An OsRPP13 protein contributes to rice resistance against herbivorous insects

Rice (Oryza sativa) is one of the world's most vital crops. Rice production faces significant threats from insect pests such as the brown planthopper (Nilaparvata lugens, BPH) and the striped stem borer (Chilo suppressalis, SSB) (Deng et al., 2024; Kuai et al., 2024). The piercing-sucking insect BPH directly damages rice plants by extracting phloem sap and transmitting various viral diseases. In field settings, severe BPH outbreaks can lead to complete crop desiccation, resulting in 'hopperburn'. The chewing insect SSB feeds on newly formed tillers and stems, causing 'dead hearts' and 'white heads'. R proteins such as BPH14, BPH9, and OsLRR2 play a critical role in insect resistance (Guo et al., 2023). While several R genes conferring BPH resistance have been cloned, there are no rice germplasms resistant to SSB. This study identifies a novel R gene, OsRPP13, that positively regulates rice resistance to BPH by regulating flavonoids and hydrogen peroxide levels. Additionally, the resulting increase in jasmonic acid (JA) positively contributes to resistance against SSB. These findings provide valuable insights into the mechanisms underlying insect resistance conferred by R genes and present a potential avenue for breeding insect-resistant rice cultivars. In this study, we first analyzed the expression profile of OsRPP13 through quantitative reverse transcription polymerase chain reaction assays. Primers refer to Supporting Information Table S1. Various tissues including the leaf blade, stem, root, and leaf sheath were analyzed, revealing that OsRPP13 was mainly expressed in the leaf sheath, which is the primary location for BPH feeding (Fig. 1a). A further analysis unveiled drastic changes in OsRPP13 expression following BPH and SSB infestation (Fig. 1b), suggesting the gene's vital role in the interaction between rice and herbivorous insects. Subcellular localization analysis showed that OsRPP13–YFP fusion protein both in the cytoplasm and in the nucleus of rice protoplasts (Fig. S1). Then, we utilized agrobacterium-mediated plant transformation and CRISPR-Cas9 technology to create transgenic OsRPP13 plants. Two OsRPP13 overexpression lines (OsRPP13OE) (Fig. 1c) and two OsRPP13 knockout lines (OsRPP13KO), which have a one-base insertion and a two-base deletion, respectively (Fig. 1d), were selected to investigate the impact on rice resistance to BPH and SSB. We employed various methods to characterize the phenotypic response of OsRPP13 transgenic plants to BPH infestation. While OsRPP13OE lines exhibited a significantly enhanced resistance (Fig. 1e), OsRPP13 knockout lines were more susceptible to BPH than the wild-type (WT) (Fig. 1f). Compared with the WT, rice seeding rates were significantly enhanced in OsRPP13OE plants, but decreased in OsRPP13KO plants (Fig. S2). To determine the effects of OsRPP13 transgenic plants on BPH, we recorded the area and intensity of honeydew, an excretion product of the pest. BPH feeding activity was significantly suppressed in OsRPP13OE plants (Fig. 1g,i) but increased in OsRPP13KO plants (Fig. 1h,j) when compared to the WT. Further examination showed that BPH survival rates were significantly lower on OsRPP13OE plants than on OsRPP13KO plants (Fig. 1k). Taken together, these results indicate that OsRPP13 positively regulates BPH resistance in rice. Previous studies have demonstrated that increased total flavonoid content in rice enhances BPH resistance, with the flavonoid pathway playing a crucial role in this defense (Dai et al., 2019). Within the flavonoid biosynthetic pathway, OsPAL8, Os4CL5, and OsF3H are known to regulate BPH resistance (Chen et al., 2022). Our analysis of total flavonoid content revealed significant increases in OsPRR13OE plants compared with OsPRR13KO plants (Fig. 1l). Additionally, the expression levels of OsPAL8, Os4CL5, OsDFR, and OsF3H in the flavonoid pathway were significantly elevated in OsPRR13OE plants compared with the WT (Fig. 1m). Reactive oxygen species (ROS) are essential molecules involved in plant innate immunity, with mitochondria constituting a significant source of ROS, particularly hydrogen peroxide (H2O2) (Gao et al., 2021). Therefore, we measured the concentration of H2O2 in WT and OsRPP13 transgenic plants at various times after infestation by BPH nymphs. Following infestation, there was a constitutively higher level of H2O2 in OsRPP13OE plants than in OsRPP13KO plants (Fig. 1n). This further confirms that OsRPP13 regulates BPH resistance by affecting flavonoid and H2O2 levels. In addition to BPH, we assessed the reaction of OsRPP13 transgenic plants to SSB. Observational results indicated that OsRPP13OE plants exhibited higher SSB tolerance, with significant reductions in caterpillar growth mass (Fig. 2a,c,e). Compared with the WT plants, OsRPP13KO plants exhibited reduced SSB resistance and accelerated caterpillar growth mass (Fig. 2b,d,f). After SSB feeding, OsRPP13KO plants exhibited severe leaf withering, whereas OsRPP13OE plants showed minimal damage (Fig. S3). These results indicate that OsRPP13 positively regulates SSB resistance in rice. During predation by herbivorous insects, plants recognize herbivore-associated molecular patterns and initiate defense-related signaling pathways. As a key resistance regulator, the JA signaling pathway responds to various rice defense mechanisms, especially those associated with chewing insects (Yao et al., 2023). Previous studies have reported that silencing OsAOS2 in rice reduces SSB resistance (Zeng et al., 2021), whereas overexpression of OsAOC and OsOPR3 enhances resistance (Guo et al., 2014). In our study, we measured the JA content in OsRPP13OE plants before and after SSB feeding, revealing a significant increase in JA levels following SSB feeding compared with the WT (Fig. 2g). The plant hormone salicylic acid (SA) is also crucial for many plant defense responses to herbivorous insects. We observed significant increases in SA levels in all plant lines after SSB feeding compared with their levels before SSB feeding, with no notable differences among the plant lines (Fig. 2h). Consistent with the higher JA accumulation in OsRPP13OE plants after SSB feeding, the expression levels of key genes in the JA pathway were also elevated in these plants following SSB feeding (Fig. 2i). However, in line with the changes in SA levels, there were no significant differences in the expression of key genes in the SA pathway between WT and OsRPP13OE plants after SSB feeding (Fig. 2j). We next treated OsRPP13KO plants with methyl jasmonate (MeJA) before SSB feeding to examine the impact of exogenous hormone applications. Compared with untreated plants, MeJA treatment successfully reduced the SSB susceptibility of OsRPP13KO plants (Fig. 2k), resulting in a significantly attenuated caterpillar growth mass (Fig. 2l). These findings suggest that OsRPP13 positively regulates SSB resistance in rice by elevating JA levels. Resistance proteins are critical for plant defense against various biotic stresses; however, the mechanisms underlying their activation and signal transduction remain poorly understood. In rice, the RPM1-like resistance gene 1 (OsRLR1) mediates the defense response through direct interaction with the transcription factor OsWRKY19 in the nucleus. OsWRKY19 binds to the promoter of OsPR10, thereby activating the defense response (Du et al., 2023). Similarly, Panicle blast 1 (Pb1), a gene associated with blast resistance, is located in both the cytoplasm and the nucleus. Pb1 interacts with WRKY45, a key transcription factor in the salicylic acid signaling pathway, indicating that blast resistance conferred by Pb1 is dependent on WRKY45. Furthermore, Pb1 protects WRKY45 proteins from ubiquitin–proteasome system-dependent degradation through a protein–protein interaction (Inoue et al., 2013). BROWN PLANTHOPPER RESISTANCE14 (BPH14), the first planthopper resistance gene isolated via map-based cloning in rice, which can increase the accumulation of OsWRKY46 and OsWRKY72 as well as OsWRKY46- and OsWRKY72-dependent transactivation activity through interacting with them (Hu et al., 2017). In Solanaceae, MED10b/MED7 of the Mediator complex, and transcription repressor JAZs interact with each other to repress the expression of jasmonate-dependent defense genes. Sw-5b interfere with the interaction between MED10b and MED7, thereby derepressing the repressor activity of MED10b–MED7–JAZ to activate immunity (Wu et al., 2023). In this study, we found OsRPP13-mediated SSB resistance by activating the JA-specific defense pathways. Considering the cytoplasmic and nuclear localization of OsRPP13 in the cytoplasm and nucleus of rice protoplasts, this suggests the potential for multiple molecular mechanisms that regulate plant immunity. Notably, in the nucleus, similar to other R proteins, OsRPP13 may interact with various transcriptional regulatory components to mediate the expression of downstream defense genes and facilitate the transduction of defense signals, such as the expression of JA biosynthesis genes and the transduction of JA signals. Collectively, our findings demonstrate that OsRPP13 significantly enhances rice resistance to two different herbivorous insects, BPH and SSB. BPH resistance was increased through the regulation of flavonoid and hydrogen peroxide levels, while SSB resistance was achieved by modulating the contents of JA (Fig. 2m). According to Methods S1, in a field setting, there was no notable difference between the T2 homozygous OsRPP13 transgenic lines and the WT (Fig. S4a,c) in terms of plant height, tiller number (Fig. S3b,d), and 100-grain weight (Fig. S4b,e). This indicates that OsRPP13 does not affect rice yield or quality. The lack of adverse effects indicates that OsRPP13 has significant potential as a valuable germplasm resource for resistance breeding in rice. We would like to thank Professor Xuexia Miao, Haichao Li, and Zhenying Shi from the Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology Chinese Academy of Sciences for providing us with help during the research. This work was supported by National Natural Science Foundation of China (32102236, 32200107, 32300116), the Faculty Startup Fund for CS from Jining Medical University and Shandong Provincial Natural Science Foundation (ZR2023QC309). FM designed the research project. FM, JC, ZL and DW worked on transgenic lines and developed materials. ZW, FM, SZ, MQ, XG, GS, JZ, MW, YW, MZ, YG, XX, YZ, XX, JZ and QW performed the experiments and analyzed the data. SC, BS and ZT wrote the paper. All authors read and approved of its content. None declared. Fig S1 Subcellular localization of the OsRPP13 protein. Fig. S2 Phenotypic identification of brown planthopper resistance in small populations of OsRPP13 transgenic plants. Fig. S3 Phenotypes of OsRPP13 transgenic plants and wild-type plants at the rice leaves. Fig. S4 Agronomic traits of OsRPP13 transgenic plants. Methods S1 Materials and methods used in this study. Table S1 Primers used in this study. 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