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.

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.

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.

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.