Research Laboratory for Biotechnology and Biochemistry

Kathmandu, Nepal

Research Laboratory for Biotechnology and Biochemistry

Kathmandu, Nepal
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Ashwin N.M.R.,Indian Council of Agricultural Research | Barnabas L.,Indian Council of Agricultural Research | Ramesh Sundar A.,Indian Council of Agricultural Research | Malathi P.,Indian Council of Agricultural Research | And 7 more authors.
Journal of Proteomics | Year: 2017

Colletotrichum falcatum, an intriguing hemibiotrophic fungal pathogen causes red rot, a devastating disease of sugarcane. Repeated in vitro subculturing of C. falcatum under dark condition alters morphology and reduces virulence of the culture. Hitherto, no information is available on this phenomenon at molecular level. In this study, the in vitro secretome of C. falcatum cultured under light and dark conditions was analyzed using 2-DE coupled with MALDI TOF/TOF MS. Comparative analysis identified nine differentially abundant proteins. Among them, seven proteins were less abundant in the dark-cultured C. falcatum, wherein only two protein species of a cerato-platanin protein called EPL1 (eliciting plant response-like protein) were found to be highly abundant. Transcriptional expression of candidate high abundant proteins was profiled during host-pathogen interaction using qRT-PCR. Comprehensively, this comparative secretome analysis identified five putative effectors, two pathogenicity-related proteins and one pathogen-associated molecular pattern (PAMP) of C. falcatum. Functional characterization of three distinct domains of the PAMP (EPL1) showed that the major cerato-platanin domain (EPL1. (increment). N1-92) is exclusively essential for inducing defense and hypersensitive response (HR) in sugarcane and tobacco, respectively. Further, priming with EPL1. (increment). N1-92 protein induced systemic resistance and significantly suppressed the red rot severity in sugarcane. Biological significance: Being the first secretomic investigation of C. falcatum, this study has identified five potential effectors, two pathogenicity-related proteins and a PAMP. Although many reports have highlighted the influence of light on pathogenicity, this study has established a direct link between light and expression of effectors, for the first time. This study has presented the influence of a novel N-terminal domain of EPL1 in physical and biological properties and established the functional role of major cerato-platanin domain of EPL1 as a potential elicitor inducing systemic resistance in sugarcane. Comprehensively, the study has identified proteins that putatively contribute to virulence of C. falcatum and for the first time, demonstrated the potential role of EPL1 in inducing PAMP-triggered immunity (PTI) in sugarcane. © 2017 Elsevier B.V.


Gupta R.,Pusan National University | Min C.W.,Pusan National University | Wang Y.,Max Planck Institute for Plant Breeding Research | Kim Y.C.,Pusan National University | And 5 more authors.
Frontiers in Plant Science | Year: 2016

Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail. © 2016 Gupta, Min, Wang, Kim, Agrawal, Rakwal and Kim.


Agrawal G.K.,Research Laboratory for Biotechnology and Biochemistry | Jwa N.-S.,Sejong University | Jung Y.-H.,Sejong University | Kim S.T.,Pusan National University | And 8 more authors.
Methods in Molecular Biology | Year: 2013

Rice is one of the most important food and cereal crop plants in the world. Rice proteomics began in the 1990s. Since then, considerable progress has been made in establishing protocols from isolation of rice proteins from different tissues, organs, and organelles, to separation of complex proteins and to their identification by mass spectrometry. Since the year 2000, global proteomics studies have been performed during growth and development under numerous biotic and abiotic environmental conditions. Two-dimensional (2-D) gel-based proteomics platform coupled with mass spectrometry has been retained as the workhorse for proteomics of a variety of rice samples. In this chapter, we describe in detail the different protocols used for isolation of rice proteins, their separation, detection, and identification using gel-based proteomics and mass spectrometry approaches. © 2013 Springer Science+Business Media, LLC.


Amalraj R.S.,Indian Council of Agricultural Research | Selvaraj N.,Indian Council of Agricultural Research | Veluswamy G.K.,Indian Council of Agricultural Research | Ramanujan R.P.,Indian Council of Agricultural Research | And 7 more authors.
Electrophoresis | Year: 2010

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2-DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2-D gel protein profiles, TCA/acetone precipitation-LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2-D gel, which was mostly overcome by the use of 2-D cleanup kit after protein extraction. Among all the methods tested, 2-D cleanup-phenol method was found to be the most suitable for producing high number of good-quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD-IT-TOF-MS/MS and nESI-LC-MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Singh R.,Sejong University | Lee M.-O.,Sejong University | Lee J.-E.,Sejong University | Choi J.,Sejong University | And 13 more authors.
Plant Physiology | Year: 2012

Mitogen-activated protein kinase (MAPK) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. Yet, our knowledge of the global physical MAPK interactome in plants remains largely fragmented. Here, we utilized the yeast twohybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localization, and kinase assay experiments in the model crop rice (Oryza sativa) to systematically map what is to our knowledge the first plant MAPK-interacting proteins. We identified 80 nonredundant interacting protein pairs (74 nonredundant interactors) for rice MAPKs and elucidated the novel proteome-wide network of MAPK interactors. The established interactome contains four membrane-associated proteins, seven MAP2Ks (for MAPK kinase), four MAPKs, and 59 putative substrates, including 18 transcription factors. Several interactors were also validated by experimental approaches (in vivo and in vitro) and literature survey. Our results highlight the importance of OsMPK1, an ortholog of tobacco (Nicotiana benthamiana) salicyclic acid-induced protein kinase and Arabidopsis (Arabidopsis thaliana) AtMPK6, among the rice MAPKs, as it alone interacts with 41 unique proteins (51.2% of the mapped MAPK interaction network). Additionally, Gene Ontology classification of interacting proteins into 34 functional categories suggested MAPK participation in diverse physiological functions. Together, the results obtained essentially enhance our knowledge of the MAPK-interacting protein network and provide a valuable research resource for developing a nearly complete map of the rice MAPK interactome. © 2012 American Society of Plant Biologists.


Hajduch M.,University of Missouri | Hajduch M.,Slovak Academy of Sciences | Hearne L.B.,University of Missouri | Miernyk J.A.,University of Missouri | And 8 more authors.
Plant Physiology | Year: 2010

Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis (Arabidopsis thaliana) seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed two-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/ transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of post-transcriptional regulation. © 2010 American Society of Plant Biologists.


PubMed | Research Laboratory for Biotechnology and Biochemistry
Type: | Journal: Reviews of environmental contamination and toxicology | Year: 2011

Ozone is now considered to be the second most important gaseous pollutant in our environment. The phytotoxic potential of O was first observed on grape foliage by B.L. Richards and coworkers in 1958 (Richards et al. 1958). To date, unsustainable resource utilization has turned this secondary pollutant into a major component of global climate change and a prime threat to agricultural production. The projected levels to which O will increase are critically alarming and have become a major issue of concern for agriculturalists, biologists, environmentalists and others plants are soft targets for O. Ozone enters plants through stomata, where it disolves in the apoplastic fluid. O has several potential effects on plants: direct reaction with cell membranes; conversion into ROS and HO (which alters cellular function by causing cell death); induction of premature senescence; and induction of and up- or down-regulation of responsive components such as genes , proteins and metabolites. In this review we attempt to present an overview picture of plant O interactions. We summarize the vast number of available reports on plant responses to O at the morphological, physiological, cellular, biochemical levels, and address effects on crop yield, and on genes, proteins and metabolites. it is now clear that the machinery of photosynthesis, thereby decreasing the economic yield of most plants and inducing a common morphological symptom, called the foliar injury. The foliar injury symptoms can be authentically utilized for biomonitoring of O under natural conditions. Elevated O stress has been convincingly demonstrated to trigger an antioxidative defense system in plants. The past several years have seen the development and application of high-throughput omics technologies (transcriptomics, proteomics, and metabolomics) that are capable of identifying and prolifiling the O-responsive components in model and nonmodel plants. Such studies have been carried out ans have generated an inventory of O-Responsive components--a great resource to the scientific community. Recently, it has been shown that certain organic chemicals ans elevated CO levels are effective in ameliorating O-generated stress. Both targeted and highthroughput approaches have advanced our knowledge concerning what O-triggerred signaling and metabolic pathways exist in plants. Moreover, recently generated information, and several biomarkers for O, may, in the future, be exploited to better screen and develop O-tolerant plants.


PubMed | Research Laboratory for Biotechnology and Biochemistry
Type: Journal Article | Journal: Proteomics | Year: 2011

Given the essential role of proteomics in understanding the biology of plants, we are establishing a global plant proteomics organization to properly organize, preserve and disseminate collected information on plant proteomics. We call this organization International Plant Proteomics Organization (INPPO; http://www.inppo.com). Ten initiatives of INPPO are outlined along with how to address them in multiple phases. As our vision is global, we sincerely hope the scientific communities around the world will come together to support and join INPPO.


PubMed | Research Laboratory for Biotechnology and Biochemistry
Type: Journal Article | Journal: Journal of proteomics | Year: 2012

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics.


PubMed | Research Laboratory for Biotechnology and Biochemistry
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

Rice is one of the most important food and cereal crop plants in the world. Rice proteomics began in the 1990s. Since then, considerable progress has been made in establishing protocols from isolation of rice proteins from different tissues, organs, and organelles, to separation of complex proteins and to their identification by mass spectrometry. Since the year 2000, global proteomics studies have been performed during growth and development under numerous biotic and abiotic environmental conditions. Two-dimensional (2-D) gel-based proteomics platform coupled with mass spectrometry has been retained as the workhorse for proteomics of a variety of rice samples. In this chapter, we describe in detail the different protocols used for isolation of rice proteins, their separation, detection, and identification using gel-based proteomics and mass spectrometry approaches.

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