Publications by authors named "Kanakachari Mogilicherla"

21 Publications

  • Page 1 of 1

systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

RNA Biol 2020 Nov 9:1-9. Epub 2020 Nov 9.

Department of Botany, Kongunadu Arts and Science College (Autonomous), Bharathiar University, Coimbatore, India.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in requires systemic RNA interference defective protein 1 (. The expression of Ce in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed in two cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic expressing were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to expression showed tissue specificity. Compared to RNAi efficiency in wild-type expressing transgenic showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The cell-type specific and tissue-specific enhancement of RNAi efficiency by in provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.
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http://dx.doi.org/10.1080/15476286.2020.1842632DOI Listing
November 2020

Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

Arch Insect Biochem Physiol 2020 Aug 11;104(4):e21690. Epub 2020 May 11.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-β catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.
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http://dx.doi.org/10.1002/arch.21690DOI Listing
August 2020

Inhibitor of apoptosis is an effective target gene for RNAi-mediated control of Colorado potato beetle, Leptinotarsa decemlineata.

Arch Insect Biochem Physiol 2020 Aug 29;104(4):e21685. Epub 2020 Apr 29.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double-stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB-specific genes to identify targets for RNAi-mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat-killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.
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http://dx.doi.org/10.1002/arch.21685DOI Listing
August 2020

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

Arch Insect Biochem Physiol 2020 Aug 15;104(4):e21678. Epub 2020 Apr 15.

Department of Entomology, University of Kentucky, Lexington, Kentucky, 40546, USA.

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.
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http://dx.doi.org/10.1002/arch.21678DOI Listing
August 2020

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

Arch Insect Biochem Physiol 2020 Aug 14;104(4):e21677. Epub 2020 Apr 14.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi-dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi-dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.
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http://dx.doi.org/10.1002/arch.21677DOI Listing
August 2020

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

Proc Natl Acad Sci U S A 2018 08 30;115(33):8334-8339. Epub 2018 Jul 30.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40546

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, and , showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing , Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.
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http://dx.doi.org/10.1073/pnas.1809381115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099913PMC
August 2018

Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR.

Sci Rep 2018 02 27;8(1):3720. Epub 2018 Feb 27.

Department of Entomology, University of Kentucky, Lexington, Kentucky, 40546, USA.

The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.
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http://dx.doi.org/10.1038/s41598-018-22035-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829169PMC
February 2018

Comparative analysis of double-stranded RNA degradation and processing in insects.

Sci Rep 2017 12 6;7(1):17059. Epub 2017 Dec 6.

Department of Entomology, College of Agriculture, Food and Environment, Agriculture Science Center North, University of Kentucky, Lexington, KY, USA.

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within the same order. We identified homologs of key RNAi genes in the genomes of some of these insects and studied their domain architecture. These data suggest that dsRNA digestion by dsRNases and its processing to siRNAs in the cells are among the major factors contributing to differential RNAi efficiency reported among insects.
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http://dx.doi.org/10.1038/s41598-017-17134-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719073PMC
December 2017

Transcript profiling of genes expressed during fibre development in diploid cotton (Gossypium arboreum L.).

BMC Genomics 2017 Aug 31;18(1):675. Epub 2017 Aug 31.

Plant Transformation Group, International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi, India.

Background: Cotton fibre is a single cell and it is one of the best platforms for unraveling the genes express during various stages of fibre development. There are reports devoted to comparative transcriptome study on fiber cell initiation and elongation in tetraploid cultivated cotton. However, in the present investigation, comparative transcriptome study was made in diploid cultivated cotton using isogenic fuzzy-lintless (Fl) and normal fuzzy linted (FL) lines belong to Gossypium arboreum, diploid species at two stages, 0 and 10 dpa (days post anthesis), using Affymetrix cotton GeneChip genome array.

Result: Scanning electron microscopy (SEM) analysis uncovered the occurrence of few fibre cell initials in the Fl line as compared to many in Normal FL at -2 and 0 dpa. However, at 10 dpa there were no fibre cells found elongated in Fl but many elongated cells were found in FL line. Up-regulation of transcription factors, AP2-EREBP, C2H2, C3H, HB and WRKY was observed at 0 dpa whereas in 10 dpa transcription factors, AP2-EREBP, AUX/IAA, bHLH, C2H2, C3H, HB, MYB, NAC, Orphans, PLATZ and WRKY were found down regulated in Fl line. These transcription factors were mainly involved in metabolic pathways such as phytohormone signaling, energy metabolism of cell, fatty acid metabolism, secondary metabolism and other signaling pathways and are related directly or indirectly in fiber development. Quantitative real-time PCR was performed to check fold up or down-regulation of these genes and transcription factors (TFs) down regulated in mutants as compared to normal at 0 and 10 dpa.

Conclusion: This study elucidates that the up-regulation of transcription factors like AP2-EREBP, C2H2, C3H, HB, WRKY and phytohormone signaling genes at 0 dpa and their down-regulation at the 10 dpa might have constrain the fibre elongation in fuzzy-lintless line. Along with this the down-regulation of genes involved in synthesis of VLCFA chain, transcripts necessary for energy and cell wall metabolism, EXPANSINs, arabinogalactan proteins (AGPs), tubulin might also be the probable reason for reduced growth of fibres in the Fl. Plant receptor-like kinases (RLKs), Leucine Rich Repeats) LRR- family protein and signal transduction coding for mitogen-activated protein kinase (MAPK) cascade, have been engaged in coordination of cell elongation and SCW biosynthesis, down-regulation of these might loss the function leads to reduced fibre growth.
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http://dx.doi.org/10.1186/s12864-017-4066-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580217PMC
August 2017

Development of RNAi method for screening candidate genes to control emerald ash borer, Agrilus planipennis.

Sci Rep 2017 08 7;7(1):7379. Epub 2017 Aug 7.

University of Kentucky, Department of Entomology, Lexington, 40546, USA.

The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, β subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.
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http://dx.doi.org/10.1038/s41598-017-07605-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547101PMC
August 2017

RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors.

Insect Biochem Mol Biol 2016 11 26;78:78-88. Epub 2016 Sep 26.

Department of Entomology, University of Kentucky, Lexington, KY 40546, USA. Electronic address:

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.
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http://dx.doi.org/10.1016/j.ibmb.2016.09.002DOI Listing
November 2016

Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

RNA Biol 2016 07 31;13(7):656-69. Epub 2016 May 31.

a Department of Entomology , College of Agriculture, Food and Environment, Agriculture Science Center North, University of Kentucky , Lexington , KY , USA.

RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.
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http://dx.doi.org/10.1080/15476286.2016.1191728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962799PMC
July 2016

Genome-wide transcriptomic and proteomic analyses of bollworm-infested developing cotton bolls revealed the genes and pathways involved in the insect pest defence mechanism.

Plant Biotechnol J 2016 06 22;14(6):1438-55. Epub 2016 Jan 22.

International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% of differentially expressed proteins were found to be involved in signalling followed by redox regulation. Further analysis showed that defence-related stress hormones and their lipid precursors, transcription factors, signalling molecules, etc. were stimulated, whereas the growth-related counterparts were suppressed during bollworm infestation. Around 26% of the significantly up-regulated proteins were defence molecules, while >50% of the significantly down-regulated were related to photosynthesis and growth. Interestingly, the biosynthesis genes for synergistically regulated jasmonate, ethylene and suppressors of the antagonistic factor salicylate were found to be up-regulated, suggesting a choice among stress-responsive phytohormone regulation. Manual curation of the enzymes and TFs highlighted the components of retrograde signalling pathways. Our data suggest that a selective regulatory mechanism directs the reallocation of metabolic resources favouring defence over growth under bollworm infestation and these insights could be exploited to develop bollworm-resistant cotton varieties.
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http://dx.doi.org/10.1111/pbi.12508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066800PMC
June 2016

Delineating the glycoproteome of elongating cotton fiber cells.

Data Brief 2015 Dec 26;5:717-25. Epub 2015 Oct 26.

Plant Transformation Group, International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi, India.

The data presented here delineates the glycoproteome component in the elongating cotton fiber cells attained using complementary proteomic approaches followed by protein and N-linked glycosylation site identification (Kumar et al., 2013) [1]. Utilizing species specific protein sequence databases in proteomic approaches often leads to additional information that may not be obtained using cross-species databases. In this context we have reanalyzed our glycoproteome dataset with the Gossypium arboreum, Gossypium raimondii (version 2.0) and Gossypium hirsutum protein databases that has led to the identification of 21 N-linked glycosylation sites and 18 unique glycoproteins that were not reported in our previous study. The 1D PAGE and solution based glycoprotein identification data is publicly available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD000178 and the 2D PAGE based protein identification and glycopeptide approach based N-linked glycosylation site identification data is available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD002849.
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http://dx.doi.org/10.1016/j.dib.2015.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652025PMC
December 2015

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

Appl Biochem Biotechnol 2016 Feb 15;178(3):433-50. Epub 2015 Oct 15.

ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India.

Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.
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http://dx.doi.org/10.1007/s12010-015-1884-8DOI Listing
February 2016

Evaluation of different carbon sources for high frequency callus culture with reduced phenolic secretion in cotton ( L.) cv. SVPR-2.

Biotechnol Rep (Amst) 2015 Sep 30;7:72-80. Epub 2015 May 30.

Department of Plant Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.

An efficient protocol was developed to control excessive phenolic compound secretion during callus culture of cotton. As cotton is naturally rich in phenolic compounds factors influencing the phenolic compound secretion, callus induction and proliferation were optimized for getting high frequency callus culture. Different carbon sources such as fructose, glucose, sucrose and maltose were tested at various concentrations to control phenolic secretion in callus culture. Among them, 3% maltose was found to be the best carbon source for effectively controlling phenolic secretion in callus induction medium. High frequency of callus induction was obtained on MSB medium supplemented with 3% Maltose, 2,4-D (0.90 μM) and Kinetin (4.60 μM) from both cotyledon and hypocotyl explants. The best result of callus induction was obtained with hypocotyl explant (94.90%) followed by cotyledon explant (85.20%). MSB medium supplemented with 2,4-D (0.45 μM) along with 2iP (2.95 μM) gave tremendous proliferation of callus with high percentage of response. Varying degrees of colors and textures of callus were observed under different hormone treatments. The present study offers a solution for controlling phenolic secretion in cotton callus culture by adjusting carbon sources without adding any additives and evaluates the manipulation of plant growth regulators for efficient callus culture of SVPR-2 cotton cultivar.
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http://dx.doi.org/10.1016/j.btre.2015.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466046PMC
September 2015

GhDRIN1, a novel drought-induced gene of upland cotton (Gossypium hirsutum L.) confers abiotic and biotic stress tolerance in transgenic tobacco.

Biotechnol Lett 2015 Apr 21;37(4):907-19. Epub 2014 Nov 21.

National Research Centre on Plant Biotechnology, LBS Building, Pusa Campus, New Delhi, 110012, India.

A novel stress tolerance cDNA fragment encoding GhDRIN1 protein was identified and its regulation was studied in cotton boll tissues and seedlings subjected to various biotic and abiotic stresses. Phylogenetic and conserved domain prediction indicated that GhDRIN1 was annotated with a hypothetical protein of unknown function. Subcellular localization showed that GhDRIN1 is localized in the chloroplasts. The promoter sequence was isolated and subjected to in silico study. Various cis-acting elements responsive to biotic and abiotic stresses and hormones were found. Transgenic tobacco seedlings exhibited better growth on amended MS medium and showed minimal leaf damage in insect bioassays carried out with Helicoverpa armigera larvae. Transgenic tobacco showed better tolerance to water-deficit and fast recovered upon rewatering. Present work demonstrated that GhDRIN1, a novel stress tolerance gene of cotton, positively regulates the response to biotic and abiotic stresses in transgenic tobacco.
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http://dx.doi.org/10.1007/s10529-014-1733-9DOI Listing
April 2015

Glycoproteome of elongating cotton fiber cells.

Mol Cell Proteomics 2013 Dec 9;12(12):3677-89. Epub 2013 Sep 9.

Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India;

Cotton ovule epidermal cell differentiation into long fibers primarily depends on wall-oriented processes such as loosening, elongation, remodeling, and maturation. Such processes are governed by cell wall bound structural proteins and interacting carbohydrate active enzymes. Glycosylation plays a major role in the structural, functional, and localization aspects of the cell wall and extracellular destined proteins. Elucidating the glycoproteome of fiber cells would reflect its wall composition as well as compartmental requirement, which must be system specific. Following complementary proteomic approaches, we have identified 334 unique proteins comprising structural and regulatory families. Glycopeptide-based enrichment followed by deglycosylation with PNGase F and A revealed 92 unique peptides containing 106 formerly N-linked glycosylated sites from 67 unique proteins. Our results showed that structural proteins like arabinogalactans and carbohydrate active enzymes were relatively more abundant and showed stage- and isoform-specific expression patterns in the differentiating fiber cell. Furthermore, our data also revealed the presence of heterogeneous and novel forms of structural and regulatory glycoproteins. Comparative analysis with other plant glycoproteomes highlighted the unique composition of the fiber glycoproteome. The present study provides the first insight into the identity, abundance, diversity, and composition of the glycoproteome within single celled cotton fibers. The elucidated composition also indirectly provides clues about unicellular compartmental requirements underlying single cell differentiation.
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http://dx.doi.org/10.1074/mcp.M113.030726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861716PMC
December 2013

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation.

BMC Genomics 2012 Nov 14;13:624. Epub 2012 Nov 14.

Plant Transformation Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

Background: Fuzzless-lintless cotton mutants are considered to be the ideal material to understand the molecular mechanisms involved in fibre cell development. Although there are few reports on transcriptome and proteome analyses in cotton at fibre initiation and elongation stages, there is no comprehensive comparative transcriptome analysis of fibre-bearing and fuzzless-lintless cotton ovules covering fibre initiation to secondary cell wall (SCW) synthesis stages. In the present study, a comparative transcriptome analysis was carried out using G. hirsutum L. cv. MCU5 wild-type (WT) and it's near isogenic fuzzless-lintless (fl) mutant at fibre initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and SCW synthesis (20 dpa) stages.

Results: Scanning electron microscopy study revealed the delay in the initiation of fibre cells and lack of any further development after 2 dpa in the fl mutant. Transcriptome analysis showed major down regulation of transcripts (90%) at fibre initiation and early elongation (5 dpa) stages in the fl mutant. Majority of the down regulated transcripts at fibre initiation stage in the fl mutant represent calcium and phytohormone mediated signal transduction pathways, biosynthesis of auxin and ethylene and stress responsive transcription factors (TFs). Further, transcripts involved in carbohydrate and lipid metabolisms, mitochondrial electron transport system (mETS) and cell wall loosening and elongation were highly down-regulated at fibre elongation stage (5-15 dpa) in the fl mutant. In addition, cellulose synthases and sucrose synthase C were down-regulated at SCW biosynthesis stage (15-20 dpa). Interestingly, some of the transcripts (~50%) involved in phytohormone signalling and stress responsive transcription factors that were up-regulated at fibre initiation stage in the WT were found to be up-regulated at much later stage (15 dpa) in fl mutant.

Conclusions: Comparative transcriptome analysis of WT and its near isogenic fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation.
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http://dx.doi.org/10.1186/1471-2164-13-624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556503PMC
November 2012

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes.

Plant Mol Biol 2012 Feb 7;78(3):223-46. Epub 2011 Dec 7.

National Research Centre on Plant Biotechnology, New Delhi, India.

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.
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http://dx.doi.org/10.1007/s11103-011-9857-yDOI Listing
February 2012

Genome-wide transcriptome and proteome analyses of tobacco psaA and psbA deletion mutants.

Plant Mol Biol 2011 Jul 14;76(3-5):407-23. Epub 2011 Feb 14.

Plant Transformation Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

Photosynthesis in higher land plants is a complex process involving several proteins encoded by both nuclear and chloroplast genomes that require a highly coordinated gene expression. Significant changes in plastid differentiation and biochemical processes are associated with the deletion of chloroplast genes. In this study we report the genome-wide responses caused by the deletion of tobacco psaA and psbA genes coding core components of photosystem I (PSI) and photosystem II (PSII), respectively, generated through a chloroplast genetic engineering approach. Transcriptomic and quantitative proteomic analysis showed the down regulation of specific groups of nuclear and chloroplast genes involved in photosynthesis, energy metabolism and chloroplast biogenesis. Moreover, our data show simultaneous activation of several defense and stress responsive genes including those involved in reactive oxygen species (ROS) scavenging mechanisms. A major finding is the differential transcription of the plastome of deletion mutants: genes known to be transcribed by the plastid encoded polymerase (PEP) were generally down regulated while those transcribed by the nuclear encoded polymerase (NEP) were up regulated, indicating simultaneous activation of multiple signaling pathways in response to disruption of PSI and PSII complexes. The genome wide transcriptomic and proteomic analysis of the ∆psaA and ∆psbA deletion mutants revealed a simultaneous up and down regulation of the specific groups of genes located in nucleus and chloroplasts suggesting a complex circuitry involving both retrograde and anterograde signaling mechanisms responsible for the coordinated expression of nuclear and chloroplast genomes.
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http://dx.doi.org/10.1007/s11103-011-9731-yDOI Listing
July 2011
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