Introduction
Rice pests cause huge yield losses every year, and rice planthoppers (main brown planthopper, BPH, Nilaparvata lugens Stål) are among the most important pests in rice production. The application of pesticides has been the traditional and most commonly used method to control the pests until now. However, this method increases production costs and decreases farmers’ incomes. What makes the situation worse is that the rice planthoppers will eventually become tolerant to the pesticides (Jena et al ., 2006, Tanaka et al ., 2000). Therefore, identifying BPH resistance genes and exploring their resistance mechanisms is one of the most fundamental and effective methods to manage this pest.
Aspartic proteases (APs) are important subfamily members of the four major proteolytic enzyme families, and they are expressed in different plant organs, including seeds, stems, leaves, and floral organs (Chenet al ., 2009). In the 1990s, APs were successively isolated from monocotyledons, such as corn, rice, and wheat; and dicotyledons, such asArabidopsis thaliana , rape, tobacco, and potato (Mutlu et al ., 1999). In recent years, many APs have been isolated from plants, particularly from Arabidopsis thaliana and rice, and their functions have been studied using in-vivo experiments. At present, 69 and 96 AP genes have been identified in the genomes of Arabidopsis thaliana and rice, respectively (Takahashi et al ., 2008, Chenet al ., 2009).
APs are mainly involved in the degradation of organelles, key enzymes in photosynthesis, and hormone-regulated plant senescence (Kato et al ., 2004). For example, OsAP25 and OsAP27 are involved in the degradation of the anther tapetum, and OsAP37 is associated with the activation of caspase-like proteases during programmed cell death (PCD) (Niu et al ., 2013). Plant APs are also involved in abiotic and biotic stress responses. Carvalhoa et al. (2001) isolated AP genes from the leaves of kidney beans and cowpeas, and found that their transcriptional levels and enzyme activities were induced by drought stress (Carvalhoa et al ., 2001). Guevara et al. (2002) found that the expression of AP genes in tubers infected withPhytophthora was significantly increased, and that APs directly inhibited the germination of fungal cysts (Guevara et al ., 2002). Alam et al. (2014) indicated that the expression of OsAP77 in rice vascular tissue was significantly increased after fungal, bacterial, or viral infection, or after treatment with salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide, and abscisic acid (ABA) (Alam et al ., 2014). Wang et al. (2022) showed that the knockout of OsAP47 could improve rice resistance to black-streaked dwarf virus and southern rice black-streaked dwarf virus diseases (Wang et al ., 2022). Although many studies have shown that AP genes are involved in several biological functions, there have been no reports of these genes being involved in insect resistance in plants. Therefore, it is necessary to explore its function in plant insect resistance to further understand the functions of AP genes.
Plant growth and defense are largely governed by diverse phytohormones such as jasmonic acid (JA), SA, and ethylene (ET). Auxins, with indole acetic acid (IAA) as the main active form in higher plants, can regulate many aspects of plant development, and abiotic and biotic stresses (Kieffer et al ., 2009, Zhao, 2010, Liu et al ., 2013). For instance, the reduction in IAA accumulation in root tips is the main driver of root growth inhibition under Al3+ stress (Wang et al ., 2016). Moreover, the IAA-amido synthetase geneOsGH3.8 impacts OsNPR1 -mediated disease resistance in rice partly through the auxin signaling pathway (Li et al ., 2016). Furthermore, it plays important roles in rice growth and basal defense by modifying the cell wall structure (Ding et al ., 2008). Many studies have suggested that changes in IAA conditions affect cell wall structure and biotic stress levels.
In this study, the AP gene OsAP79 was found to be very highly expressed in the BPH resistance line, RBPH16, after insect infestation, compared with the susceptible line. To clarify its role in BPH resistance in rice, transgenic lines of overexpression (OE) and knockout of OsAP79 were generated and surveyed to detect BPH resistance and the mechanisms by which it occurs. As a result, OsAP79 was transcriptionally expressed in all organs tested, particularly in the roots at the adult stage. The knockout of OsAP79 impaired rice resistance to BPH, whereas OE lines showed improved resistance to BPH by increasing plant survival rates, and reducing BPH weight gain and honeydew excretion. Observation of the transverse section of rice shoots indicated that the OE lines of OsAP79 had significantly thicker sclerenchyma. Meanwhile, a significantly longer or shorter root was detected in the knockout or OE lines, respectively, which was positively correlated with IAA content. Hormone measurement and metabolomic analysis suggested that IAA biosynthesis, the citric acid cycle, and glycolysis biosynthesis contributed significantly toOsAP79 -induced rice resistance against BPH. Collectively,OsAP79 was continuously induced to express and enhance BPH resistance by reducing IAA content, which restricts root growth and increases shoot sclerenchyma thickness. This study demonstrates the previously unappreciated importance of the role of AP genes in rice in combating piercing-sucking insect herbivores.