Chitinase-E from Chitiniphilus shinanonensis generates chitobiose from chitin flakes.

Enzymatic deconstruction of chitin to chitobiose is of significant interest in view of its various biological applications. Here we report detailed insights into the chitin degradation by chitinase-E from a chitinolytic bacterium Chitiniphilus shinanonensis (CsChiE). CsChiE was optimally active at 50 °C in 50 mM sodium phosphate pH-7.0. It showed a kcat and overall catalytic efficiency (Kcat/Km) as 3.9 × 103 s-1 and 0.6 × 103 s-1 mg-1 mL, respectively, on colloidal chitin (CC). CsChiE efficiently hydrolyzed crystalline polymers like α-, ß- and CC and released chitobiose as the predominant product (11.3 mM on CC). Further, CsChiE displayed substantial activity towards the unmilled crab shell chitin waste (chitin-flakes) and generated chitobiose. Activity studies on chitooligosaccharides revealed that CsChiE produced chitobiose as the major product. Our results indicate that the multi-modular CsChiE is a non-processive exo-chitinase which is more suitable to generate chitobiose from a variety of chitinous substrates including unprocessed chitin-flakes.

Partner-triggered proteome changes in the cell wall of Bacillus sonorensis and roots of groundnut benefit each other.

Plant growth promoting rhizobacteria (PGPR) promote plant growth and activate defense response against phytopathogens. At the subcellular level plant-PGPR interaction is less understood, which would be essential for future improvement(s) of PGPR formulations. In a rigorous screening process, that also involved efficient PGPR strains, Bacillus sonorensis RS4 was selected to study partner-triggered interactions. The potential of B. sonorensis RS4 to improve growth of groundnut, efficiency to colonize roots, and influence on root topology was assessed. Twenty four cell wall proteins of B. sonorensis RS4 [in presence of groundnut root exudates (REs)], and 22 groundnut root proteins (in RS4-bacterized plants) were differentially expressed. The alterations in cell wall proteins of B. sonorensis RS4 were primarily related to the amino acids synthesis, chemotaxis, antioxidant-metabolism, carbohydrate metabolism, transporters, and antibiosis-related secondary metabolites. Root proteins that were differentially expressed during the interaction may be involved in plant growth, defense responses, and in transportation. The changes in B. sonorensis RS4 cell wall proteome and groundnut root proteome, suggest that at least a part of the proteome changes triggered by each of the partners appear to play a significant role in helping each other akin to symbiosis.

HarpinPss encapsulation in chitosan nanoparticles for improved bioavailability and disease resistance in tomato.

HarpinPss, an elicitor from Pseudomonas syringae pv. syringae, induces systemic acquired resistance in non-host plants, providing resistance to phytopathogens. Poor assimilation of harpinPss is a major constraint in foliar application as biopesticide. We, therefore, prepared harpinPss-loaded chitosan nanoparticles (H-CSNPs) to improve permeability and bio-availability of harpinPss in tomato. H-CSNPs showed high encapsulation efficiency (90%), improved stability (p < 0.01) and bioavailability of harpinPss (p < 0.01). Treatment with H-CSNPs resulted in sustained induction of peroxidase, phenylalanine ammonia lyase and decreased Rhizoctonia solani infection (p < 0.05). Transcripts of several genes involved in defense response were differentially expressed in harpinPss, CSNPs and H-CSNPs treatments. While, genes involved in jasmonic acid (JA) metabolism were up-regulated during harpinPss and H-CSNP spray treatments, indicating the role of JA pathway in triggering harpin-mediated defense responses. Furthermore, the entry of CSNPs into the cell and localization of harpinPss into chloroplast was tracked using rhodamine-labelled CSNPs encapsulated with GFP tagged harpinPss. The results of this study indicate use of H-CSNPs is effective for sustained-release of harpinPss and provides resistance for prolonged duration.

Exploring Combined Effect of Abiotic (Soil Moisture) and Biotic (Sclerotium rolfsii Sacc.) Stress on Collar Rot Development in Chickpea.

Plants being sessile are under constant threat of multiple abiotic and biotic stresses within its natural habitat. A combined stress involving an abiotic and a biotic factor reportedly increases susceptibility of the plants to pathogens. The emerging threat, collar rot disease of chickpea (caused by Sclerotium rolfsii Sacc.) is reported to be influenced by soil moisture condition (SMC). Hence, we studied the influence of differential SMC viz. upper optimum (100%), optimum (80%), lower optimum (60%), and limiting (40%) soil moisture conditions on colonization and collar rot development over the course of infection in two chickpea cultivars, Annigeri (susceptible to collar rot) and ICCV 05530 (moderately resistant to collar rot). Disease incidence was found to be directly proportional to increase in soil moisture (R2 = 0.794). Maximum incidence was observed at 80% SMC, followed by 100 and 60% SMC. Expression of genes (qPCR analysis) associated with host cell wall binding (lectin) and degradation viz. endopolygalacturonase-2, endoglucosidase, and cellobiohydrolase during collar rot development in chickpea were relatively less at limiting soil moisture condition (40%) as compared to optimum soil moisture condition (80%). As compared to individual stress, the expression of defense response genes in chickpea seedlings were highly up-regulated in seedlings challenged with combined stress. Our qPCR results indicated that the expression of defense-related genes in chickpea during interaction with S. rolfsii at low SMC was primarily responsible for delayed disease reaction. Involvement of moisture and biotic stress-related genes in combined stress showed a tailored defense mechanism.

Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites.

Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.

Accumulation of transcription factors and cell signaling-related proteins in the nucleus during citrus-Xanthomonas interaction.

The nucleus is the maestro of the cell and is involved in the modulation of cell signaling during stress. We performed a comprehensive nuclear proteome analysis of Citrus sinensis during interaction with host (Xanthomonas citri pv. citri-Xcc) and non-host (Xanthomonas oryzae pv. oryzae-Xoo) pathogens. The nuclear proteome was obtained using a sequential method of organelle enrichment and determined by nano-LC-MS/MS analysis. A total of 243 proteins accumulated differentially during citrus-Xanthomonas interaction, belonging to 11 functional groups, with signaling and transcription-related proteins dominating. MADS-box transcription factors, DEAD-box RNA helicase and leucine aminopeptidase, mainly involved in jasmonic acid (JA) responses, were in high abundance during non-host interaction (Xoo). Signaling-related proteins like serine/threonine kinase, histones (H3.2, H2A), phosphoglycerate kinase, dynamin, actin and aldolase showed increased accumulation early during Xoo interaction. Our results suggest that there is a possible involvement of JA-triggered defense responses during non-host resistance, with early recognition of the non-host pathogen.

Root exudate-induced alterations in Bacillus cereus cell wall contribute to root colonization and plant growth promotion.

The outcome of an interaction between plant growth promoting rhizobacteria and plants may depend on the chemical composition of root exudates (REs). We report the colonization of tobacco, and not groundnut, roots by a non-rhizospheric Bacillus cereus (MTCC 430). There was a differential alteration in the cell wall components of B. cereus in response to the REs from tobacco and groundnut. Attenuated total reflectance infrared spectroscopy revealed a split in amide I region of B. cereus cells exposed to tobacco-root exudates (TRE), compared to those exposed to groundnut-root exudates (GRE). In addition, changes in exopolysaccharides and lipid-packing were observed in B. cereus grown in TRE-amended minimal media that were not detectable in GRE-amended media. Cell-wall proteome analyses revealed upregulation of oxidative stress-related alkyl hydroperoxide reductase, and DNA-protecting protein chain (Dlp-2), in response to GRE and TRE, respectively. Metabolism-related enzymes like 2-amino-3-ketobutyrate coenzyme A ligase and 2-methylcitrate dehydratase and a 60 kDa chaperonin were up-regulated in response to TRE and GRE. In response to B. cereus, the plant roots altered their exudate-chemodiversity with respect to carbohydrates, organic acids, alkanes, and polyols. TRE-induced changes in surface components of B. cereus may contribute to successful root colonization and subsequent plant growth promotion.

Warriors at the gate that never sleep: non-host resistance in plants.

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.