Proteomics of Two Thermotolerant Isolates of Trichoderma under High-Temperature Stress.

Several species of the soil borne fungus of the genus Trichoderma are known to be versatile, opportunistic plant symbionts and are the most successful biocontrol agents used in today's agriculture. To be successful in field conditions, the fungus must endure varying climatic conditions. Studies have indicated that a high atmospheric temperature coupled with low humidity is a major factor in the inconsistent performance of Trichoderma under field conditions. Understanding the molecular modulations associated with Trichoderma that persist and deliver under abiotic stress conditions will aid in exploiting the value of these organisms for such uses. In this study, a comparative proteomic analysis, using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/time-of-flight (MALDI-TOF-TOF) mass spectrometry, was used to identify proteins associated with thermotolerance in two thermotolerant isolates of Trichoderma: T. longibrachiatum 673, TaDOR673 and T. asperellum 7316, TaDOR7316; with 32 differentially expressed proteins being identified. Sequence homology and conserved domains were used to identify these proteins and to assign a probable function to them. The thermotolerant isolate, TaDOR673, seemed to employ the stress signaling MAPK pathways and heat shock response pathways to combat the stress condition, whereas the moderately tolerant isolate, TaDOR7316, seemed to adapt to high-temperature conditions by reducing the accumulation of misfolded proteins through an unfolded protein response pathway and autophagy. In addition, there were unique, as well as common, proteins that were differentially expressed in the two isolates studied.

Efficient One-Pot Synthesis and pH-Dependent Tuning of Photoluminescence and Stability of Au18(SC2H4CO2H)14 Cluster.

Developing efficient ways to control the nanocluster properties and synthesize atomically precise metal nanoclusters are the foremost goals in the field of metal nanocluster research. In this article, we demonstrate that the direct synthesis of atomically precise, hydrophilic metal nanoclusters as well as tuning of their properties can be achieved by an appropriate selection of reactants, binding ligand, and their proportions. Thus, a facile, single-step method has been developed for the direct synthesis of Au18(SC2H4CO2H)14 nanocluster in an aqueous medium under ambient conditions. The synthesis does not require any pH or temperature control and postsynthesis size-separation step. The use of a hydrophilic, bifunctional short carbon-chain capping ligand, HSC2H4CO2H, allows tuning of cluster properties such as the photoluminescence and stability in an aqueous medium via the variation of pH of the cluster solution. By using a phase transfer catalyst, the nanoclusters can also be transferred into toluene solvent, which further enhances the nanocluster photoluminescence. The formation, composition, and purity of the product clusters have been characterized by using a number of methods such as the polyacrylamide gel electrophoresis, UV-visible and FTIR spectroscopies, transmission electron microscopy, energy dispersive X-ray analysis, thermogravimetric analysis, X-ray photoelectron spectroscopy, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Gold nanoclusters with properties such as water solubility, water-to-organic phase-transfer ability, and tunable stability and photoluminescence are promising for various studies and applications. The work reveals a few principles that can be helpful in the development of a general toolbox for the rational design of size-selective synthesis and properties tuning of the metal nanoclusters.

Comparative proteomics reveals differential induction of both biotic and abiotic stress response associated proteins in rice during Xanthomonas oryzae pv. oryzae infection.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice and brutally affects the yield up to 50 % of total production. Here, we report a comparative proteomics analysis of total foliar protein isolated from infected rice leaves of susceptible Pusa Basmati 1 (PB1) and resistant Oryza longistaminata genotypes. Two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) approaches identified 29 protein spots encoding unique proteins from both the genotypes. Identified proteins belonged to a large number of biological and molecular functions related to biotic and abiotic stress proteins which are potentially involved during Xoo infection. Biotic and abiotic stress-related proteins were induced during Xoo infection, indicating the activation of common stress pathway during bacterial blight infection. Candidate genes conferring tolerance against bacterial blight, which include germin-like protein, putative r40c1, cyclin-dependent kinase C, Ent-isokaur-15-ene synthase and glutathione-dependent dehydroascorbate reductase 1 (GSH-DHAR1), were also induced, with germin-like proteins induced only in the resistant rice genotype O. longistaminata. Energy, metabolism and hypothetical proteins were common among both the genotypes. Further, host defence/stress-related proteins were mostly expressed in resistant genotype O. longistaminata, indicating possible co-evolution of the pathogen and the wild rice, O. longistaminata.

Serum proteomic profiling in granumomatosis with polyangiitis using two-dimensional gel electrophoresis along with matrix assisted laser desorption ionization time of flight mass spectrometry.

AIM: The present study is a proteomic approach to find differentially expressed proteins in sera of limited and systemic subsets of active disease versus their remitting state in patients with granulomatosis with polyangiitis (GPA) and their correlation with disease activity. METHODS: Eighteen patients with GPA in active as well as in remitting state and four healthy controls (HC) were included in the study. For proteomics analysis, two-dimensional gel electrophoresis along with matrix-assisted laser desorption ionization time-of-flight mass spectrometry were performed. A total of 14 gels were run from pooled patients' sera from active GPA and remission as well as pooled HC serum. RESULTS: There was significant differential expression of proteins in limited versus systemic GPA and between active systemic versus remitting patients of systemic disease. We identified nine maximally differentially expressed and five proteins which were not detected in HC. Among nine proteins, one (Prolow density lipoprotein receptor-related protein 1) was downregulated and four proteins (haptoglobin Hp, Hp2, vitamin D binding protein, killer cell lectin-like receptor subfamily F member 2), were up-regulated in both limited and systemic active disease, two proteins like Ig gamma-4 chain C region protein and serum albumin were up-regulated in limited active GPA and two proteins, that is, cysteine rich secretory protein LCCL domain-containing 2 precursor and serine-threonine-protein kinase A-Raf were up-regulated in systemic active disease. Levels of interleukin-17 and vitamin-D binding protein (VDBP) by enzyme-linked immunosorbent assay could distinctly demarcate active disease versus remission. CONCLUSION: Our study provides potential protein markers of active disease versus remission in GPA.

Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata.

A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in Vigna radiata (L.) Wilczek.

To understand the complex drought response mechanism in crop plants, a systematic root proteomics approach was adopted to identify and analyze the expression patterns of differentially expressed major root proteins of Vigna radiata during short-term (3 days) and consecutive long-term water-deficit (6 days) as well as during recovery (6 days after re-watering). Photosynthetic gas exchange parameters of the plant were measured simultaneously during the stress treatment and recovery period. A total of 26 major protein spots were successfully identified by mass spectrometry, which were grouped according to their expression pattern during short-term stress as significantly up-regulated (9), down-regulated (10), highly down-regulated, beyond detection level of the software (2) and unchanged (5). The subsequent changes in the expression patterns of these proteins during long-term stress treatment and recovery period was analyzed to focus on the dynamic regulation of these functionally important proteins during progressive drought and recovery period. Cytoskeleton-related proteins were down-regulated initially (3d) but regained their expression levels during subsequent water-deficit (6d) while glycoprotein like lectins, which were primarily known to be involved in legume-rhizobia symbiosis, maintained their enhanced expression levels during both short and long-term drought treatment indicating their possible role in drought stress response of legumes. Oxidative stress-related proteins including Cu/Zn superoxide dismutase, oxidoreductase and aldehyde reductase were also up-regulated. The analyses of the dynamic regulation of these root proteins during short- and long-term water-deficit as well as recovery period may prove crucial for further understanding of drought response mechanisms in food legumes.