Proteomic Analysis Reveals Salicylic Acid as a Pivotal Signal Molecule in Rice Response to Blast Disease Infection

Rice blast disease caused by a fungus, Magnaporthe grisea, is one of the most destructive diseases in rice production worldwide, and salicylic acid (SA) can efficiently decrease the damage of M. grisea. Here, we combined the 2-Dimensional-Liquid Chromatography and the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (2D-LC-MALDI-TOF-TOF MS) techniques to compare and identify differentially expressed labelled proteins by the isobaric tags for relative and absolute quantitation (iTRAQ) between the blast-resistant cultivar Minghui and the susceptible rice cultivar Nipponbare in response to blast fungus infection. The group samples were treated with salicylic acid and compared to control samples. A total of 139 DEPs from the two cultivars showed either more than a two-fold change or alternating regulation patterns. Protein functionality analysis also exhibited that these proteins are involved in a wide range of molecular functions including: energy-related activity (30%), signal transduction (11%), redox homeostasis (15%), amino acid and nitrogen metabolism (4%), carbohydrate metabolism (5%), protein folding and assembly (10%), protein hydrolysis (9%), protein synthesis (12%), and other unknown functions (4%). Specifically, we demonstrated that exogenous treatment with salicylic acid promoted recovery in both rice cultivars from Magnaporthe grisea infection by enhancing: the regulation of signal transduction, increasing energy conversion and production through the regulation of the glycolytic pathway, and other various biochemical processes. These findings may facilitate future studies of the molecular mechanisms of rice blast resistance.


Introduction
Rice is a staple crop of economic importance in many countries, as well as being a model monocot species for plant research [1]. With an estimated production capacity of at least 600 million tons per annum, its production is affected by both biotic and abiotic factors. Infectious diseases are among the sternest threats to rice crop production, reducing rice productivity by 25% worldwide [2]. The Magnaporthe grisea (Hebert) Barr (anamorph, Pyricularia grisea Sacc.), an ascomycete filamentous fungus, one of the most infamous causative agents of blast disease, continuously threatens rice production globally [3], affecting the Poaceae family including rice [4]. Visible disease symptoms are characterized by white to gray-green lesions or spots with darker borders. However, with the enhancement of agronomic practices, several cultural strategies such as the use of genetically resistant varieties and the application of fungicides have been implemented to control blast disease in rice [5,6]. Recently, Zhang et al. [7] reported on a tripartite interaction among rice plants, insects, and pathogens in the control of rice blast disease. They have demonstrated that pre-infestation by herbivores on rice crops reduces the disease incidence of rice blast by increasing the accumulation of salicylic acid (SA) [7], emphasizing a known fact that phytohormones accumulation including SA improves basal crop resistance to blast disease in rice through reducing plant biochemical processes such as: plant photosynthesis rate and regulating the antioxidant defense activities [8,9].
Measuring rice proteomic changes in response to salicylic acid (SA) has provided new insights into the molecular mechanisms of SA-induced rice defense to blast disease. It has been shown that SA treatment after rice blast infection elevates rice blast disease resistance. Sun et al. [10] identified 33 protein spots that were differentially expressed after SA treatment, evidencing that SA improves basal resistance to rice blast through decreasing plant photosynthesis, regulating the antioxidant defense activities. They concluded that enhanced blast disease resistance in rice plants is coordinated by SA-induced complex cellular activities. Although, full comprehension of the mechanisms of regulation is still yet to be attained. Cell-level research has also demonstrated that SA signaling is mediated by a series of downstream factors such as OsNPR1 and WRK45 acting in parallel, and OsAAA-ATPase1 [11][12][13].
The integration of various powerful fields such as proteomics has also enabled the investigation of rice blast disease at the translational level, disclosing several key genes and proteins associated with rice blast resistance [14]. Hence, utilizing proteomic techniques such as two-dimensional liquid chromatography mass combined with the matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for both qualitative and quantitative characterizations of protein mixtures and post-translational modifications has extended the breadth and depth of proteome analysis [15][16][17]. The 2D-LC proteome analysis has been widely used in the identification of protein expression patterns, to address the mechanisms by which Magnaporthe oryzae regulates G-proteins (GTP-binding protein) and signaling proteins (RGS) that orchestrate blast disease incidence [18]. Likewise, several researchers have also used the MALDI-TOF/TOF MS in the identification and quantification of protein expression patterns in SA-added rice plants resisting the blast disease.
First developed in 2004, the Isobaric Tags for Relative and Absolute Quantification (iTRAQ) has also been shown to pose unique advantages over other conventional proteomics techniques [19]. It enables the identification and quantification of many proteins from specific biological environments using labeled peptides as internal standards identifiable by sensitive mass spectrometers. Furthermore, it utilizes bioinformatics tools and statistical packages to visualize obtained data. The incidence of the iTRAQ approach has enabled many researchers to identify proteins involved in pathogen resistance in several plant species. Ma et al. [20] implemented the iTRAQ to compare and detect differentially expressed proteins (DEPs) from a blast-resistant variety and a susceptible variety. They detected 46 common DEPs among the two varieties involved in plant-pathogen interaction, plant hormone signal transduction, fatty-acid metabolism, and peroxisome biosynthesis [20]. Furthermore, iTRAQ has been implemented in several kinds of research that may include the quantification of rice grains proteins [19], assessing the effect of high night temperature stress on rice [21], assessing the impact of ion pollution and detoxification in rice [22], and assessing the expression patterns of rice proteins under several abiotic stresses [23,24].
Although the iTRAQ approach has been utilized in the past to assess proteins related to rice blast resistance, few proteins have been successfully identified. In this present study, we compared the posttranslational responses of rice cultivars (blast-resistant Minghui and blast-susceptible Nipponbare cultivars) sprayed with salicylic acid against the controls to Magnaporthe grisea fungus infection using the iTRAQ approach to label the peptides and the combined 2D-LC/MALDI-TOF-TOF MS technique for protein separation and identification, with an intent to comprehend the molecular mechanisms underlying blast resistance in rice. This study may facilitate and provide valuable molecular data for rice blast-resistant breeding purposes.

Identification and Relative Quantification of Proteins
In present study, differentially expressed proteins (DEPs) expressed in response to Magnaporthe grisea (M) infection were identified using the isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique coupled with the 2-D Liquid Chromatography-Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (2D-LC-MALDI-TOF-TOF MS) analysis. The proteomic differences were contrasted and analyzed from the two rice cultivars, the blast-resistant Minghui (M) and blast susceptible Nipponbare (R) varieties, samples were grouped in four sub-pairs as M-M vs. M-CK, M-SA vs. M-CK, R-M vs. R-CK, and R-SA vs. R-CK. The schematic workflow of the experimental design is shown in Figure 1. A total of 1230 proteins were obtained, which were further separated using the Liquid Chromatography (LC), and 1163 proteins were successfully identified using the MASCOT program. Expression analysis of the observed proteins exhibited that 139 proteins had more than 2.0-fold change and/or less than 0.5-fold difference with p < 0.05 between samples, and these were considered to be significantly altered, as shown in Table 1.
These DEPs were shown to be involved in a wide range of molecular functions, 30%, which was the majority, was constituted in energy-related activities, 11% in signal transduction, 15% in redox homeostasis, 4% in amino acid and nitrogen metabolism, 5% in carbohydrate metabolism, 10% in protein folding and assembly, 9% in protein hydrolysis, 12% in protein synthesis, and the remaining 4% in other unknown functions ( Figure 2). This suggests that biological processes with high protein constituency such as energyrelated activities are likely to enhance blast disease resistance in rice varieties. A deeper analysis in the energy-related activity showed that 65 of the total DEGs were upregulated, to which 36 and 29 were enriched in Minghui and Nipponbare, respectively. Specifically, the 23 kDa polypeptide of photosystem II, chloroplastic glutamine synthetase, and enoyl-CoA hydratase/isomerase proteins were highly upregulated among the significantly expressed protein in the R-SA group and downregulated in the R-M group in the Nipponbare variety.

Bioinformatics Analysis of Differentially Expressed Proteins
To define the key proteins associated with rice blast resistance and SA-dependent recovery, we did bioinformatics analyses of the four pair samples, M-M vs. M-CK, M-SA vs. M-CK, R-M vs. R-CK, and R-SA vs. R-CK. The differentially expressed analysis of most significant protein changes between the two varieties showed that three common DEPs were expressed in all the groups (Figure 3). This implies that these DEPs may play an important role in all rice cultivars of study and also that there is less in common in the regulation of blast disease infection between the two varieties of study. As expected, the SA-added groups, M-SA and R-SA, made up the largest part of the differentially expressed proteins, 24 and 20, respectively, compared to the control groups. This shows that salicylic acid had an important effect on fungal infection rates. On the other hand, the R-M group also constituted a larger group of DEPs (24) as compared to the M-M group, suggesting that the R-group (Nipponbare variety) was negatively affected by blast disease. Comparisons between two cultivars showed that more DEPs were expressed in the susceptible Nipponbare cultivar as compared to the blast-resistant Minghui cultivar, evidencing the fact SA exhibited positive regulatory roles against the M. grisea infection. Hence, these results can be summarized as that salicylic acid upregulated the expression of DEPs in both varieties.

Comprehensive Inventory of the Differentially Expressed Proteins
To classify the DEPs in Minghui and Nipponbare varieties, putative proteins induced by M. grisea were separated by the 2Dimensional-Liquid Chromatography (2D-LC) ( Figure 4). All the identified DEPs were classified into three GO terms; biological process (BP), molecular function (MF), and cell component (CC). The majority of the identified proteins in the M-M group were involved in single-organism metabolic processes (BP analysis), cytoplasm (CC analysis), and nucleoside phosphate binding (MF analysis). The identified proteins in the M-SA group were concentrated in the organonitrogen compound metabolic process (BP analysis), cytoplasm (CC analysis), oxidoreductase activity, and catalytic activity (MF analysis). In the R-M group, the majority of identified proteins were involved in the small molecule metabolic process (BP analysis), cytoplasm (CC analysis), and catalytic activity (MF analysis). Lastly, the identified proteins in the R-SA group were concentrated in the organonitrogen compound metabolic process (BP analysis), chloroplast and cytoplasm (CC analysis), oxidoreductase activity, and nucleoside phosphate binding (MF analysis). Wholly, these results show that DEPs from different rice cultivars and exposed to different treatments exhibited different functional characteristics, and that more genes were assigned in the susceptible Nipponbare cultivar as compared to the blast-resistant Minghui cultivar.

Specific Metabolic Processes in Minghui and Nipponbare Are Involved in the Response against M. grisea
To determine specific metabolic processes in Minghui and Nipponbare following M. grisea inoculation and salicylic acid spray, we used the KEGG database ( Figure 5). We noted that only three metabolic pathways were significant at both p-values (p < 0.01 and p < 0.05), including carbon fixation and metabolism, carbon metabolism, glyoxylate and dicarboxylate metabolism. Carbon fixation and metabolism exhibited the highest −log(Pvalue) change in both groups of the Minghui cultivar, while the carbon metabolism, glyoxylate and dicarboxylate metabolism were highly upregulated in the R-M and R-SA groups, respectively. However, significant expression differences were observed when comparing two distinct varieties with the same treatment. In SA-treated cultivars we noted that 50% of the biochemical processes in the M-SA group were not significant at p-value < 0.05 as compared to the R-SA group, to which only two of the investigated processes were insignificant when p-value < 0.05. In the M. grisea-treated groups, similar findings were observed, although all the processes investigated in the R-M group had significant differences when p-value < 0.05, except for the Glycine, serine and threonine metabolism in the M-M group. Based on the results of the KEGG analysis, the salicylic acid added groups from both varieties had higher upregulation expression as compared to the control groups.

Protein-Protein Interactions
To comprehend the protein network interaction, we constructed protein to protein interactions (PPI) using the STRING database and viewed using Cytoscape software ( Figure 6). Generally, the results exhibited that the identified proteins were densely connected and regulated several pathways. In the M-M ( Figure 6A) group, the majority of the investigated proteins with high fold change were involved in metabolic pathways, moderately upregulating its enrichment. The carbon fixation in photosynthetic organisms and carbon metabolism pathways were highly enriched with fewer moderately upregulated proteins. The glyoxylate and dicarboxylate metabolism was lowly enriched by several upregulated proteins. Likewise, the glycine, serine, and the threonine metabolism were upregulated and downregulated by two proteins, 2,3-diphosphoglycerate-independent and betaine aldehyde dehydrogenase, respectively. Similar enrichment patterns were observed in the M-SA group ( Figure 6B), with fewer proteins involved in the metabolic pathways, and highly upregulated as compared to the M-M group. Specifically, the metabolic pathways were densely connected with several upregulated proteins. However, some of the pathways were absent in the M-M group and were regulated by several proteins. For instance, the pentose phosphate pathway was present in M-SA but absent in R-SA and was lowly enriched by two upregulated proteins. Intriguingly, vitamin B6 metabolism was not linked to any of the pathways within the network, and only one protein was highly upregulating its enrichment. The R-M group ( Figure 6C) was also densely connected with moderately expressed proteins. Carbon metabolism was highly enriched among the observed pathways, with fewer proteins involved as compared to the metabolic pathways. Nonetheless, the metabolic pathways were moderately enriched with two highly expressed proteins involved. The remaining pathways in the R-M group, oxidative phosphorylation, pyruvate metabolism, and arginine biosynthesis, were lowly enriched and had two, three, and four downregulated proteins, respectively, linking their enrichment. The R-SA group was sparsely connected ( Figure 6D), with some proteins highly expressed while some were lowly expressed. The majority of the highly expressed proteins with a high fold change moderately enriched the regulation of metabolic pathways, with a few downregulating their enrichment. Nonetheless, the carbon fixation in photosynthetic organisms and glyoxylate and dicarboxylate metabolisms were highly enriched with two and three proteins, respectively, linked.

Discussion
Developments in proteomics research over the past decades have enabled the comprehension of plant disease resistance at the protein level. In this study, the proteomics approach was employed to comparatively identify the expressed proteins from the blastsusceptible Nipponbare and the blast-resistant Minghui rice cultivars. To reveal the mechanism of resistance to Magnaporthe grisea infection, the two rice cultivars were treated with the biotic stress hormone, salicylic acid, after infection. A total of 139 DEPs were identified among all samples and were further shown to be involved in energy metabolism, signal transduction, carbohydrate metabolism, nitrogen metabolism, protein hydrolysis, and the synthesis process.

Differentially Expressed Proteins Related to Energy
Energy deficits are a general symptom of photosynthetic plants under stress due to reductions in photosynthesis and/or respiration, ultimately resulting in cell death and growth arrest. However, plants counter energy shortages by enhancing inherent pathways of carbohydrate metabolism, induction of alternative pathways of glycolysis, and maintaining energy supply and carbon skeletons for key metabolic processes. Sucrose is the primary translocated carbohydrate in the majority of plants; thus, its metabolism is vital in the regulation of plant growth in response to stresses [25][26][27]. We identified 41 energy-related proteins (spots 1-41) involved in the response to blast fungus infection in rice. These proteins mainly included 23 kDa polypeptide of photosystem II, chloroplastic glutamine synthetase, NADP-dependent malic enzyme, oxalate oxidase-like protein, uroporphyrinogen decarboxylase, 6-phosphogluconolactonase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, and ATP synthase (Table 1). In plants, these proteins are involved in the glycolysis pathway, tricarboxylic acid cycle, chlorophyll synthesis, pentose phosphate pathway, photosynthesis process energy, and so on.
ATP synthase is widely distributed in the mitochondrial membrane and chloroplasts thylakoid [28], participating in oxidative phosphorylation and photophosphorylation, under the impetus of the transmembrane proton power potential, resulting in ATP hydrolysis and the release of energy, which is the most direct energy source in plants [29,30]. We observed that the levels of ATP synthase proteins significantly declined under the blast fungus infection in Nipponbare (susceptible cultivar) and that treatment with salicylic acid failed to promote significant recovery of ATP synthase protein levels in Nipponbare. Nonetheless, in Minghui (resistant variety) the three levels of ATP synthase proteins spots (13, 14, and 16) were slightly downregulated under blast fungus infection, and treatment with salicylic acid significantly elevated the levels of these proteins. These results demonstrated that Magnaporthe grisea infection significantly reduces energy production in Nipponbare compared to Minghui and that salicylic acid exogenous treatment promotes more energy recovery in Minghui than in Nipponbare after infection.
Thirteen differentially expressed protein spots (1, 5, 24, 28, 30-36, 39, 40) were involved in photosynthesis metabolism, including chlorophyll synthesis; in Minghui under blast fungus infection these proteins were upregulated but not in Nipponbare. Treatment with salicylic acid afterwards further improved the expression of these proteins in Minghui. We identified five proteins (spots 3,20,22,25,26) involved in the glycolytic pathway. Glycolysis is the process of degrading glucose to produce ATP, providing partial energy for cell activities. In this study, the expression of proteins involved in the glycolytic pathway was downregulated in Nipponbare infected by Magnaporthe grisea. However, in Minghui two of the five proteins (spots 22 and 26) were upregulated significantly. These results showed that the glycolytic pathway was inhibited by blast infection in Nipponbare.

Proteins Associated with Signal Transduction
Stress signal perception and transmission are important for plant environmental stress response [31]. We identified 15 signal transduction-related proteins, including brain-specific protein, calreticulin precursor, and GTP-binding protein ( Table 1). In Nipponbare, most of the expressed proteins were downregulated, while in Minghui the expressed proteins were upregulated. This implies that active regulation of signal transduction is associated with blast infection resistance in Minghui.
Integrins belong to a family of cell surface glycoproteins, which connect the extracellular matrix to the actin cytoskeleton and serve in cell signaling across the plasma membrane to confer cell survival. Overexpressed 14-3-3 beta protein interacts with cytosol B1 integrin promoting cell migration and signal transmission [32,33]. We analyzed the protein level of 14-3-3 beta (spot 50) and noted that its stability was constant in Minghui, while it was downregulated in Nipponbare under blast fungus infection. Spot 59 is a spermidine synthase important in plant stress responses. It is involved in signal transduction associated with plant stress resistance [34]. The expression of the protein was significantly upregulated only in Nipponbare after treatment with salicylic acid. Wholly, this result showed that SA enhances the regulation of signal transduction by up-and downregulating proteins involved. However, the correlation between proteomic changes due to rice blast resistance may deserve further investigation.

Differentially Expressed Proteins with Redox Homeostasis
There is active redox homeostasis in a plant cell in response to stress stimuli. Oxidants mainly include reactive oxygen and nitrogen species, and reductants (antioxidant molecules). It has been shown that many enzymes are involved in redox homeostasis. Normal cell functions require the balance of oxidation and reduction processes [35,36]. We identified 21 proteins (spots 57-77) associated with redox homeostasis under blast fungus infection, which mainly included glutathione S-transferase, putative glyoxalase I, manganese superoxide dismutase, lactoylglutathione lyase, and superoxide dismutase (Table 1). In general, most of these proteins in Nipponbare were downregulated under the blast fungus infection. In Minghui, 5 out of the 21 proteins were upregulated and 3 downregulated under the blast infection. Treatment with salicylic acid did not change their expression patterns, suggesting the presence of an SA-mediated defense in the resistant cultivar subjected to blast infection.
Glutathione S-transferase is the key enzyme of glutathione binding reaction, and catalyzes the initial steps of the glutathione-binding reaction. It can catalyze the binding reaction of nucleophilic glutathione with various electrophilic exogenous chemicals. Many exogenous chemicals are easily separated from some bioactive intermediates in the biotransformation of the first phase reaction; they can covalently combine with the important components of cell biological macromolecules and cause cell damage. Glutathione can prevent the occurrence of such covalent binding for detoxification [37,38]. In this study, the expression of these proteins in both varieties decreased, but the Minghui cultivars showed a recovery after treatment with salicylic acid (spot 43). This suggests that exogenous treatment with salicylic acid is necessary to induce the glutathione-binding reaction process in Minghui after infection.
Manganese superoxide dismutase (spot 60) is an important antioxidation enzyme [39], alongside Betaine aldehyde dehydrogenase (spot 66) involved in the synthesis of betaine, which is the substance in methylation reactions and detoxification of homocysteine [40]. PDI-like protein (Spot 74) is also crucial; it catalyzes the formation of disulfides and also regulates specific biochemical processes [41]. The expression of these three proteins was significantly upregulated in Minghui infected with Magnaporthe grisea. By contrast, in Nipponbare, expressions of the proteins were significantly downregulated under blast fungus infection.

Proteins Associated with Amino Acid and Nitrogen Metabolism
Previously, rice blast fungus infection was shown to alter expression patterns of proteins involved in amino acid metabolism, nitrogen assimilation, nucleotide anabolism, and lipid oxidation [42]. In this study, we observed that cysteine synthase, glutamate ammonia ligase, ketol-acid reductoisomerase, and ornithine acetyltransferase expressions were altered in response to blast fungus infection ( Table 1). The activities of cysteine synthase determine the level of cysteines, an important amino acid in protein synthesis. The enzyme plays a very important role in antioxidation and stress resistance in plants [43,44]. In Nipponbare, Cysteine synthase was downregulated after blast fungus infection, and recovery was observed after treatment with salicylic acid. In Minghui, the expression of cysteine synthase remained stable under fungus infection.
Glutamate ammonia ligase (spot 81) plays a role in ammonia and glutamate detoxification, acid-base homeostasis, cell signaling, and cell proliferation. Ketol-acid reductoisomerase (spot 82) is one of the key enzymes in the biosynthesis of amino acids with branched chains in plants; however, interruption of the synthesis can cause plant death [45]. Ornithine acetyltransferase (spot 83) regulates the abundance of L-arginine, which plays an important role in maintaining cell functions [46]. Generally, the expressions of these three proteins were inhibited in Nipponbare under the Magnaporthe grisea infection; additional treatment with SA improved the expression of these proteins in blast-infected Nipponbare, while in Minghui their levels remained stable under infection.

Proteins Associated with Carbohydrate Metabolism
Glucose, a monosaccharide metabolized in plants, is one of the most important carbohydrates in plant cell metabolism [47]. We observed that the expression patterns of seven proteins involved in carbohydrate metabolism were downregulated in response to blast fungus infection in Nipponbare. Additional treatment with SA elevated the expression of these proteins, evidencing the vital role of SA during blast fungus infection. Furthermore, the cytoplasmic malate dehydrogenase (spot 86) plays an important role in the shuttle system of the cytoplasm and organelles, nucleic acid selective channels, gluconeogenesis, and malic acid metabolism [48]. In Nipponbare, this enzyme was significantly downregulated after Magnaporthe grisea infection, suggesting inhibition of corresponding biological processes. In comparison, enzyme levels remained unchanged in Minghui.
S-adenosylmethionine synthetase 2 (spot 90) plays an important role in cell metabolism in all organisms. We observed that the level of S-adenosylmethionine synthetase was downregulated only in Nipponbare infected by Magnaporthe grisea, and later recovered with salicylic acid treatment. Takusagawa et al. [49] have shown the abundance of spot 90 in more than one hundred different kinds of methyltransferase catalytic reaction of a donor in the synthesis of glutathione (GSH) precursor molecules of the sulfur transfer process and polyamine synthesis to aminopropyl process, and also related activity to a variety of enzymes [49].

Differentially Expressed Proteins Related to Protein Metabolism
Thirty-three proteins (spots 91-133) were shown to be mainly involved in the processes of protein metabolism including protein synthesis, protein folding, and assembly. Proteins involved in protein folding and assembly mainly included heat shock proteins (HSPs), HSP 70 kDa and 90 kDa. Heat shock proteins (HSPs) are synthesized under various stresses [50]. These proteins mainly serve as molecular chaperones to stabilize target proteins subjected to stresses, thereby playing a protective role [51]. In Nipponbare, these proteins were significantly downregulated after infection with Magnaporthe grisea. However, in Minghui the expression of these proteins was upregulated. Proteins involved in protein hydrolysis mainly include aminopeptidase, proteasome, hydrolase, and so on. These enzymes mainly function to degrade target proteins. In Nipponbare, these proteins showed a downward regulation trend in general, whereas the opposite was true in Minghui: of the two species, only a few were exceptions. Similar to the proteins involved in protein folding and assembly discussed above, the levels of the proteins may also indicate the differential response of the two rice cultivars under infection.
Proteins involved in protein synthesis mainly include ribosomal protein (RP), binding protein, assembly protein, and so on. Ribosomal protein is ubiquitous in every cell and forms ribosomes with RNA to function in protein synthesis [52]. In Nipponbare, the proteins were downregulated in response to blast fungus infection, but an afterward treatment with salicylic acid significantly improved the levels of the proteins. By contrast, in Minghui, the proteins were upregulated after infection.

Plant Materials
Two rice cultivars, blast-resistant Minghui (M) and blast-susceptible Nipponbare (R), were comparatively assessed for differential expression of proteins (DEPs) in this study. The 20 seeds disinfected with 70% alcohol and rinsed with distilled water were planted into a 10 cm-diameter plant pot in a greenhouse under optimum conditions and supplemented with the nutrient solution. The experiment was designed in two blocks (2 cultivars), 3 treatments (including the control-CK) and 3 replicates. Nine two-week-old rice plants from Minghui were randomly selected and divided into three replicate groups designated as the Minghui set as a control (M-CK), Minghui infected with Magnaporthe grisea fungus (M-M), and Minghui sprayed with salicylic acid (M-SA). Likewise, 3 Nipponbare replicate groups were randomly selected and designated as the Nipponbare set as a control (R-CK), Nipponbare infected with Magnaporthe grisea fungus (R-M), and Nipponbare sprayed with salicylic acid (R-SA). The groups, R-M, R-SA, M-M, and M-SA, were sprayed with a fungus strain of M. grisea, Guy11, at an intensity of 5 × 10 4 /mL. The groups R-CK and M-CK were sprayed with water and served as controls. To investigate the effect of salicylic acid, the groups R-SA and M-SA were sprayed with salicylic acid with a concentration of 8 mmol/L. Then, the rice plants were left for another week to grow. One-week-old treated leaves were harvested and stored at −80 • C before further experiments.

Protein Extraction
The trichloroacetic acid (TCA)/acetone precipitation method was used to extract proteins [18]. Leaf tissues were ground into a fine powder in liquid nitrogen. The resulting leaf powder was suspended in 10% (w/v) TCA/acetone supplemented with 0.1% DTT, and stored at −20 • C for 2 h for promoting perception. The suspension was centrifuged at 15,000 rpm for 20 min. The resulting pellet was re-suspended in 10% (w/v) TCA/acetone supplemented with 1 mM PMSF and 0.07% (w/v) β-mercaptoethanol, and was stored at −20 • C for 1 h. The suspension was further centrifuged at 15,000 rpm for 20 min to precipitate proteins, which were then subjected to vacuum freeze-drying processes. The resulting protein powder was dissolved in 800 µL lysis solution containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT, 1 mM PMSF, and 0.5% (v/v) violates. The protein concentration of the sample was quantified according to the Bradford method [53].

Database Search and iTRAQ Quantification
The spectra of proteins were used for the NCBInr database search with the online MASCOT program (http://www.matrixscience.com, accessed on 2 January 2019). The searching parameters included 0.15 Da mass tolerance for peptides and 0.25 Da mass tolerance for TOF-TOF fragments, one trypsin miscleavage allowed, carbamidomethyl of Cys as fixed modification, and oxidation of Met, pyro-Glu formation of N-terminal Gln and Glu as variable modification. Only significant hits, as defined by the MASCOT probability analysis (p < 0.05), were accepted [59]. Quantification of peptides and proteins was based on signature peak areas (m/z: 113, 114, 115, 116, 117, and 118). Relative quantification ratios of the identified proteins were calculated, averaged, and corrected for any systematic errors caused by iTRAQ™ labeling of peptides. The accuracy of each protein ratio was given by a calculated error factor in the analysis software. The standard of a 95% uncertainty range was adopted [60]. Finally, a more than 2.0-fold or less than 0.5-fold difference (p < 0.05) in expression values was considered as a significant difference in expression.

Conclusions
The M. grisea (Hebert) Barr continuously threatens rice production globally, and exogenous application with salicylic acid enhances rice resistance to blast disease infection. In this study, the iTRAQ approach was utilized to investigate the alteration of global protein expression abundance affected blast infection and SA application in rice varieties with different resistance to blast. A total of 139 DEPs were identified and were mainly involved in a wide range of biological processes, including energy-related activity, signal transduction, redox homeostasis, amino acid and nitrogen metabolism, carbohydrate metabolism, protein folding and assembly, protein hydrolysis, protein synthesis, and others. Comparing their expression patterns in two rice cultivars, susceptible Nipponbare and resistant Minghui, treated with SA and untreated with SA, it was found that expression patterns recovered from disease effects after SA exogenous application, which evidenced the potential role of SA to increase rice blast disease resistance. Nonetheless, there are still gaps within the comprehension of the SA mechanism in increasing blast resistance in rice. By contrast, SA-medicated pathways appeared to be constitutively expressed in the resistant Minghui cultivar, which enhances resistance to blast fungus infections.