Quantitative proteomic analysis deciphers mechanisms of sheath blight resistance in novel rice landrace against Rhizoctonia solani

doi:10.21203/rs.3.rs-4316464/v1

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Quantitative proteomic analysis deciphers mechanisms of sheath blight resistance in novel rice landrace against Rhizoctonia solani

https://doi.org/10.21203/rs.3.rs-4316464/v1

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Sheath blight (ShB) disease, instigated by Rhizoctonia solani Kuhn, inflicts a significant economic threat to rice production, trailing closely behind blast. Acknowledging the limited understanding of ShB resistance proteomics in highly resistant germplasm, our study aimed to unravel the proteomic intricacies underlying the interaction between resistant landrace Nizam Shait and R. solani. Utilizing Nizam Shait and BPT-5204 as representatives of ShB resistance and susceptibility, a comparative proteome analysis was performed using Orbitrap-Fusion mass spectrometry. The analysis unveiled 5133 differentially expressed proteins, with 118 significantly upregulated and 172 significantly downregulated at a0.05 p-value. Notably, the 14-3-3 like protein GF-E exhibited the highest upregulation, indicating its pivotal role in the brassinosteroid signaling pathway and defense modulation. Proteins associated with systemic acquired resistance, R gene-mediated resistance, and cell death were downregulated, while those linked to jasmonic acid-induced systemic resistance (JA-ISR) showed notable upregulation, confirming ISR induction in response to ShB infection. Key signaling pathways involved in ShB resistance in Nizam Shait encompassed PTI via JA-ISR, cell wall strengthening, and brassinosteroid and abscisic acid-mediated resistance. Validation of the proteome data through qRT-PCR corroborated the findings, underscoring the significance of this research for future proteome assisted breeding efforts aimed at developing ShB resistant rice varieties.

Biological sciences/Molecular biology

Biological sciences/Plant sciences

Sheath blight

Rhizoctonia solani

Comparative proteomics

Orbitrap - fusion mass spectrometry

Differentially expressed proteins

14-3-3 like protein GF-E

Sheath blight (ShB) poses a significant threat to rice cultivation, inflicting widespread devastation as one of the most pervasive fungal afflictions. Across various cropping regions, its impact is felt profoundly, with yield losses ranging from 5.9 to a staggering 69 percent, contingent upon diverse environmental factors and cropping practices^1–2. The proliferation of this malady finds solace in intensive farming practices, the adoption of high-yielding dwarf cultivars, and the dense canopy formed by crops. Its causal agent, the soil-borne necrotrophic pathogen Rhizoctonia solani Kuhn (Teleomorph: Thanatephorus cucumeris (Frank) Donk) AG1-IA survives as mycelium within debris and the formation of sclerotia in the absence of a host³. Given its enduring presence, broad spectrum of hosts, and the scant natural resistance exhibited by rice cultivars, grappling with the management of this disease proves to be a formidable challenge.

Currently, disease management in farmers' fields heavily relies on chemical interventions, which unfortunately carry adverse effects on the environment, beneficial microbes, and human health. The optimal approach to combat ShB lies in the development of resistant cultivars, either through conventional or molecular breeding techniques. However, the complex polygenic nature of resistance coupled with the absence of major resistance genes in the primary gene pool poses significant challenges in the breeding program⁴. Despite its global devastation, the precise molecular mechanisms governing the interactions between R. solani and rice remain elusive, with scant information available⁵. In such a perplexing scenario, proteomics emerges as a valuable tool, shedding light on the crop's adaptability to the extreme pressure of pathogen which in turn aids the breeding community to identify the resistant proteins formed before and after infection and develop the resistant cultivars⁶.

Proteomic analysis stands as a beneficial tool for unraveling the intricate interactions between plants and pathogens, owing to the direct implication of proteins in molecular processes and biological functions⁷. Unlike other omics branches, proteomics offers distinct advantages; the proteome closely mirrors the phenotype, unlike the genome or transcriptome. Moreover, evidences suggest a low correlation between protein and transcript expression levels across various organisms^6,8.

The era of proteomics dawned with the conventional gel-based approaches and advanced further with gel-free, labelled and label-free approaches as a result of advancements in mass spectroscopy (MS). Conventional techniques such as two dimensional gel electrophoresis (2DGE) and difference gel electrophoresis (DIGE) are effective, but suffer from limitations such as restricted coverage, low throughput and less sensitivity and hence, labelled and label-free proteomics have emerged as the preferred methods for studying the entire proteome. The rapid progress in MS-based proteomics, coupled with advancements in bioinformatics, has empowered the analysis of a larger proportion of proteins, offering a comprehensive understanding of the host system⁹. In scenarios where comprehensive insights are crucial, MS-based comparative proteomic approaches prove invaluable enabling the examination of proteome alterations in hosts or pathogens triggered by specific events at particular or different intervals of time⁷. In recent years, the integration of linear ion trap with an Orbitrap analyzer has emerged as a gold standard approach in MS, particularly for characterizing the whole proteome¹⁰. Here, we have employed Orbitrap fusion mass spectroscopy (Orbitrap-MS), which affords high-resolution MS spectra acquisition at rapid sequencing speeds, without compromising on depth of analysis.

Due to the scarcity of highly resistant germplasm sources, a panel of 450 rice landraces of Karnataka, India were screened against R. solani and identified few highly resistant landraces (Grade 1 with 1–20% RLH) as per Standard evaluation system¹¹. Subsequent independent investigations employing association mapping and bulk segregant approaches revealed the presence of novel Quantitative Trait Loci (QTLs) associated with ShB resistance in these landraces^12,13. In this background, present study was aimed to delve into the mechanisms underlying resistance in these novel landraces utilizing a comparative proteomic approach and to pinpoint the key proteins responsible for ShB resistance using Orbitrap-MS.

Plant material and pathogen inoculation

The landrace Nizam Shait was used as highly resistant source for sheath blight which is maintained in the repository of University of Agricultural Sciences,Dharwad,Karnataka,India^11,12. Permission was obtained from the University of Agricultural Sciences,Dharwad to use Nizam Shait for research purpose.The variety BPT-5204 is a popular variety of India but highly susceptible to disease.The seeds of Nizam Shait and BPT-5204 were sown in earthen pots filled with autoclaved soil and fertilized as recommended. Each pot contained three robust and healthy plants, two of which were deliberately inoculated with the pathogen, while the third served as a control¹². The experiment was meticulously replicated thrice to minimize potential errors. The ten-days-old culture of highly virulent isolate of Rhizoctonia solani RS4 (GenBank Accession No MK213724), maintained on potato dextrose agar (PDA) was utilised for this study¹⁴. Inoculation was conducted when the plants reached 50 days after sowing, employing the agar block method¹⁵. This involved delicately opening the leaf sheath and placing agar blocks containing sclerotia and mycelia using forceps. The inoculated area was covered with wet cotton and aluminum foil to prevent moisture loss, and the pots were enclosed in polythene bags to uphold humidity levels conducive to disease development.

Sample collection

The leaf sheath samples were collected from Nizam Shait, BPT-5204 plants and their respective controls in triplicates at five days post inoculation (dpi) for protein isolation (Fig. 1).The polythene cover, aluminium foil and moist cotton were removed from the inoculated plants and the inoculation site was gently wiped with cotton soaked in 70 per cent alcohol in order to eliminate any pathogen structures. Using sterilized scissors, the leaf sheath portion above the lesion was carefully excised, collected in aluminum foil, promptly wrapped, and immersed in liquid nitrogen to halt enzymatic activity before being stored at -80°C for further analysis.

Protein isolation and quantification

Protein isolation was conducted using the phenol extraction method as outlined by Amalraj et al. with slight modification¹⁶. The protein content in the test samples was quantified using the Bradford protocol¹⁷. Bovine serum albumin (BSA) stock solution (1.0 mg/ml) was utilized to prepare protein standards. Later, final volume was adjusted to 30 µl by adding 10 µl sample buffer. Dilutions of different protein samples were prepared by dissolving 2.5 µl of protein samples with 7.5 µl of sample buffer, followed by the addition of 20 µl of deionized water to each dilution. Subsequently, 1 ml Bradford’s reagent was added to both protein standards and samples and allowed to incubate for 20 minutes in dark conditions. Absorbance readings were recorded at 595 nm using the Eppendorf BioSpectrometer (Eppendorf, Germany). A linear standard curve and regression equation was generated in Microsoft excel (Microsoft, USA) based on the absorbance values of BSA standards. The protein sample's concentration was determined by comparing its absorbance value to the standards and using the regression equation.

Trypsin digestion

The samples were diluted with solubilization buffer to achieve a final protein concentration of 50 µg. Then, 10 µl of the diluted sample was mixed with double-distilled water to a total volume of 20 µl. To this mixture, Tris(2-carboxylethyl)phosphine (TCEP) (0.8 µl, 20mM) (Sisco Research Laboratories, India) was added and incubated at 37°C for 60 minutes, followed by addition of Iodoacetamide (IAA) (1.6 µl, 40 mM) (Bio-Rad Laboratories, USA) and further incubation in darkness for 30 minutes. Subsequently, 179.2 µl of 50 mM ammonium bicarbonate (pH 8.0) (HiMedia, India) in a 1:8 ratio was added to dilute urea to a final concentration of 1M. Afterwards, pierce trypsin protease (ThermoFisher Scientific, USA) was introduced in a 1:50 ratio (4 µl of trypsin for 200 µl of total mixture) and incubated at 37°C for 12 hours. The digested samples were then stored at -80°C before undergoing lyophilization using the Scanvac Coolsafe Touch 110-4 Freeze dryer (LaboGene, Denmark). Finally, the lyophilized samples were subjected to orbitrap-MS analysis at the Mass Spectrometry Facility at IIT Bombay (MASSFIITB), India.

Protein identification and data analysis

The proteome database for Oryza sativa was accessed from UniProt, followed by protein identification using Proteome Discoverer Software 2.4. The analysis entailed treatments comprising pathogen-treated resistant germplasm (Nizam Shait) and pathogen-treated susceptible check (BPT-5204). Subsequently, the mean of protein abundances was calculated, and relative abundance ratios were determined by dividing the two treatments of interest. These ratios were then converted into log₂-fold change values. Values below − 1.5 indicated protein downregulation in the treatment compared to the control, while positive values exceeding 1.5 folds indicated upregulation. The p-value was computed using the standard t-test formula. The proteins with log₂-fold change values surpassing 1.5 and the p-value below 0.05 were designated as significantly upregulated proteins, whereas those with log2-fold change values below − 1.5 and a p-value below 0.05 were deemed significantly downregulated¹⁸. Principal component analysis (PCA) was conducted on the average abundance values of each protein for every treatment using the R package to confirm the findings.

In silico analysis

The gene ontology, encompassing biological processes, molecular functions and cellular components, was elucidated using db2db (bioDBnet - Biological Database Network (ncifcrf.gov). The enrichment analysis was conducted using ShinyGO 0.76.1¹⁹. The functional classification of significantly upregulated and downregulated proteins was accomplished using Panther database (pantherdb.org) in conjunction with a thorough literature search. Additionally, the interplay among defense-related proteins was scrutinized using String version 11.5 at string-db.org.

Validation of proteomic data through quantitative real time polymerase chain reaction (qRT-PCR )

Validation commenced with the isolation of RNA from samples collected at 5 dpi. Following this, total RNA extraction from both inoculated and uninoculated leaf sheath samples was carried out using the TRizol method, with subsequent quantification performed using the Eppendorf BioSpectrometer (Eppendorf, Germany). The cDNA synthesis was executed utilizing PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). For the assessment of selected gene transcript levels, qRT-PCR was employed in a total voume of 10 µl containing 0.2 µl of each primer (100 µM), 1 µl of the cDNA, 5 µl of TB greenPremix Ex Taq™ II (Takara, Japan), ROX reference dye 0.4 µl (Takara, Japan) and 3.2 µl of nuclease free water in QuantStudio™ 5 Real-TimePCR system (Thermo Fisher Scientific, USA). Cycling conditions comprised an initial denaturation step at 95°C for 5 minutes, followed by 45 cycles of amplification (denaturation at 95°C for 15 seconds, annealing as per primer specifications for 30 seconds, and extension at 72°C for 45 seconds).

Key defense related proteins, including 14-3-3-like protein GF14-E, Acetylserotonin O-methyltransferase 2 (ASMT), Probable glutathione S-transferase GSTF2 (GST), Chitinase 8, Non-expressor of pathogenesis related gene (NPR4) and photosynthesis related protein Chlorophyll a-b binding protein 2 (CBP) were selected (Supplementary Table S1). The data were normalized using rice OsEF-Iα gene specific primer OSEF-1F/OSEF-1R. For each treatment, three technical replicates were used. The relative expression of different expressed genes was determined using ΔΔCT method²⁰.

Proteomic analysis

Nizam Shait and BPT-5204 plants were infected with R. solani at maximum tillering stage, and leaf sheath samples were collected at 5 dpi for protein extraction. The linear standard curve and the regression equation (y = 0.0341x + 0.054) based on BSA standards indicated an approximate protein concentration of 5 mg/ml or 5 µg/µl in the extracted samples (Supplementary Fig. S1). The proteomic dataset detailing rice-R. solani interaction yielded 5,133 proteins in the present analysis. Subsequent statistical analysis revealed 373 proteins to be upregulated, with 118 significantly upregulated in pathogen-inoculated Nizam Shait compared to the BPT-5204 sample. Similarly, 393 proteins were found to be downregulated in pathogen-inoculated resistant Nizam Shait compared to susceptible BPT-5204, among which 172 proteins were significantly downregulated, as illustrated in Fig. 2. PCA differentiated between pathogen-inoculated BPT-5204, Nizam Shait, and control Nizam Shait, supporting the sequencing results (Fig. 3). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier “PXD040634”.

GO enrichment analysis of DEPs

Significantly upregulated proteins showed the highest fold enrichment in monocarboxylic acid metabolic process and carboxylic acid metabolic process in terms of biological processes. Chloroplast and ribosome were the most enriched cellular components, while oxidoreductase and structural activities dominated in molecular functions (Fig. 4a). In case of significantly downregulated proteins, the predominant biological processes were translation and peptide biosynthesis. These proteins were found to be enriched in ribosome and endoplasmic reticulum lumen in cellular components, with translation being the most enriched molecular function (Fig. 4b).

Functional annotation of DEPs

The significantly upregulated and downregulated proteins were segregated into different functional categories based on biological process using Panther database (pantherdb.org). The identified one hundred and eighteen significantly upregulated proteins were classified into seven functional categories: interspecies interaction between organisms, biological regulation, cellular process, localization, metabolic process, response to stimulus and defense related proteins (Fig. 5 (Left)). Among these, 28 proteins were found to be directly engaged in defense mechanisms, encompassing well known plant defense proteins such as chitinase 8 (Q7XCK6) (2.63 folds), phenylalanine ammonia lyase (PAL) (P14717) (2.19 folds), peroxiredoxin Q (P0C5D5) (2.10 folds), peroxiredoxin-2E-2 (Q7F8S5) (1.69 folds), alongside some less familiar yet pivotal defense proteins like 14-3-3-like protein GF14-E (Q6EUP4) (5.80 folds), cysteine synthase (Q9XEA6) (4.43 folds), sucrose synthase 7 (Q7XNX6) (3.93 folds), pyruvate dehydrogenase E1 component subunit beta-2 (Q0J0H4) (3.13 folds), succinate-semialdehyde dehydrogenase (B9F3B6) (2.95 folds), glycolate oxidase (2.90 folds), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (Q6K8J4) (2.78 folds), gamma-amminobutyrate transaminase 1 (Q01K11) (2.74 folds), monodehydroascrobate reductase 3 (Q652L6) (2.73 folds), glutathione S-transferase (O82451) (2.72 folds), chalcone synthase (A2ZEX7) (2.60 folds), lactoylgluthionase lyase (Q948T6) (2.27 folds), and others (Supplementary Table S2g). Similarly, the significantly downregulated proteins were grouped into eight biological functions: biological regulation, cellular process, developmental and multi-organismal process, localization, metabolic process, response to stimulus, signalling and defense related proteins (Fig. 5 (Right), Supplementary Table S3).

Protein-protein interaction studies

The analysis yielded a comprehensive diagram elucidating the interrelation of proteins concerning biological processes, molecular functions, cellular components, and KEGG pathways. Within this framework, 8 proteins crucial to jasmonic acid-based induced systemic resistance (JA-ISR) and 5 proteins contributing to cell wall fortification were pinpointed. Specifically, the JA-ISR ensemble comprised allene oxide cyclase (A2XID3), allene oxide synthase 2 (Q7XYS3), allene oxide synthase 1 (Q7Y0C8), probable linoleate 9S-lipoxygenase 4 (Q53RB0), probable lipoxygenase 8 (Q84YK8), putative 12-oxophytodienoate reductase 4 (Q69TH8), putative 12-oxophytodienoate reductase 5 (Q69TI0) and putative 12-oxophytodienoate reductase 11 (B9FSC8) (Supplementary Table S4a). Meanwhile, the cellular reorganization and strength was associated with UDP-arabinopyranose mutase 3 (Q6Z4G3), Probable inactive UDP-arabinopyranose (Q7FAY6), Expansin-A4 (A2Y5R6), Expansin B4 (Q94LR4) and Expansin B3 (Q336T5) (Supplementary Table S4b). Among the pool of 41 defense-associated proteins consisting of 28 defense proteins, 8 JA-ISR related proteins and 5 cell wall strengthening proteins, the string analysis successfully identified 36.

A network of proteins orchestrating cellular biosynthesis processes emerged, featuring Probable lipoxygenase 8 (Q84YK8), Allene oxide synthase 2 (Q7XYS3), Allene oxide synthase 1 (Q7Y0C8), Putative 12-oxophytodienoate reductase 4 (Q69TH8), Putative 12-oxophytodienoate reductase 5 (Q69TI0), Putative 12-oxophytodienoate reductase 11 (B9FSC8), Probable linoleate 9S-lipoxygenase 4 (Q53RB0) (Supplementary Fig. S2a). This assembly, along with 9-cis-epoxycarotenoid dioxygenase NCED1 (Q6YVJ0), Glycolate oxidase 1 (Q10CE4) and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (Q6K8J4) contributed to the monocarboxylic acid metabolic process (Supplementary Fig. S2b). The same proteins in addition to Pyruvate dehydrogenase E1 subunit beta-1 (Q6Z1G7), Pyruvate dehydrogenase E1 subunit beta-2 (Q0J0H4), Succinate dehydrogenase (Q6ZDY8), Malate dehydrogenase (Q7XDC8) and ATP-citrate synthase beta chain protein 1 (Q93VT8) support an increased influx of carbon for secondary metabolite production during the defense process (Fig. 6). Additionally, a protein network comprising Peroxiredoxin Q (P0C5D5), Peroxiredoxin-2E-2 (Q7F8S5), Catalase isozyme A (Q0E4K1) and Monodehydroascrobate reductase 3 (Q652L6) formed a part of cellular oxidant detoxification mechanism (Supplementary Fig. S2c).

Validation of key proteins at transcript level by transcriptomic/ qRT-PCR approach

Validation of DEPs at transcript level was performed to ascertain the fidelity of the proteomic findings. The Oryza elongation factor gene (OsEF-Iα) served as internal reference or housekeeping gene. Each gene Ct value was normalized with the Ct value of housekeeping gene. Subsequently, all genes, except for NPR4, exhibited upregulation at the transcript level in Nizam Shait relative to BPT-5204 post ShB inoculation. Remarkably, the most pronounced upregulation was observed in case of 14-3-3-like protein GF14-E (26.61 folds), followed by CBP (22.47 folds), Chitinase 8 (5.00 folds), GST (1.88 folds) and ASMT (1.36 folds). Conversely, the NPR4 gene displayed a downregulation of 0.49 folds in pathogen-inoculated Nizam Shait compared to BPT-5204. Encouragingly, the qRT-PCR data aligned closely with the outcomes derived from proteomic analyses, affirming the robustness and coherence of the findings across methodologies (Fig. 7).

Utilizing the landrace Nizam Shait as a resistance source, we aimed to decipher the proteins pivotal to the resistance response and establish a comprehensive proteome database for the Rice- R. solani interaction. Sample collection on the 5th day post-inoculation (DPI) was chosen, aligning with the onset of visible symptoms on Nizam Shait. Leaf sheath samples were specifically targeted, recognizing their role as the primary barrier against the R. solani invasion²¹. In proteomics, meticulous sample preparation is paramount as it significantly impacts the quality of data generated by mass spectrometry (MS)²².The efficacy of a protein extraction method depends upon tissues type, age of plant species and presence or absence of non-proteinaceous interfering compounds²³. Therefore, standardizing protein extraction protocols across different plant samples is imperative. Moreover, the abundance of dietary fiber in cereal crops poses challenges in cell wall disruption and contaminant removal during protein extraction. Successful data retrival hinges on the purity of sample extracted with the best isolation protocol. Among the array of protein isolation methods, phenol and TCA (Trichloro acetic acid)- acetone precipitation methods are regarded as the most reliable. In our investigation, proteins were extracted from the rice leaf sheath sample using phenol extraction method, ensuring robust data acquisition¹⁶.

Previous proteomic studies focusing on the rice-sheath blight interaction predominantly relied on 2DGE, where differential spots were eluted from SDS-gels and subsequently analyzed via MS²⁴. In contrast, the present study adopts Orbitrap-MS, a cutting-edge technique renowned for its superior peptide coverage and high-throughput capabilities. By leveraging this method, we mitigate the risk of false positive detections, thereby maximizing data recovery and enhancing the reliability of our findings. In-depth analysis of the identified significantly upregulated and downregulated proteins was conducted to obtain a panormaic understanding of proteomic alterations in both resistant and susceptible germplasms.

Proteins involved in cell redox homeostasis

Cell redox homeostasis is pivotal for regulating the levels of reactive oxygen species (ROS) within a cell, wherein ROS serves a dual role in plant biology. It acts as a signaling molecule during plant defense, while excessive concentration of ROS intermediates can induce cell death through direct oxidation²⁵. In our investigation, we identified 26 proteins associated with cell redox homeostasis, revealing that, apart from Peroxiredoxin Q and Peroxiredoxin-2E-2, the majority of peroxiredoxins, glutaredoxins, and thioredoxins, were downregulated. Recent research has associated the resistance mechanism in ShB resistant varieties with the suppression of ROS burst induced by R solani²⁶. The identification of these cell redox and antioxidant-related proteins suggests that Nizam Shait effectively maintains ROS levels during its fight against R. solani.

Protein involved in cell death

The hypersensitive response (HR) or programmed cell death is a crucial defense mechanism against biotrophic pathogens, yet it paradoxically benefits necrotrophic pathogens by facilitating their survival and proliferation²⁷. In our study, the key enzymes associated with the cell death, viz., superoxide dismutases [Cu-Zn] (SOD) and its copper chaperone, fumarylacetoacetase, DEAD-box ATP-dependent RNA helicase 38 and aspartyl protease, were observed to be downregulated following R. solani infection in resistant Nizam Shait compared to the susceptible BPT-5024. SOD plays a dual role, acting positively by removing superoxide radicals, while also inducing NO-associated cell death. By scavenging O₂^– to produce hydrogen peroxide, SOD facilitates the elimination of NO by minimizing NO-O₂^– interaction²⁸.

Proteins involved in carbohydrate metabolism

Carbohydrate metabolism pathways generate energy in the plant to fight against the pathogen²⁹. Among these pathways, the tricarboxylic acid cycle (TCA), glycolysis and pentose phosphate pathway (PPP) are prominent for oxidizing carbohydrate molecules and producing ATP. In our experiment, the key proteins involved in the glycolysis, TCA and PPP such as fructose-1, 6-bisphosphatase, fructose-bisphosphate aldolases, ATP-citrate synthase, malate dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenases, glyceraldehyde-3-phosphate and 6-phosphogluconate dehydrogenase, were significantly upregulated as a result of pathogen attack. These findings sync with the result demonstrated by Mutuku and Nose³⁰. The upregulation of glycolysis in R. solani-infected rice plants results in the overproduction of erythrose-4-phosphate by transketolase, which combines with phosphoenolpyruvate to produce high amount of lignin, serving as the first line of defense against ShB pathogen³¹. Importantly, the production of these enzymes in response to pathogen inoculation was more pronounced in Nizam Shait compared to BPT-5204, indicating a faster utilization of carbohydrate reserves in Nizam Shait to impede pathogen invasion.

In addition to glycolysis and TCA pathway, other pathways crucial for energy production, such as monocarboxylic acid metabolic processes and carboxylic acid metabolic processes, exhibited significant enrichment in ShB inoculated Nizam Shait compared to BPT-5204. Moreover, glycolysis and the TCA pathway contribute essential precursors, including tyrosine and phenylalanine, to the lignin and phenylpropanoid pathways, which are integral components of plant defense mechanisms³².

Proteins involved in cell wall remodelling

The necrotrophs are the weak pathogens and resistance to such pathogen is intricately linked to the cell wall composition³³. In our investigation, the proteins crucial for remodeling and fortifying the cell wall displayed upregulation in pathogen-inoculated Nizam Shait compared to BPT-5204. The cell wall strengthening proteins identified in our study can be broadly categorized into four groups, viz., UDP-arabinopyranose mutase, expansins, sucrose synthase and cinnamyl alcohol dehydrogenase. Among these, the cytosolic enzyme UDP-arabinopyranose mutase plays a pivotal role, converting UDP-arabinopyranose to UDP-arabinofuranose, a principal component of both primary and secondary cell walls³⁴.

Expansins, responsible for loosening the cell wall, facilitate pH dependent extension of the plant cell and are induced in response to PAMP-triggered immunity. Brasileiro et al. demonsrated that overexpression of expansin protein enhances groundnut plant tolerance against pathogens like Sclerotinia sclerotiorum, Meloidogyne incognita through cell wall modification and induction of abscissic acid (ABA) and jasmonic acid (JA) signaling pathways³⁵.

Proteins involved in defense

The 14-3-3-like protein GF14-E, highly upregulated in pathogen-inoculated Nizam Shait, is paramount in the brassinosteroid signaling pathway and acts as an adapter for phosphorylated proteins. It catalyzes the activation of other defense-related proteins and serves as a transcriptional factor, regulating signaling pathways based on environmental cues. Apart from this, it interacts with transcriptional factors such as BAD ( Bcl-2-antagonist of cell death) and the FKHRL-1 to inhibit the cell death^36,37. The protein was reported to be associated with effector triggered immunity (ETI) as well as PAMP-triggered immunity (PTI)³⁸. Durgadevi et al. have previously noted its upregulation in response to R. solani during tripartite interactions³⁹. Furthermore, it was reported to bolster ShB resistance in transgenic rice inoculated with R. solani by activating the MAPK cascade, highlighting its role in fortifying plant defense mechanisms⁴⁰.

Chalcone synthase and PAL are the vital enzymes in the phenylpropanoid pathway which orchestrates the synthesis of flavonoid and isoflavonoid compounds crucial for plant defense and acts as the primary gatekeeper in the production of phytoalexin. The qRT-PCR studies on defense-associated gene transcripts of R. solani-infected bean seedlings have revealed the upregulation of these genes, affirming the significance of phytoalexin production in combating R. solani⁴¹. Chitinase, also recognized as PR3 protein, exhibits a propensity for breaking down fungal chitin into simpler subunits. There are several reports available in which overexpression of chitinase gene has led to the enhanced plant resistance in case of ShB^26,42,43,44. Acetylserotonin O-methyltransferase, a constituent of melatonin and aromatic secondary metabolite production pathways, contributes to the synthesis of melatonin in plants. While melatonin primarily governs plant development, its association with innate immunity has also been documented⁴⁵.

As per the biological database network (bioDBnet), the defense protein, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase is produced in response to biotic stress, leading to the production of terpenoid secondary defense compounds. Zhang et al. documented that this key protein involved in terpenoid backbone synthesis exhibits significant accumulation in the resistant cucumber cultivar ZJ compared to the susceptible cultivar following Pseudoperonospora cubensis infection, suggesting that the accumulation of pathogen-induced terpenoids plays a pivotal role in conferring resistance against ShB infection ⁴⁶.

The two key proteins involved in the gamma-aminobutyric acid (GABA) shunt mediated defense, Succinate-semialdehyde dehydrogenase and Gamma-aminobutyrate transaminase were upregulated in Nizam Shait versus BPT-5204 after inoculation. This shunt prevents the accumulation of ROS intermediates and subsequent cell death⁴⁷. Moreover, it influences the virulence of the necrotrophic pathogen Fusarium graminearum in cereals by affecting the mitochondrial respiration⁴⁸. Therefore, the upregulation of GABA shunt proteins in Nizam Shait likely delayed cell death process and prevented ROS accumulation, contributing to its novel resistance against ShB. Additionally, proteins linked to PTI and ABA synthesis were found to be upregulated in response to R. solani infection, indicating that the resistance mechanism was mediated by PTI, with ABA acting as a signaling molecule. These findings corroborate similar results reported by previous researchers in various host-pathogen systems^{49, 50, 51}.

Validation of key DEPs at transcript level using qRT-PCR

The qRT validation results aligned with the proteomic data, confirming its accuracy. All selected genes, except NPR, exhibited upregulation. Notably, the 14-3-3-like protein GF14-E, which displayed the highest significant upregulation in the proteomic data, also demonstrated the highest fold change of 26.61 in qRT validation, indicating robust gene expression at both transcript and protein levels. Feng et al. observed several glutathione S-transferases (1.62–2.90 folds), chitinases (1.52–3.86 folds) and Chlorophyll a-b binding proteins (0.08–0.36 folds) to be differentially expressed in R. solani inoculated ShB resistant variety, YSBR1 compared to the control. However, transcriptome-level validation for these genes was not conducted²⁶. While previous reports highlighted the upregulation of 14-3-3 protein in R. solani- BPT-5204-Bacillus subtilis tripartite interaction, qRT-PCR validation was lacking³⁹. Therefore, this study marks the first instance where proteomic findings were validated at the transcriptome level in the rice-sheath blight interaction.

Putative model to explain resistance mechanism of resistant germplasm Nizam Shait in response to R. solani infection

When Rhizoctonia solani isolate RS4 encounters Nizam Shait, the non-specific lipid transfer proteins 2B (PRR) on the rice plant's surface detect the PAMP, initiating PTI. This triggers four defense processes, first, activation of cell wall strengthening enzymes such as expansions, UDP-arabinopyranose mutase, and cinnamyl alcohol dehydrogenase and second, JA-ISR signaling pathway, third, ABA defense signaling and fourth, the most important, Brassinosteroid (BR) signaling pathway. JA-ISR signaling pathway commences with conversion of α-linolenic acid to 13-hydroperoxyoctadecadienoic acid (HPOD) using lipoxygenase enzyme (9S-LOX). Subsequently, allene oxide synthase 1 (AOS 1) and allene oxide synthase 2 (AOS 2) convert it to 12–13 epoxy octadecatrienoic acid (12–13 EOT), which further forms oxyphtodienoic acid (OPDA) and jasmonic acid (JA) via allene oxide cyclase (AOC) and oxophytodienoate reductase 11 (OPR 11), respectively. The JA-ISR signaling pathway was identified in BPT-5204 also, so the prime reason for high resistance in Nizam Shait is associated with BR-signaling pathway, particularly, 14-3-3 protein, which exhibited highest upregulation in our study. The induction of 14-3-3 protein signaling contributes to the increased expression of defense-related proteins in Nizam Shait compared to BPT-5204 following ShB inoculation (Fig. 8).

In conclusion, current study elucidates the proteome of resistance in Nizam Shait against sheath blight compared to the highly susceptible BPT-5204. The substantial upregulation of 14-3-3 proteins suggests its pivotal role in the defense mechanism, highlighting the contribution of the brassinosteroid signaling pathway, JA-ISR, PTI, and cell wall strengthening. This information on key proteins could be utilized to develop a peptide-based marker panel for breeding sheath blight-resistant rice varieties.

Acknowledgement

The first author acknowledges financial support in the form of Senior Research Fellowship awarded by ICAR, New Delhi. Government of India.

Author Contribution

PSK conceptualized the idea and supervised all the experiments; AM conducted the experiments and AM, AYP and RA did statistical analysis: PSK, AM interpreted the results and formulated the manuscript. SN, YH and PUK critically read the manuscript and provided valuable suggestions.

Competing interests

The authors declare no competing interests

Data Availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier “PXD040634”. "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request".

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No competing interests reported.

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Quantitative proteomic analysis deciphers mechanisms of sheath blight resistance in novel rice landrace against Rhizoctonia solani

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Abstract

Figures

Introduction

Materials and methods

Plant material and pathogen inoculation

Sample collection

Protein isolation and quantification

Trypsin digestion

Protein identification and data analysis

Results

Proteomic analysis

GO enrichment analysis of DEPs

Functional annotation of DEPs

Protein-protein interaction studies

Validation of key proteins at transcript level by transcriptomic/ qRT-PCR approach

Discussion

Proteins involved in cell redox homeostasis

Protein involved in cell death

Proteins involved in carbohydrate metabolism

Proteins involved in cell wall remodelling

Proteins involved in defense

Validation of key DEPs at transcript level using qRT-PCR

Declarations

Acknowledgement

Author Contribution

Competing interests

Data Availability

References

Additional Declarations

Status:

Version 1