Comparative Proteomic Analysis of Rhizoctonia solani Isolates Identifies the Differentially Expressed Proteins with Roles in Virulence

Sheath blight of rice is a destructive disease that could be calamitous to rice cultivation. The significant objective of this study is to contemplate the proteomic analysis of the high virulent and less virulent isolate of Rhizoctonia solani using a quantitative LC-MS/MS-based proteomic approach to identify the differentially expressed proteins promoting higher virulence. Across several rice-growing regions in Odisha, Eastern India, 58 Rhizoctonia isolates were obtained. All the isolates varied in their pathogenicity. The isolate RS15 was found to be the most virulent and RS22 was identified as the least virulent. The PCR amplification confirmed that the RS15 and RS22 belonged to the Rhizoctonia subgroup of AG1-IA with a specific primer. The proteomic information generated has been deposited in the PRIDE database with PXD023430. The virulent isolate consisted of 48 differentially abundant proteins, out of which 27 proteins had higher abundance, while 21 proteins had lower abundance. The analyzed proteins acquired functionality in fungal development, sporulation, morphology, pathogenicity, detoxification, antifungal activity, essential metabolism and transcriptional activities, protein biosynthesis, glycolysis, phosphorylation and catalytic activities in fungi. A Quantitative Real-Time PCR (qRT-PCR) was used to validate changes in differentially expressed proteins at the mRNA level for selected genes. The abundances of proteins and transcripts were positively correlated. This study provides the role of the proteome in the pathogenicity of R. solani AG1-IA in rice and underpins the mechanism behind the pathogen’s virulence in causing sheath blight disease.


Introduction
Rhizoctonia solani Kuhn (Teleomorph: Thanatephorus cucumeris (Frank) Donk) is a soilborne necrotrophic fungal pathogen that affects many food crops that are important to humankind. Fourteen anastomosis groups (AG1-AG13 and AGBI) have been assigned based on hyphal fusion [1]. The rice sheath blight causal organism, R. solani subgroup of AG1-IA, leads to severe economic losses to rice growers worldwide. Under favorable conditions, the pathogen could cause significant yield losses, affecting as much as 50% of the entire yield [2]. The severity of the disease depends on the variety, plant age, climatic factors and excessive use of nitrogenous fertilizers. R. solani AG1-IA produces necrotic, dark to reddish-brown color, oval or elliptical-shaped lesions on the leaf sheath, blade and culm of rice [3,4]. Unavailability of resistant donors, pathogen variability, prolonged existence as sclerotia in soil and lack of promising crop protection strategies make it challenging to manage sheath blight disease [5].
fewer experimental errors and a convenient approach in proteomics studies [28,29]. With this background information, the present study is undertaken to identify the differentially expressed proteins among R. solani isolates using a quantitative LC-MS/MS-based proteomic approach.

Isolation of Sheath Blight Pathogen
Infected rice plant sheaths and leaves exhibiting typical symptoms of sheath blight disease were gathered across various rice-growing areas of Odisha (40 locations from seven districts), Eastern India, during 2019-2020 and brought to the laboratory to isolate the causal organism. From each location 3-4 rice fields were selected randomly when the crop was at tillering to maturity stage. The specimens were gently rinsed in tap water, cut into small pieces (approximately 1 cm) and then sterilized for 1 min using 1% sodium hypochlorite solution, followed immediately by three sterile distilled water washes and blotted out between sterilized filter papers. The dried tissue bits were placed on a water agar medium and incubated at 27 ± 1 • C for 1-2 days. The emerged hyphal tips were examined under a microscope and then transferred to a Petri dish containing a medium of potato dextrose agar (PDA). The pure cultures in PDA slants were later stored at 4 • C.

Pathogenicity and Disease Assessment
At the ICAR-National Rice Research Institute, Cuttack, India (85 • 55 48" E longitudes and 20 • 26 35" N latitude), the experiment was implemented. The sheath blight susceptible cultivar, Tapaswini, was used for the experiment. Seeds were sterilized with a 2% sodium hypochlorite solution and washed with sterile distilled water before sowing. Then, seeds were sown in a pot (45 × 60 cm) with autoclaved pot mixture. The 25-day-old rice seedlings were transplanted at two seedlings per hill with three hills per pot (45 × 60 cm). The 58 R. solani isolates were grown on PDA medium with seven-day-old mycelial plugs embedded beneath the leaf sheath of 60-day-old plants. The inoculated sheaths were covered with absorbent cotton to maintain humidity. The plants without pathogen inoculation were maintained as healthy controls. The experiment employed a completely randomized block design. Every treatment had three replications with three pots in each and the experiment was repeated twice. Disease progress was documented on the 7th, 14th, 21st and 28th day after pathogen inoculation. The relative lesion height (RLH = lesion height/Plant height × 100) was recorded as described by Sharma et al. [30]. The disease severity was calculated based on the RLH percentage. The data of RLH was converted into a disease index based on a disease score with a 0 to 9 rating scale [31], where 0 represented no infection; 1, lesion was limited to 20%; 3, 21-30%; 5, 31-45%; 7, 46-65%; 9, more than 66-100% of plant height [32]. Based on disease reaction, isolates were categorized into four groups: highly virulent (8)(9), moderately virulent (4-7.9), less virulent (1-3.9) and avirulent (0). The virulence index was calculated by averaging the disease score. The percentage disease index (PDI) was calculated following Wheeler [33]. PDI = Sum of all rating/(Total no. of observations × Maximum rating scale) × 100.

Virulence Test on Other Rice Genotypes
The selected highly virulent (RS15) and less virulent (RS22) isolates were inoculated on other genotypes viz., Pusa Basmati-1, Vanaprabha, Hazaridhan, Swarna, Lunishree, Savitri, Sadabahar and TN1. The preparation of inoculums, method of artificial inoculation and disease assessments were similar to those in Section 2.2. The experiment was conducted with three replications in a completely randomized manner. Each replication had five pots with three hills (two seedlings/hill) in each pot. The experiment was repeated twice.

Molecular Characterization
The highly virulent and less virulent isolate of R. solani was characterized at the molecular level using ITS and AG1-1A primers. The fungal culture was grown on potato dextrose broth (PDB) for three days at 27 ± 1 • C. The fungal mycelium was harvested and finely ground with liquid nitrogen. The fungal genomic DNA was extracted as per the procedure by the manufacturer (Qiagen fungal DNA extraction kit). A thermal cycler (Eppendorf Master Cycler Gradient, (St. Louis, MO, USA) was used to amplify the ITS by applying the primer pair ITS1:CCTGTGCACCTGTGAGACAC and ITS4: TGTCCAAGT-CAATGGACTAT with the following PCR conditions: 94 • C for 5 min; 35 cycles of 94 • C for 1 min; 56 • C for 1 min and 72 • C extension for 1.5 min; and a final extension at 72 • C for 10 min [34]. Furthermore, the AG1-1A specific primers 5 -CTCAAACAGGCATGCTC-3 and 5 -CAGCAATAGTTGGTGGA-3 were used to identify R. solani as the subgroup of AG1-1A. A total of 25 µL PCR reaction mixture contained 12.5 µL Mix (Bangalore Genei, India), 0.5 µL of forward primer, 0.5 µL of reverse primer, 0.5 µL Taq enzyme, 1 µL of fungal DNA and 10 µL Nanopure water. The PCR reaction was executed with the following program: 94 • C for 4 min; 30 cycles of 94 • C for 1 min and 54 • C for 2 min; 72 • C for 3 min; and the final extension at 72 • C for 7 min [35]. The 25 µL of PCR products were analyzed on 1.5% agarose gel (Sigma-Aldrich, St. Louis, MO, USA), and a gel documentation system (Biorad, Berkley, CA, USA) was used to analyze the ethidium bromide-stained DNA bands. The PCR product was purified using a QIA quick gel extraction kit (Qiagen, Inc., Chatsworth, CA, USA) as per the manufacturer's instruction. The purified PCR products of ITS and AG1-1A were sent for DNA sequencing at AgriGenome Labs Pvt Ltd., Kochi, India. The DNA sequences were compared with existing R. solani sequences available in the GenBank database (www.ncbi.nlm.nih.gov), and submitted to GenBank applying the Bankit sequence submission tool.

Protein Extraction
R. solani isolates, RS15 and RS22, were cultured on PDB for three days at 27 ± 1 • C. Sterile filter paper was used to collect the mycelial mat, washed thrice with sterile distilled water and liquid nitrogen was used to obtain a fine powder. The TCA (sigma-76-05-1)acetone (Merck-100014) method was employed to precipitate the proteins. The samples were brought up to 10% TCA and incubated at −20 • C overnight. The samples were centrifuged for 10 min at 13,000 rpm to obtain an intact pellet and the supernatant was separated. Acetone wash (added 500 µL of acetone to the pellet and incubated for 10 min at −20 • C and again centrifuged for 10 min at 13,000 rpm) of the pellet was performed thrice. Finally, the air-dried pellet was dissolved in a 50 mM ammonium bicarbonate (Fluka analytical FL40867) buffer. The samples were solubilized in 100 µL of 50 mM ammonium bicarbonate (NH 4 HCO 3 ) with 1% SDS, mixed well by vortexing followed by 20 min sonication and centrifuged for 10 min at 10,000 rpm. The supernatant was quantified using the Bicinchoninic Acid assay, in which proteins were reduced from Cu +2 to Cu +1 in an alkaline solution, resulting in the formation of the bicinchoninic acid-induced purple color. The protein samples were incubated along with the standard at 37 • C for 60 min and the spectrophotometer reading was noted at 562 nm. From all the samples, an equal amount of protein was loaded on a 12% 1D SDS-PAGE, silver-stained gels were observed for bands and an Epson Expression 11000XL Scanner was used to scan them.

In Solution Protein Digestion
The extracted protein (100 µg) was digested, treated for 1 h at 95 • C with 100 mM dithiothreitol (DTT), and exposed for 45 min at room temperature in the dark to 250 mM iodoacetamide. With the addition of 4 µg of trypsin, digestion of the protein samples was achieved at 37 • C overnight. The peptides were resuspended in 0.1% formic acid (50 µL) and incubated for 45 min at 37 • C. The supernatants were transferred into a separate tube after centrifugation at 10,000× g. The speed vacuum concentrator (Basic Concentraor, Eppendorf, Hamburg, Germany) was used to vacuum dry (45 • C for 6 h at 800 Pa in VAQ mode) the resulting samples and was dissolved in 0.1% formic acid in water (20 µL) for LC-MS/MS.

LC-MS/MS Analysis
An ACQUITY UPLC system (Waters, UK) was used to perform liquid chromatography by injecting 10 µL of the protein samples. The ACQUITY UPLC BEH C18 column (150 mm × 2.1 mm × 1.7 µm) (Waters, UK) separated the samples. Three runs will be performed on each sample to achieve label-free quantification. A gradient elution program was performed for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). Mass spectrometric detection was performed with an electrospray ionization (ESI) source equipped with an SYNAPT G2 QTOF (Waters, UK). Sample analysis was performed in a positive mode.

Peptide Identification and Data Analysis
MassLynx 4.1 WATERS was used to process raw data. The protein identification on PLGS (Protein Lynx Global Server) software 3.0.2, WATERS involved matching MSMS spectra of individual peptides to the database sequence. The sample runs were processed following specific search parameters in the software: Peptide tolerance (ppm): 50; min no. of fragment matches for peptides: 2; fragment tolerance (ppm): 100; min no. of peptide matches for proteins: 2; min no. of fragment matches for proteins: 5; missed cleavages: 1. The sample cysteine sites were modified during processing to carbamidomethylated cysteine, and the methionine sites were considered as a variable modification to the mass, which was prone to oxidation. The Swissprot protein database for R. solani was used to search for proteins present in the sample, and a 5% false discovery rate was used. The expression score was normalized using PLGS software, and the mean abundance of a particular protein in two samples was determined by ratio calculation. A t-test determined the differences between isolates RS15 and RS22. Protein abundance RS15 to RS22 with at least two-fold differences was considered for up-regulation and 0.5-fold changes for down-regulation. The differentially abundant proteins were classified according to their molecular functions, biological processes and protein class using the Panther online tool (http://www.pantherdb.org/pathway/ (accessed on 10 January2022)).

Quantitative Real-Time PCR Analysis
R. solani isolates RS15 and RS22 were cultured on PDB for 3 days at 27 ± 1 • C, and the mycelial mat was collected from three biological replicates and immediately ground with liquid N 2 for RNA extraction. Three technical replicates per biological replicates were analyzed during qRT-PCR. Total RNA was extracted using the RNeasy Mini kit (QIA-GEN, Hilden, Germany). The isolated RNA quality was checked in 1.5% agarose gel and quantified by a NanoDrop ® ND-1000 UV-Vis spectrophotometer (Thermo SCIENTIFIC, Walthem, MA, USA). The residual DNA was removed with an RNase-free DNase I enzyme (QIAGEN, Hilden, Germany). According to the manufacturer's instructions, the complementary DNAs were synthesized from 1 µg of total RNA using the QuantiTect ® Reverse Transcription Kit (QIAGEN) in a 20 µL reaction mixture. The qRT-PCR specific primers were designed using the Primer Quest TM tool (Integrated DNA Technologies, Coralville, IA, USA) and all primer information is listed in Supplementary Table S1. The RT-PCR was performed on a BIORAD CFX96 Real-Time system, with a total of 10 µL PCR reaction mixture containing 1.0 µL of diluted template cDNA, 5.0 µL of 2× buffer SYBR Green (QIAGEN, Germany), 0.5 µL of each forward and reverse primer and 3.0 µL of sterilized nanopure water. The RT-PCR amplification conditions were as follows: pre-incubation for 95.0 • C for 15 min, and three-step amplification at 94.0 • C for 0.15 s, 60 • C for 0.30 s and 72 • C for 0.30 s and 39 cycles, followed by 95.0 • C for 0.05 s, 65 • C for 0.05 s and 95 • C for 0.5 s. The Rhi-18S rRNA (Forward: ATGATAACTCGACGGATCGC; Reverse: CTTGGATGTGGTAGCCGT) was used as the internal control to normalize the expression of each gene, and the specificity of amplicons was verified by a melting curve analysis using the peak values [36]. Additionally, specificity was confirmed by running a 2.5% agarose gel. Fluorescence was measured at the end of every 72 • C extension phase. The final threshold cycles (Ct) values were the means of three values, including three biological replicates and three technical replicates per biological replicates. The relative expression level of each gene was calculated using the 2 -∆∆Ct method. The Ct values for the housekeeping gene was subtracted from the gene of interest to obtain a Ct value. The Ct value of the mock-inoculated control sample was subtracted from the ∆Ct value to obtain the ∆∆Ct value = Ct Target-Ct Reference. Each fold change in expression level relative to that of the control was expressed as 2 -∆∆Ct and all experiments were repeated thrice.

Statistical Analysis
The IRRISTAT v.92-1 program (Biometric Unit, International Rice Research Institute, Los Baños, Laguna, Philippines) was utilized for the data analysis. Data were subject to an analysis of variance (ANOVA, New Providence, NJ, USA), and the data in percentages were arcsine transformed before the analysis. The treatment means were compared by the Duncan's multiple range test (DMRT) [37].

Isolation, Pathogenicity and Molecular Characterization
Overall, 58 isolates were identified from various rice-growing areas of Odisha, Eastern India. Artificial inoculation on the rice cultivar Tapaswini demonstrated that typical symptoms of sheath blight disease were produced in all isolates, which displayed differences in virulence, disease index and disease progress. RS15 recorded 100% PDI among the isolates, followed by RS34 (92.59%). The isolates RS16, RS17, RS22, RS26 and RS27 had the lowest PDI of 11.11%. Based on disease reaction, the 58 isolates were categorized into highly virulent (2), moderately virulent (31) and less virulent (25). The virulent index varied from 1 to 9 (Table 1). The RLH was recorded on the 7th, 14th, 21st and 28th days after pathogen inoculation. Among the isolates, RS15 demonstrated the maximum percentage of RLH, and RS22 demonstrated the most negligible percentage of RLH at different time intervals (Figure 1).
Isolates RS15 and RS22 were characterized at the molecular level by sequencing the rDNA internal transcribed spacer region using ITS and AG1-1A specific primers. The DNA sequences were matched with the NCBI GenBank BLAST analysis (http://www. ncbi.nlm.nih.gov (accessed on 5 March 2021)). The resultant sequences were submitted to GenBank, and the accession numbers of MW762960, MW757241 for RS15 and MW757249 and MW757239 for RS22 were obtained, respectively.

Determination of Virulence on Other Rice Genotypes
The virulence spectrum of RS15 and RS22 was evaluated on eight rice genotypes viz., Pusa Basmati-1, Vanaprabha, Hazaridhan, Swarna, Lunishree, Savitri, Sadabahar and TN1. The plants challenged with RS15 demonstrated higher RLH % on different genotypes than the RS22-inoculated plants on the 7th, 14th, 21st and 28th day, after pathogen inoculation. The disease progress was significantly increased in RS15-inoculated plants, which were low in RS22-inoculated plants (Figure 2).

Proteomic Analysis
A comparative proteomic analysis was performed for virulent (RS15) and less virulent (RS22) isolates using the LC-MS/MS analysis to identify the proteins related to virulence. The results demonstrated that the virulent isolate comprised 48 disparately abundant proteins, out of which 27 proteins had higher abundance and 21 had lower abundance in the virulent isolate. The proteomic information generated has been archived in the PRIDE database and can be accessed with PXD023430 [38,39]. Among the differentially abundant proteins, JmjC domain-containing histone demethylation protein 1, sorting nexin-4, rRNA biogenesis protein RRP36, Exportin-T, Heat shock 70, topoisomerase 1, cyanate hydratase, pyranose 2-oxidase, actin-like protein ARP6, phenylalanine ammonia-lyase and squalene synthase were the most predominant with an increased abundance in the virulent isolate. Similarly, GMP synthase, elongation factor G, glycylpeptide N-tetradecanoyl transferase, sulphate adenylyltransferase, 5 -3 exoribonuclease 2, glyceraldehyde-3-phosphate dehydrogenase, protein phosphatase methylesterase and protein CFT1 were found to be depleted ( Table 2).

Pathway Analysis
A pathway analysis using the Panther online tool revealed the molecular functions, biological processes and protein class in which differently abundant proteins were involved, as depicted in Figure 3.

Validation of Differentially Expressed Proteins by qRT-PCR
qRT-PCR was performed to investigate the changes of differentially expressed proteins at mRNA levels in RS15 and RS22. The six up-regulated proteins viz., phenylalanine ammonia-lyase (P10248), squalene synthase (Q92459), cyanate hydratase (A8NV38), actinlike protein ARP6 (P0CM05), topoisomerase 1-associated factor 1 (P0CR92) and JmjC domain-containing histone demethylation protein 1 (P0CO40), and five down-regulated proteins, such as GMP synthase [glutamine-hydrolyzing] (Q4P763), glycylpeptide Ntetradecanoyltransferase (P34809), sulfate adenylyltransferase (Q4P460), glyceraldehyde-3phosphate dehydrogenase 1 (P32635) and pentafunctional AROM polypeptide (P0CM22), were randomly selected for gene expression analysis. The relative mRNA levels of each transcript were checked in RS15 and RS22. Each gene's mRNA levels were normalized to the expression of Rhi-18S rRNA as a control gene in each sample. The qRT-PCR results demonstrated a positive correlation of the mRNA expression level with the changes in protein abundance levels for all eleven genes (Figure 4).  The relative fold changes of mRNA and protein level were positively correlated for eleven genes with a 95% correlation (r = 0.95) that indicated a strong agreement between the qRT-PCR analysis and the proteomic data. This result confirmed our proteomics data and implied that the proteomics results were reliable.

Discussion
Across different rice ecosystems in Odisha, eastern India, 58 isolates of R. solani were isolated. Koch's postulates were proven for the sheath blight pathogen on the rice cultivar, Tapaswini. The isolates varied in their virulence from 11.11% to 100%. The virulence diversity among R. solani isolates have been well documented by many researchers [13,[40][41][42][43]. We have used both universal ITS and species-specific R. solani AG1-IA primers to identify at the molecular level. The PCR amplification and sequencing results revealed that RS15 and RS22 belonged to the R. solani AG1-IA. These results were supported by Lore et al. [13] and Wang et al. [9], who differentiated R. solani isolates using species-specific AG1-IA primers. Similarly, Yu et al. [44] used AG1-IA specific primers and identified that 220 strains of R. solani isolates collected from 11 provinces of China belonged to R. solani AG1-IA.
Several researchers have well documented the morphological and genetic diversity of R. solani isolates [45][46][47]. However, much work has not been published on the fungal proteomics of this specific pathogen. Variations in protein expression levels among the isolates could be studied from the protein profiles of pathogens. It helps identify proteins commonly present within the population of certain pathogens. Fungal proteomics has become a suitable tool to obtain molecular maps of pathogenicity and virulence factors; thus, new insights were opened to detect and protect plant pathogens. Fungal-secreted proteins contribute to virulence and fungal pathogenicity control [20]. In this aspect, virulence-responsive protein identification is most important to uncover the mechanism of the pathogenic process in plants. Hence, we attempted to compare the proteomic profiles of the highly virulent RS15 isolate with the less virulent RS22 isolate using a quantitative LC-MS/MS-based proteomic approach to identify differentially expressed proteins that promote virulence. A total of 27 proteins were differentially expressed with higher abundance in the virulent isolate RS15, of which there existed a greater abundance of protein P0CR62; sorting nexin-4 was highly up-regulated in RS15 compared to RS22. Deng et al. [48] reported that sorting nexin is essential for Magnaporthe conidiation and pathogenicity. The Snx41 mutant strain exhibited reduced aerial hyphal growth and defects in conidiation and pathogenicity of M. oryzae. Similarly, Zheng et al. [49] reported that sorting nexin MoVps17 null mutation is responsible for defective growth, development and pathogenicity in M. oryzae. Our results suggest that higher expression of sorting nexin might support fungal development and virulence of R. solani (RS15).
In our study, the proteins P0CO40 and P0CO41 were identified as the histone demethylation protein 1 containing the JmjC domain. These play a vital role in histone demethylation and transcriptional regulation of secondary metabolite gene clusters in Aspergillus nidulans and phytopathogenic fungus Fusarium graminearum [50,51]. Huh et al. [52] conducted a functional analysis of a putative JmjCdomain-containing histone demethylase in Magnaporthe oryzae. The fungal development requires MoJMJ1-encoding JmjC histone demethylase, especially in the pre-penetration phase of M. grisea [52]. From these supportive findings, our results suggest that the over-expression of P0CO40 and P0CO41 proteins enhanced the pathogenicity of virulent isolates.
The protein P18694 was identified as Heat shock 70 kDa (Hsp70) protein 2, which was overexpressed in RS15. Hsp70 is highly conserved and has a prominent role in the fungal system for growth, morphogenesis and various stress conditions [53,54]. Dnj1 in Ustilago maydis is imperative for pathogenicity and was characterized as part of a conserved cellular response to ER stress [55]. Yang et al. [56] revealed that heat shock proteins MoSsb1, MoSsz1 and MoZuo1 and their functional mechanisms are involved in regulating the conidiation, pathogenicity, vegetative growth and mating of rice blast fungus. Similarly, FgHsp90 is required for the virulence sexual and asexual development of the plant pathogenic fungus, Fusarium graminearum [57]. Chen et al. [54] demonstrated that FpHsp70 genes were upregulated when infected with F. pseudograminearum, implying their significance in the virulence of F. pseudograminearum. This supporting information suggested an up-regulation of Hsp70 in virulent isolates that plays a significant role in sexual development, sporulation and virulence.
Another up-regulated protein was P0CR92, Topoisomerase 1, which relaxes the supercoiled DNA and enables numerous basic cellular processes, including transcription, replication and recombination [58]. In wheat ear infection assays, top1 mutants of F. graminearum and F. culmorum demonstrated exceptionally reduced virulence. The particularly mutated strain F. graminearum negatively affected the pathogenicity and conidia formation of the fungi [59]. When functionally analyzed, the TOP1 gene from the Basidiomycete plant pathogen Ustilago maydis demonstrated that vegetative growth did not require it, but a pigmentation defect was exhibited by its mutant [60]. From this information, our study revealed that Topoisomerase 1 was necessary for RS15 virulence. The proteins P0CM04 and P0CM05 were linked with actin-like protein ARP6, which were overexpressed in the virulent isolate. Actin is one of the most fundamental and abundant proteins in eukaryotic cells. ARP6, an actin-related protein, is essential for nucleolar function and structure. Further, it has a vital role in transcriptional regulation and DNA repair [61,62]. This finding supports that actin-like protein ARP6 up-regulation in the virulent isolate was vital for essential metabolism and transcriptional activities. Squalene synthase (Q92459) was over-expressed in the virulent isolate RS15. Squalene is a polyunsaturated terpene, an intermediate in the ergosterol biosynthetic pathway, playing a significant role in fungal cell structure. Squalene levels influence ergosterol biosynthesis [63]. Primarily, the squalene synthase Erg9 enzyme used two molecules of farnesyl-PP to form squalene, the precursor of all steroids. All eukaryotic organisms have essential molecules called sterols, and various genetic mutations that eliminate the enzymatic steps in sterol biosynthesis were proven to be lethal [64]. Numerous antifungal agents, including azoles, target the sterol biosynthesis pathway, which results in the production of ergosterol, a significant component of the fungal plasma membrane [65]. The secondary metabolite compounds ergosterol and their biological function were identified from R. solani [66][67][68]. Moreover, overexpressed squalene synthase enzyme in virulent isolate may promote the proper function of critical molecular events, particularly in ergosterol synthesis.
The protein A8NV38, cyanate hydratase, is over-expressed in virulent isolate, which plays a vital role in nitrogen assimilation or cyanate detoxification in bacteria, fungi and plants [69][70][71]. Cyanate, an important cyanide derivative, is formed by the oxidation of a toxic substance, cyanide. The whole-genome sequencing and secretome analysis of Thermomyces lanuginosus demonstrated the presence of cyanase (Tl-Cyn), and the overexpressed Tl-Cyn gene was evaluated for cyanate detoxification activity [72][73][74][75][76]. In our study, the virulent R. solani had overexpressed cyanases and hypothesized that virulent R. solani could tolerate plant-produced and another microbial cyanate in the soil environment. The protein Q6QWR1 is linked with pyranose 2-oxidase (Pox2), a flavin-dependent oxidoreductase [77]. It oxidizes D-glucose as well as other monosaccharide substrates of hydrogen peroxide. Pox is typically found in basidiomycetes fungi, where it is associated extracellularly with membrane-bound vesicles or other membrane structures in the periplasmic space of hyphae [78,79]. The hydrogen peroxide (H 2 O 2 ) secreted by the fruiting bodies of Tricholoma matsutake, produced by pyranose oxidase, strongly inhibited mycelial growth of the phytopathological fungus R. solani. This data suggested a high-level expression of pyranose oxidase that plays a significant role in the antifungal activity, thereby dominating the microbial communities [80]. The role of some up-regulated proteins, such as Protein EFR, Pre-mRNA-splicing factor, Lon protease homolog, O-acetyltransferase, rRNA biogenesis protein, mRNA cleavage and polyadenylation factor CLP1 on the virulence or pathogenicity of R. solani, is unknown.

Conclusions
A total of 58 R. solani isolates were obtained from rice-growing areas of Odisha, Eastern India. They displayed differences in virulence on the rice cultivar, Tapaswini. The isolate RS15 was found to be the most virulent and RS22 was identified as the least virulent. The virulence of RS15 and RS22 was further confirmed on other rice genotypes viz., Pusa Basmati-1, Vanaprabha, Hazaridhan, Swarna, Lunishree, Savitri, Sadabahar and TN1. Further, we employed an LC-MS/MS-based proteomic approach to profile the proteomic differences between the virulent RS15 isolate and less virulent RS22 isolate. A total of 48 differentially expressed proteins were identified, of which 27 proteins were up-regulated in virulent isolate. These up-regulated proteins were more responsible for pathogenicity, detoxification, antifungal activity, sporulation, fungal development, morphology, essential metabolism and transcriptional activities. The important proteins, such as sorting nexin-4, JmjC domain-containing histone demethylation protein 1, topoisomerase 1, squalene synthase, pyranose 2-oxidase, Hsp70 and cyanate hydratase, may have major roles in the pathogenicity and virulence of R. solani RS15. The proteomic information generated in this study contributes knowledge on the virulence mechanism responsible for the sheath blight of rice disease. Further research is required to explore the importance of these proteins and their possible roles during sheath blight disease establishment.
Author Contributions: S.R.P., conceptualization, methodology, writing-original draft preparation. C.P., data analysis and interpretation. S.B.S., methodology and validation. R.N., data curation and conceptualization. A.M., methodology and software. U.K., writing-reviewing and editing. M.K.Y., writing-reviewing and editing. A.A., data curation and investigation. P.P. and M.K.B., data analysis and interpretation. P.C.R., supervision. All authors have read and agreed to the published version of the manuscript.

Funding:
The work was carried out at ICAR-NRRI with the regular funds from the Indian Council of Agricultural Research (ICAR).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.