Browsing by Author "Kumar, Karunesh"

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Differential expression of peroxidase and ABC transporter as the key regulatory components for degradation of azo dyes by Penicillium oxalicum SAR-3
(Springer, 2014) Saroj, Samta; Kumar, Karunesh; Prasad, Manoj; Singh, R. P.
Fungal species are potential dye decomposers since these secrete spectra of extracellular enzymes involved in catabolism. However, cellular mechanisms underlying azo dye catalysis and detoxification are incompletely understood and obscure. A potential strain designated as Penicillium oxalicum SAR-3 demonstrated broad-spectrum catabolic ability of different azo dyes. A forward suppression subtractive hybridization (SSH) cDNA library of P. oxalicum SAR-3 constructed in presence and absence of azo dye Acid Red 183 resulted in identification of 183 unique expressed sequence tags (ESTs) which were functionally classified into 12 functional categories. A number of novel genes that affect specifically organic azo dye degradation were discovered. Although the ABC transporters and peroxidases emerged as prominent hot spot for azo dye detoxification, we also identified a number of proteins that are more proximally related to stress-responsive gene expression. Majority of the ESTs (29.5%) were grouped as hypothetical/unknown indicating the presence of putatively novel genes. Analysis of few ESTs through quantitative real-time reverse transcription polymerase chain reaction revealed their possible role in AR183 degradation. The ESTs identified in the SSH library provide a novel insight on the transcripts that are expressed in P. oxalicum strain SAR-3 in response to AR183.
Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein
(Landes Bioscience, 2011) Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S.; Prasad, Manoj
The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.
Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions
(Springer, 2013) Kumar, Karunesh; Muthamilarasan, Mehanathan; Prasad, Manoj
Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF-1α and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.
Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor
(PLOS, 2015) Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj
14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides interesting information on their gene structure, protein domains, phylogenetic and evolutionary relationships, and expression patterns during abiotic stresses and hormonal treatments, which could be useful in choosing candidate members for further functional characterization. In addition, demonstration of interaction between Si14-3-3 and SiRSZ21A provides novel clues on the involvement of 14-3-3 proteins in the splicing events.