Proteomic signatures uncover phenotypic plasticity of susceptible and resistant genotypes by wall remodelers in rice blast.

Molecular communication between macromolecules dictates extracellular matrix (ECM) dynamics during pathogen recognition and disease development. Extensive research has shed light on how plant immune components are activated, regulated and function in response to pathogen attack. However, two key questions remain largely unresolved: (i) how does ECM dynamics govern susceptibility and disease resistance, (ii) what are the components that underpin these phenomena? Rice blast, caused by Magnaporthe oryzae adversely affects rice productivity. To understand ECM regulated genotype-phenotype plasticity in blast disease, we temporally profiled two contrasting rice genotypes in disease and immune state. Morpho-histological, biochemical and electron microscopy analyses revealed that increased necrotic lesions accompanied by electrolyte leakage governs disease state. Wall carbohydrate quantification showed changes in pectin level was more significant in blast susceptible compared to blast resistant cultivar. Temporally resolved quantitative disease- and immune-responsive ECM proteomes identified 308 and 334 proteins, respectively involved in wall remodelling and integrity, signalling and disease/immune response. Pairwise comparisons between time and treatment, messenger ribonucleic acid expression, diseasome and immunome networks revealed novel blast-related functional modules. Data demonstrated accumulation of α-galactosidase and phosphatase were associated with disease state, while reactive oxygen species, induction of Lysin motif proteins, CAZymes and extracellular Ca-receptor protein govern immune state.

Combining extracellular matrix proteome and phosphoproteome of chickpea and meta-analysis reveal novel proteoforms and evolutionary significance of clade-specific wall-associated events in plant.

Extracellular matrix (ECM) plays central roles in cell architecture, innate defense and cell wall integrity (CWI) signaling. During transition to multicellularity, modular domain structures of ECM proteins and proteoforms have evolved due to continuous adaptation across taxonomic clades under different ecological niche. Although this incredible diversity has to some extent been investigated at protein level, extracellular phosphorylation events and molecular evolution of ECM proteoform families remains unexplored. We developed matrisome proteoform atlas in a grain legume, chickpea and performed meta-analyses of 74 plant matrisomes. MS/MS analysis identified 1,424 proteins and 315 phosphoproteins involved in diverse functions. Cross-species ECM protein network identified proteoforms associated with CWI maintenance system. Phylogenetic characterization of eighteen matrix protein families highlighted the role of taxon-specific paralogs and orthologs. Novel information was acquired on gene expansion and loss, co-divergence, sub functionalization and neofunctionalization during evolution. Modular networks of matrix protein families and hub proteins showed higher diversity across taxonomic clades than among organs. Furthermore, protein families differ in nonsynonymous to synonymous substitution rates. Our study pointed towards the matrix proteoform functionality, sequence divergence variation, interactions between wall remodelers and molecular evolution using a phylogenetic framework. This is the first report on comprehensive matrisome proteoform network illustrating presence of CWI signaling proteins in land plants.

Proteomic insights of chitosan mediated inhibition of Fusarium oxysporum f. sp. cucumerinum.

Fusarium oxysporum f. sp. cucumerinum (FOC) infects cucumber plants, causing significant yield losses. Chitosan is a natural biodegradable compound that has antifungal properties. To understand the inhibitory mechanism of chitosan against FOC, a comprehensive proteomic study was carried out for the identification of chitosan responsive proteins (CRPs) from the mycelia of chitosan-treated FOC. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of 62 differentially abundant CRPs. Functional classification of these CRPs revealed that most proteins were involved in metabolism and defense. Gene Ontology analysis revealed that the majority of the proteins were assigned in proteolysis and hydrolase activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that among the biologically active pathways in chitosan-treated FOC mycelia, 'carbohydrate metabolism' was enriched for most of the proteins. This study gives a snapshot of the molecular basis of fungal inhibition by chitosan resulting in disease resistance in cucumber plants after inoculation with chitosan-treated FOC by explaining how chitosan restricted disease severity (i.e., down-regulating the plant cell wall degrading enzymes, FOC self-attack, hindering FOC structural and functional protein biosynthesis and DNA biosynthesis and affecting FOC transporter proteins). This study contributes to putting more weight on using the bioactive natural compound chitosan as an antifungal material instead of applying chemical fungicides in agriculture. SIGNIFICANCE: Chitosan has been used as one of the safe and effective alternatives to fungicides in controlling cucumber vascular wilt disease caused by Fusarium oxysporum f. sp. cucumerinum (FOC) that is responsible for severe production losses. Chitosan application showed a significant decrease in wilt disease severity compared to chitosan untreated FOC and showed an efficiency of 91.7% in reducing pathogenicity. A comprehensive proteomic investigation of chitosan-responsive proteins (CRPs) from the mycelia of chitosan-treated FOC was carried out in order to better understand the inhibitory mechanism of chitosan against FOC which led us to identify 62 differentially expressed CRPs. Our proteomic study revealed CRPs in FOC involved in a variety of functions, including disease inhibition in cucumber. This study depicts what happens inside the fungus following treatment with chitosan and how chitosan played the role of the maestro in influencing the synthesis of proteins responsible for the virulence of FOC and their respective pathways, rendering FOC unable to infect the cucumber plant and lose its pathogenic potential to cause wilt disease. The efficiency of chitosan in inhibiting certain proteins or specific pathways of FOC gives a golden opportunity in controlling vascular wilt, so we highly recommend applying chitosan in disease management under greenhouse conditions or in the open field.

Chitosan-triggered immunity to Fusarium in chickpea is associated with changes in the plant extracellular matrix architecture, stomatal closure and remodeling of the plant metabolome and proteome.

Pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan-triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho-histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan-treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan-triggered immune-responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor-like kinases, and 65 chitosan-triggered immune-responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune-related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan-responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.

Extracellular Matrix Proteome: Isolation of ECM Proteins for Proteomics Studies.

Understanding molecular mechanisms and cellular metabolism in varied plant processes necessitates knowledge of the expressed proteins and their subcellular distribution. Spatial partitioning of organelles generates an enclosed milieu for physiochemical reactions designed and tightly linked to a specific organelle function. Of which, extracellular matrix (ECM)/cell wall (CW) is a dynamic and chemically active compartment. The ECM proteins are organized into complex structural and functional networks involved in several metabolic processes, including carbon and nitrogen metabolism. Organellar proteomics aim for comprehensive identification of resident proteins that rely on the isolation of highly purified organelle free from contamination by other intracellular components. Extraction and isolation of plant ECM proteins features key caveats due to the lack of adjoining membrane, the presence of a polysaccharide-protein network that traps contaminants, and the existence of high phenolic content. Furthermore, due to diverse biochemical forces, including labile, weakly bound and strongly bound protein in the protein-polysaccharide matrix different elution procedures are required to enrich ECM proteins. Here, we describe a method that allows efficient fractionation of plant ECM, extraction of ECM proteins and protein profiling from variety of crop plants, including rice, chickpea and potato. This method can easily be adapted to other plant species for varied experimental conditions.

Comparative Nuclear Proteomics Analysis Provides Insight into the Mechanism of Signaling and Immune Response to Blast Disease Caused by Magnaporthe oryzae in Rice.

Modulation of plant immune system by extrinsic/intrinsic factors and host-specific determinants fine-tunes cellular components involving multiple organelles, particularly nucleus to mount resistance against pathogen attack. Rice blast, caused by hemibiotrophic fungus Magnaporthe oryzae, is one of the most devastating diseases that adversely affect rice productivity. However, the role of nuclear proteins and their regulation in response to M. oryzae remains unknown. Here, the nucleus-associated immune pathways in blast-resistant rice genotype are elucidated. Temporal analysis of nuclear proteome is carried out using 2-DE coupled MS/MS analysis. A total of 140 immune responsive proteins are identified associated with nuclear reorganization, cell division, energy production/deprivation, signaling, and gene regulation. The proteome data are interrogated using correlation network analysis that identified significant functional modules pointing toward immune-related coinciding processes through a common mechanism of remodeling and homeostasis. Novel clues regarding blast resistance include nucleus-associated redox homeostasis and glycolytic enzyme-mediated chromatin organization which manipulates cell division and immunity. Taken together, the study herein provides evidence that the coordination of nuclear function and reprogramming of host translational machinery regulate resistance mechanism against blast disease.

Quantitative Extracellular Matrix Proteomics Suggests Cell Wall Reprogramming in Host-Specific Immunity During Vascular Wilt Caused by Fusarium oxysporum in Chickpea.

Extracellular matrix (ECM) is the unique organelle that perceives stress signals and reprograms molecular events of host cell during patho-stress. However, our understanding of how ECM dictates plant immunity is largely unknown. Vascular wilt caused by the soil borne filamentous fungus Fusarium oxysporum is a major impediment for global crop productivity. To elucidate the role of ECM proteins and molecular mechanism associated with cell wall mediated immunity, the temporal changes of ECM proteome was studied in vascular wilt resistant chickpea cultivar upon F. oxysporum infection. The 2DE protein profiling coupled with mass spectrometric analysis identified 166 immune responsive proteins (IRPs) involved in variety of functions. Our data suggest that wall remodeling; protein translocation, stabilization, and chitin triggered immunity; and extracellular ATP signaling are major players in early, middle, and later phases of ECM signaling during fungal attack. Furthermore, we interrogated the proteome data using network analysis that identified modules enriched in known and novel immunity-related prognostic proteins centered around nascent aminopolypeptide complex (NAC), amine oxidase, thioredoxin, and chaperonin. This study for the first time provides an insight into the complex network operating in the ECM and impinges on the surveillance mechanism of innate immunity during patho-stress in crop plant.

Molecular Dissection of Extracellular Matrix Proteome Reveals Discrete Mechanism Regulating Verticillium Dahliae Triggered Vascular Wilt Disease in Potato.

Plants exposed to patho-stress mostly succumb due to disease by disruption of cellular integrity and changes in the composition of the extracellular matrix (ECM). Vascular wilt, caused by the soil borne hemibiotrophic filamentous fungus Verticillium dahliae, is one of the most significant diseases that adversely affect plant growth and productivity. The virulence of the pathogen associated with the ECM-related susceptibility of the host plant is far from being understood. To better understand ECM-associated disease responses that allow the pathogen to suppress plant immunity, a temporal analysis of ECM proteome was carried out in vascular wilt susceptible potato cultivar upon V. dahliae infection. The proteome profiling led to the identification of 75 patho-stress responsive proteins (PSRPs), predominantly involved in wall hydration, architecture, and redox homeostasis. Two novel clues regarding wilt disease of potato were gained from this study. First, wall crosslinking and salicylic acid signaling significantly altered during patho-stress. Second, generation of reactive oxygen species and scavenging proteins increased in abundance leading to cell death and necrosis of the host. We provide evidence for the first time that how fungal invasion affects the integrity of ECM components and host reprogramming for susceptibility may function at the cell surface by protein plasticity.

Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms.

The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling.