 dpi, immediately frozen in liquid nitrogen, and stored at −80 • C for further use. The BPH assay experiments were performed with 3 independent biological replicates.
For the BPH bioassay in the chemical MAPK inhibition experiment, 40 third instars of TN1-BPH or IR56-BPH were transferred into a single pot having 10 rice plants of each rice variety. The assays were performed in a confined space by the use of a plastic cage as described above. BPH survival rates were calculated for each day of infestation by counting the number of alive BPH nymphs and dividing this number by the total number transferred. The BPH survival rates were determined for 1-8 days of infestation. The BPH assay experiments were performed with 3 independent biological replicates.

Chemical Treatments
Exogenous phytohormone treatments were performed as described in Nahar et al.

RNA Isolation and cDNA Synthesis
Plant samples collected at the respective time points were ground to powder in a pre-chilled sterilized mortar and pestle with sufficient liquid nitrogen, and were used to isolate total RNA using the TransZol Up reagent (Transgen, Beijing, China) according to the manufacturer's instructions.
The concentration and quality of isolated RNA were determined by a NanoDrop ND-1000 spectrophotometer (Thermo-Fischer Scientific, Waltham, MA, USA) and by 1% (w/v) agarose gel electrophoresis. First-strand cDNA was synthesized from isolated RNA by using a Transcript one-step gDNA removal and cDNA synthesis supermix kit (Transgen) as per the manufacturer's instructions.

Quantitative Real-Time PCR (qPCR)
Seventeen OsMPKs sequences were retrieved by using their RAP-DB IDs (Table S6) and by following the nomenclature prescribed by Rohila and Yang [19]. Primers for all 17 OsMPKs and other defense-related genes were designed using Primer-BLAST. The qPCR was performed with a total volume of 10 µL of reaction mixture containing 5 µL of SYBR Green PCR mix (Transgen), 0.2 µL each of the specific forward and reverse primers (10 µM) (Table S6), 0.2 µL of passive reference dye (ROX) (Thermo-Fischer Scientific, Waltham, MA, USA), 2 µL of template cDNA, and 3.4 µL of nuclease-free water. The qPCR reaction was carried out on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instruction. Three independent biological samples for each reaction, and three technical replicates for each biological sample, were used for the qPCR analysis. The constitutively expressed housekeeping gene OsUbq from rice was used as an endogenous control [50]. The relative expression was evaluated using the 2 −∆∆Ct method [64].

Enzyme Activity Assays
The enzyme activities of POD (EC 1.11.1), SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), and GST (EC 2.5.1.18) were determined in BPH-infested and control plants by using POD, SOD, CAT, and GST-ST kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by following the manufacturer's instructions. One unit of POD activity was defined as the amount of enzyme required for a change of 0.001 in absorbance per minute. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of the photochemical reduction of nitroblue tetrazolium. One unit of CAT activity was defined as the amount of enzyme required to change the absorbance value by 0.001 per minute at 405 nm. One unit of GST activity was defined as the amount of enzyme required to produce 1 mmol of 2,4-dinitrobenzene-glutathione conjugate per minute. All of the enzymatic assays were performed with three independent biological replicates, and each replicate included three technical replicates.

Statistical Analysis
The statistical analyses were carried out using Data Processing System software [65]. Data are reported as mean ± SE. For the OsMPK expression experiment, data were analyzed by three-way ANOVA (using rice variety, BPH population, and infestation period as the main factors), and for all other experiments, data were analyzed by two-way ANOVA followed by Tukey's HSD post-hoc test. The statistical significance level was set for p-values <0.05 or 0.01.

Conclusions
In the current work, the differential roles of OsMPKs in two contrasting rice genotypes under BPH infestation at different virulence levels have been investigated. The findings from the study suggested that multiple factors, including rice variety, BPH population type, and infestation period have significant effects on the expression of the OsMPKs. Further, five OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) were found to be exclusively involved in the incompatible rice-BPH interaction. OsMPK4, OsMPK8, and OsMPK9 exhibited induced expression when infested with cross-population BPH. An analysis of the expression of phytohormone signaling genes revealed that rice resistance to BPH might be mediated by the SA and ET signaling pathways. Further, an analysis of the transcription of phenylpropanoid biosynthesis genes suggested that BPH resistance might be realized by the boosted production of secondary metabolites. Although ROS play a vital role in insect herbivory resistance, our findings suggested that ROS accumulation in response to BPH feeding is an early response in rice. Additionally, an analysis of ROS-related enzyme activities in BPH-infested plants further strengthened this hypothesis. Lastly, we have proposed a conceptual model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions, respectively. Additional functional characterizations by deducing the interaction dynamics, and a loss of function analysis, will enable more insights into the role of OsMPKs in rice-BPH interactions.

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