Transcriptomics studies of rice have provided a wealth of information and a global view of mixed gene regulation in response to various biotic stressors (Anderson and Mitchel-Olds 2011). With the emergence of advanced methods for the validation of proteins and metabolites, progress has been made in elucidating the subsequent systematic changes that follow transcription in rice (Table 1 and Table 2).
Response of Rice to Bacteria
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is the most important disease of rice caused by bacterial pathogens. Studies investigating global changes in rice proteins in response to Xoo infection have been performed. Central carbon catabolism is reduced, whereas signal transduction associated with disease resistance, pathogenesis, and the regulation of cell metabolism are upregulated, including several putative resistance (R) genes, putative receptor-like kinases, and PR (Mahmood et al. 2006; Yu et al. 2008; Mahmood et al. 2009b; Sana et al. 2010; Li et al. 2012a). Particularly, thaumatin-like protein (PR5), probenazole (PBZ), Domain of Unknown Function 26 (DUF26), and β-1,3-glucanase were reported as the key findings in the early studies (Mahmood et al. 2006; Wang et al. 2013). A secretome analysis against Xoo identified virulence-associated factors and plant-specific proteins such as proteases or peptidases and proteins involved in host defense, the transport system, and maintaining redox balance (González et al. 2012; Wang et al. 2013; Kim et al. 2014b). A similar analysis was performed using a suspension of Oryza meyeriana, a wild species that is strongly resistant to Xoo. Upregulation of the signal transduction protein, LysM receptor-like kinase and defense protein, and downregulation of a ROS enzyme (peroxidase) and cell wall modifications (via expansins and pectin acetylesterase) were reported (Chen et al. 2016). Besides, phosphosites identification suggested that phosphorylation of TFs, kinases, epigenetic controlling factors, and disease resistant proteins may be functionally relevant to Xoo resistance in IRBB5 (Hou et al. 2015). Furthermore, a metabolomics study revealed differences between resistant and susceptible phenotypes against Xoo in the accumulation of metabolites before and after infection (Sana et al. 2010). Particularly, XA21-expressing plants differed from the wild-type (WT) plants, with higher levels of sugar alcohols, tricarboxylic acid cycle (TCA) intermediates, and miscellaneous compounds in the absence of treatment. After treatment, XA21 plants contained more responsive metabolites, including rutin, pigments, fatty acids and lipids, and arginine, which are likely required for polyamine biosynthesis and alkaloid metabolism. Notably, the virulence signal acetophenone was depressed in XA21.
Plant growth-promoting rhizobacteria (PGPR), whose growth is stimulated by root exudates, assist plants via nutrient uptake and phytohormone production. In proteomics analysis, photosynthesis and defense related proteins were found to accumulate by Pseudomonas fluorescens and Sinorhizobium meliloti (Kandasamy et al. 2009; Chi et al. 2010). Metabolite profiling was first performed in 2013, when two rice cultivars were infected with rice-associated Azospirillum species (Azospirillum lipoferum 4B and Azospirillum sp. B510). In that study, changes in phenolic compounds, such as flavonoids and hydroxycinnamic derivatives, were found to differ depending on the cultivar-PGPR strain interaction (Chamam et al. 2013). Additionally, Nipponbare inoculated with 10 PGPR strains presented common metabolomics signatures, including reduced alkylresorcinol [5-tridecyl resorcinol, 5-pentadecyl resorcinol, 5 (12-heptadecyl) resorcinol] levels and the differential induction of two antimicrobial compounds, N-p-coumaroylputrescine and N-feruloylputrescine, but in different manners (Valette et al. 2020). Pseudomonas is a PGPR used as a biocontrol agent against rice disease, due to its antagonism towards other bacteria and fungi. Analysis of roots and root exudates of rice infected with Pseudomonas putida by High-performance liquid chromatography (HPLC) revealed the induction of SA (Kandaswamy et al. 2019). In another study, Pseudomonas aeruginosa was found to produce compounds associated with systemic acquired resistance (SAR), including siderophores (1-hydroxy-phenazine, pyocyanin, and pyochellin), and antibacterial compounds, including 4-hydroxy-2-alkylquinolines and rhamnolipids (Yasmin et al. 2017). This newly isolated strain can also induce defense-related enzymes in rice plants, suggesting that it may have potential for improving rice plant performance against pathogens.
Response of Rice to Fungi
Magnaporthe oryzae, the causal agent of rice blast disease, has caused huge losses in rice (Dean et al. 2012). Recently, proteomics and metabolomics studies investigating rice response to M. oryzae infection were reviewed (Meng et al. 2019; Azizi et al. 2019). In addition to the influence of M. oryzae on the basic biological processes of the host, such as photosynthesis and primary metabolism, global studies over the past decade have elucidated the detailed interaction between rice and M. oryzae considering whole proteins and metabolites (for review, see Azizi et al. 2019; Meng et al. 2019). Accordingly, metabolomics studies emphasize the difference between biotrophic and necrotrophic stages, such as the accumulation of metabolic photosynthetic sinks at biotrophic stage or phenolic compounds at necrotrophic stage (for review, see Azizi et al. 2019). In addition, a class of SMs also help to prevent fungal invasion via the antimicrobial activity of phytoalexins, including N-benzoyl tryptamine, N-cinnamoyl tryptamine, sakuranetin, and phenylamides, or the ROS-scavenging activity of serotonin. The induction or suppression of these compounds is tightly related to phytohormones such as ABA, JA, SA, and auxin. Furthermore, proteomics studies have revealed the roles of DUF26, nucleotide binding-leucine rich repeat, PRs, ROS production-scavenging enzymes, heat shock proteins (HSPs), nuclear reorganization-related proteins, TFs, and phytohormone signaling in rice resistance, whereby proteins related to pathogen perception and signal transduction are important during the early stage of infection (for review, see Meng et al. 2019). A recent study using iTRAQ found that probenazole-inducible protein 1 (PBZ1) and phenylpropanoid accumulated in both resistant and susceptible cultivars, which was in contrast to reports from previous studies utilizing the 2DE approach (Ma et al. 2020b). Interestingly, a metabolomic assay using HPLC identified a rice saponin, Bayogenin 3-O-cellobioside, which is the first saponin found in rice (Norvienyeku et al. 2020). Accordingly, the accumulation of Bayogenin 3-O-cellobioside is well corelated with blast resistance. Thus, improvements in these methods provide a more precise view of the interaction between rice and rice blast fungus on a case-by-case basis.
Rhizoctonia solani is a necrotrophic fungus that causes sheath blight in rice. In contrast to M. oryzae, this fungus causes cell death from the early stage of infection; thus, studies of R. solani infection have identified many distinct changes. The most notable changes have been observed in photosynthesis and sugar metabolism. The reduction of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) large subunits was observed in an early study (Lee et al. 2006). Two metabolomics studies reported the induction of glycolysis and TCA cycle intermediates (succinate, pyruvate, and aconitate) and decreased levels of sugar metabolites (sucrose, glucose, fructose, glucosone, galactose, hexopyranose, turanose, maltose, and glucopyranose), suggesting that respiration in the infected tissue was enhanced for energy production. ROS, SA, JA, aromatic aliphatic amino acids, and phenylpropanoid intermediates also accumulated, accompanied by the suppression of myo-inositol, indicating the loss of antioxidant activity, which is consistent with the formation of lesions by necrotrophic pathogens (Suharti et al. 2016b; Ghosh et al. 2017). The increase of SA, known as an inducer of defense responses against biotrophic pathogens, is consistent with the following research which found the hemobiotrophic nature of R. solani (Kouzai et al. 2018). Global proteome and metabolome studies also suggested the distinct responses of resistant and susceptible phenotypes. Resistant cultivars were found to produce higher levels of antifungal proteins (β-1–3 glucanase and chitinase), ROS scavenging machinery, and 3β-hydroxysteroid dehydrogenase (3β-HSD) for the synthesis or regulation of plant steroids. Additionally, 14–3-3 protein, which is involved in protein interactions, and the chaperonin 60 β precursor were reduced (Lee et al. 2006). Glycolysis or gluconeogenesis and fatty acid β-oxidation for adenosine triphosphate (ATP) production were upregulated, whereas energy consumption was reduced in resistant plants via the reassimilation of photorespiratory ammonia or the regulation of energy metabolism. A high abundance of proteins related to glycolysis, α-amino acid biosynthesis, and stress response have also been observed in R. solani resistance through the analysis of transgenic rice expressing AtNPR1, a key regulator of SAR (Karmakar et al. 2019). Consistent with JA, lignification and signaling were found to be stable in resistant plants (Suharti et al. 2016c). Additionally, differences in ROS regulation between resistant and susceptible plants were noted (Ma et al. 2020a). Consistent with this, pipecolic acid, which regulates SAR and induces necrotic symptoms, was upregulated in susceptible plants (Suharti et al. 2016b). However, some studies reported unexpected findings, including the downregulation of Casparian strip membrane domain-like protein 2B1, which is passively required for lignin deposition in resistant cultivars, and the upregulation of spermidine hydroxycinnamoyl transferase 1, which is involved in the biosynthesis or modification of alkaloids, terpenoids, and phenolics in susceptible plants (Prathi et al. 2018). A study investigating protein changes after infection with another necrotrophic fungus, Cochliobolus miyabeanus, which causes brown spot disease in rice, suggested a pattern similar to that seen in response to R. solani infection (Kim et al. 2014a). Proteins involved in the Calvin cycle (fructose bisphosphate aldolase, sedoheptulose-1, 7-bisphosphatase, and RuBisCO) were reduced; however, oxaloacetate aspartate and aminotransferase, which are required for amino acid biosynthesis, and enzymes involved in redox homeostasis (peroxiredoxins, glutathione reductase, and NADP-dependent isocitrate dehydrogenase) were found to be accumulated.
Those studies in necrotrophic pathogens suggested a distinct response compared with hemibiotrophic pathogens at an early stage, which was exemplified by the reduction in photosynthesis, increase in energy production, and accumulation of ROS. However, current data from a limited number of proteomics and metabolomics studies indicate that different cultivars may use different mechanisms to respond to fungal pathogens.
Response of Rice to Virus
Viruses transmitted to rice by either plants or insects induce physiological changes such as suppressed photosynthesis and chlorosis (for review, see Alexander and Cilia 2016). The key changes introduced by viral infection through insects as the vector are those involving carbon metabolism. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, has emerged as a multifunctional protein in several non-metabolic processes and is increased in several plant species following viral infection (for review, see Alexander and Cilia 2016; Chen et al. 2013). Additionally, fluctuations in amino acids, which provide materials for viral replication, plant defense, ROS accumulation for chlorotic damage, and respiration in terms of energy supply, are also important responses to viral infection. These findings have been confirmed and clarified by proteome and metabolome studies. For example, chloroplast proteins are degraded, chlorophyll a and chlorophyll b synthesis is inhibited, and 26S proteasome is enhanced during rice stripe virus (RSV, Tenuivirus genus) infection (Wang et al. 2015). Furthermore, enhanced H2O2 production by rice black-streaked dwarf virus (RBSDV, Reoviridae family, Fijivirus genus) may diminish light absorption, resulting in reduced photosynthesis (Xu et al. 2013). Studies on uninfected and virus-infected rice, or on resistant and susceptible cultivars, have also identified a class of proteins and metabolites that underly viral resistance. Accordingly, RBSDV induces the production of ABA and cytokinins and reduces the production of indole-3-acetic acid, gibberellins, JA, and SA by suppressing expression of the related genes (Huang et al. 2018). In that study, gibberellic acid (GA) was able to rescue the typical dwarfing symptom, and pre-application of SA was able to reduce the severity of the disease. ROS, which has a dual function in chlorosis and in viral defense mechanisms, was found to accumulate at different levels in susceptible and resistant cultivars (Xu et al. 2013). In line with this, treatment with the antiviral bioactive SM, cytosinpeptidemycin, was shown to upregulate peroxidase, superoxide dismutase, and catalase in response to southern black-streaked dwarf virus infection (Yu et al. 2018). Additionally, the increased production of PR proteins, including PR5, PR10, and Bet v1 allergen or HSPs, has also been associated with resistance to virus in rice (Yang et al. 2013; Wang et al. 2015; Yu et al. 2018).
Response of Rice to Insects
Interactions between plants and insects include dynamic defense mechanisms in plants and weapons in insects to enable successful invasion. Plants avoid being eaten in two ways, both involving a dominant role of SMs: repelling ovipositing herbivores along with attracting enemies and causing herbivore mortality. Insects may overcome this by secreting effectors in salivary proteins or capitalizing SMs (Lu et al. 2018). Although the strategies used by plants to defend against each kind of insect may vary (Harun-Or-Rashid et al. 2018), common mechanisms involve JA signaling, detoxification, cell wall modifications, photosynthesis, phytohormones, and defensive SMs (for review, see Ling et al. 2019; Zogli et al. 2020).
The brown planthopper (BPH, Nilaparvata lugens Stål, Hemiptera: Delphacidae) is a typical monophagous vascular feeder. Proteomics and metabolomics studies on rice response to BPH infection have revealed the occurrence of dynamic changes. Lipid transport and metabolism, SM biosynthesis, amino acid transport and metabolism, and phytohormone signaling are commonly induced by BPH in both susceptible and resistant cultivars (Wei et al. 2009; Dong et al. 2017; Zhang et al. 2018; Zhang et al. 2019; Zha and You 2020). Notably, studies that have utilized resistant and susceptible cultivars to observe changes on protein and metabolite levels have identified markers and features associated with resistance to BPH infection. This has also been demonstrated in time-course studies during each stage of BPH infection. A metabolomics study in leaf sheath and honeydew revealed enhanced fatty acid oxidation, glyoxylate cycle, gluconeogenesis, and γ-aminobutyric acid (GABA) shunt in susceptible cultivars, whereas glycolysis was upregulated in resistant cultivars, resulting in the production of substrates for SM synthesis via the shikimate pathway (Liu et al. 2010; Peng et al. 2016). Lower levels of amino acids in nymphs at the early, but not the late, stage of infection were reported in resistant cultivars compared with susceptible cultivars, indicating the rapid adaptation of BPH (Liu et al. 2017). In another study, there were clear differences in the response of resistant and susceptible cultivars to small BPH (Laodelphax striatellus Fallén, Homoptera: Delphacidae) during the early stage, when the resistant cultivar displayed less tissue damage due to the upregulation of ROS removal machinery and SA (Dong et al. 2017). A lipidomics study reported that the resistance conveyed by Bph6 gene involves wax biosynthesis (for example fatty acid methyl esters) (Zhang et al. 2018). Recently, Kang et al. (2019) reported that primary metabolism was inhibited in all cultivars at the early stage (24 h) but only recovered in the late stage (96 h) in resistant cultivars. In that study, amino acids, organic acids, and fatty acids were also found to be stable in resistant cultivars. Higher levels of flavonoid glycosides (schaftoside, iso-schaftoside, rhoifolin, and apigenin 6-C-α-l-arabinoside-8-C-β-l-arabinoside) were induced in resistant rice compared with susceptible rice (Uawisetwathana et al. 2019).
Phytohormones play an important role in the interaction between rice and hopper. The function of SA in this interaction appears ambiguous, as it has been reported to be upregulated in both susceptible (Peng et al. 2016) and resistant (Dong et al. 2017) cultivars. Enhanced JA metabolism is the main difference in the resistance of wild rice Oryza officinalis compared to BPH-susceptible O. sativa (Zhang et al. 2019). Interestingly, both SA and JA signaling was enhanced in the endophytic strain Bacillus velezensis YC7010, which induces resistance to BPH infection in rice (Harun-Or-Rashid et al. 2018).
Studies on the response of rice to other insects have also revealed specific responses. The response of a resistant rice line (cultivar Qingliu) to rice leafroller (Cnaphalocrocis medinalis) involved the activation of the Calvin cycle and the light reaction of photosynthesis, followed by the biosynthesis of amino acids and other metabolites (Cheah et al. 2020). Furthermore, resistance was determined by flavonoid biosynthesis at a specific rate and time. Rice also defends against the rice stem borer (Chilo suppressalis) using a similar mechanism. An integrated transcriptomics and metabolomics study suggested increased photosynthesis via the accumulation of monosaccharides and not of oligosaccharides, galactinol, and various amino acids (Liu et al. 2016). Conversely, the resistance of rice to rice gall midge was found to involve differences in fatty acids before and after infection, whereas glutamine and 23-oxotetracosanoic acids were associated with susceptibility (Agarrwal et al. 2014).
Response of Rice to Nematodes
Nematodes are universally present in nature and include species that are parasitic to plants, including rice (Sato et al. 2019). Studies investigating rice-parasitic nematode interactions have generally involved mutants and transcriptome analyses, with a notable lack of proteomics and metabolomics studies. In 1996, a group of researchers used HPLC to evaluate differences in the phenolic profiles of five resistant and two susceptible deep-water rice upon Ditylenchus angustus infection. They reported changes in SMs, such as chlorogenic acids and phytoalexin sakuranetin, which were mainly identified in the resistant rice (Gill et al. 1996). A previous proteomics study on the rice and root-knot-nematode (Meloidogyne graminicola) interaction revealed new proteins as well as changes in existing proteins (Xiang et al. 2020). Importantly, proteins involved in stress, metabolic pathways, and SM biosynthesis were found to accumulate at the early stage of infection, and this continued to the later stage of infection. Additionally, an integration of transcript analyses revealed that four specific proteins related to α-linolenic acid metabolism, phenylpropanoid biosynthesis, glutathione metabolism, and plant–pathogen interaction pathways were downregulated in the susceptible cultivar but upregulated in the resistant cultivar (Xiang et al. 2020).