Publications by authors named "Dhandapani Gurusamy"

25 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 2021 09 9;18(9):1291-1299. 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354605PMC
September 2021

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

Insect Biochem Mol Biol 2020 11 19;126:103455. Epub 2020 Aug 19.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States. Electronic address:

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.
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http://dx.doi.org/10.1016/j.ibmb.2020.103455DOI Listing
November 2020

Polymer-Coated Hydroxyapatite Nanocarrier for Double-Stranded RNA Delivery.

J Agric Food Chem 2020 Jun 16;68(25):6811-6818. Epub 2020 Jun 16.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, United States.

Conventional synthetic insecticides have limited success due to insect resistance and negative effects on off-target biota and the environment. Although RNA interference (RNAi) is a tool that is becoming more widely utilized in pest control products, naked dsRNA has limited success in most taxa. Nanocarriers have shown promising results in enhancing the efficacy of this tool. In this study, we used a layer-by-layer electrostatic assembly where we synthesized poly(acrylic acid) (PAA)-coated hydroxyapatite (HA) nanoparticles (PAA-HA NPs) as inorganic nanocarriers, which were then coated with a layer of a cationic poly(amino acid), 10 kDa poly-l-arginine (PLR), to allow for binding of a layer of negatively charged dsRNA. Binding of PLR-PAA-HA NPs to dsRNA was found to increase as the mass ratio of NPs to dsRNA increased. In vitro studies with transgenic SF9 cells (from ) expressing the firefly gene showed a significant gene silencing (35% decrease) at a 5:1 NP-to-dsRNA ratio, while naked dsRNA was ineffective at gene silencing. There was a significant concentration-response relationship in knockdown; however, cytotoxicity was observed at higher concentrations. Confocal microscopy studies showed that dsRNA from PLR-PAA-HA NPs was not localized within endosomes, while naked dsRNA appeared to be entrapped within the endosomes. Overall, polymer-functionalized HA nanocarriers enabled dsRNA to elicit gene knockdown in cells, whereas naked dsRNA was not effective in causing gene knockdown.
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http://dx.doi.org/10.1021/acs.jafc.0c02182DOI Listing
June 2020

Transport of orally delivered dsRNA in southern green stink bug, Nezara viridula.

Arch Insect Biochem Physiol 2020 Aug 22;104(4):e21692. Epub 2020 May 22.

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

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24-72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. P-labeled dsRNA injected into SGSB was processed into siRNA, but fed P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.
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http://dx.doi.org/10.1002/arch.21692DOI Listing
August 2020

RNAi in Sf9 Cells via Nanomaterial Mediated Delivery of dsRNA: A Comparison of Poly-l-arginine Polyplexes and Poly-l-arginine-Functionalized Au Nanoparticles.

ACS Appl Mater Interfaces 2020 Jun 28;12(23):25645-25657. Epub 2020 May 28.

This work focused on the delivery of dsRNA either complexed with poly-l-arginine (PLR-polyplex) or loaded onto poly-l-arginine functionalized 20 nm Au nanoparticles (PLR/Au NPs). RNA interference (RNAi) of a gene expressed in Sf9 stable cell line () was used as a model system. The binding affinity of dsRNA with two modes of functionalization of Au NPs was investigated: the electrostatic binding of PLR on citrate stabilized Au NPs (e-PLR/Au NPs) via the layer-by-layer method or the covalent-linking reaction of the polymer with NHS groups on the Au NPs surface (c-PLR/Au NPs) with excess groups quenched with either hydroxylamine (c-PLR/Au/Hyd NPs) or bovine serum albumin (c-PLR/Au/BSA NPs). The formation of PLR-polyplex particles resulting from the complexation of PLR and dsRNA were revealed by transmission electron microscope (TEM), scanning transmission electron microscope (STEM), and elemental mapping by energy dispersive X-ray spectroscopy (EDS). gene knockdown was evaluated after exposure of Sf9 cells for 72 h to 600 ng of dsRNA. Gene knockdown (GK) was found to be more efficient by exposing Sf9 cells to nanoscaled (size <100 nm) PLR-polyplex (58% GK), in contrast to microscaled (size >1 μm) PLR-polyplex (20% GK) or covalent PLR/Au/Hyd (7% GK) particles. The replacement of hydroxylamine by bovine serum albumin as ligand has significantly enhanced the efficiency of GK (31% GK). Investigation of endosomal escape, a key physiological barrier for dsRNA delivery, with CypHer5E labeled dsRNA showed the good ability for the dsRNA conjugated to the different nanosystems to enter the cells compared to the unconjugated one. This study provides valuable information concerning the required properties of materials used for dsRNA delivery in lepidopteran cells such as nanoparticle size, stability in the cell culture media, the polymer molecular weight and binding strength to the nanoparticle, and the nature of ligands on the surface.
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http://dx.doi.org/10.1021/acsami.0c06234DOI Listing
June 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

Double-stranded RNAs targeting inhibitor of apoptosis gene show no significant cross-species activity.

Arch Insect Biochem Physiol 2020 Aug 29;104(4):e21683. Epub 2020 Apr 29.

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

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double-stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species-specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.
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http://dx.doi.org/10.1002/arch.21683DOI 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

A simple and efficient Agrobacterium-mediated in planta transformation protocol for horse gram (Macrotyloma uniflorum Lam. Verdc.).

J Genet Eng Biotechnol 2020 Mar 24;18(1). Epub 2020 Mar 24.

Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu, 641046, India.

Background: Recalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized.

Results: Germinated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 μM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture.

Conclusions: It concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.
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http://dx.doi.org/10.1186/s43141-020-00023-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090105PMC
March 2020

Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito, Aedes aegypti.

Sci Rep 2019 06 19;9(1):8775. Epub 2019 Jun 19.

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

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.
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http://dx.doi.org/10.1038/s41598-019-45019-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584730PMC
June 2019

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

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

PLoS One 2018 16;13(3):e0194150. Epub 2018 Mar 16.

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

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194150PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856398PMC
July 2018

Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

Insect Biochem Mol Biol 2017 11 23;90:53-60. Epub 2017 Sep 23.

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

RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects.
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http://dx.doi.org/10.1016/j.ibmb.2017.09.011DOI Listing
November 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

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

Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate.

Plant Cell Rep 2015 May 24;34(5):721-31. Epub 2014 Dec 24.

National Research Centre on Plant Biotechnology, Lal Bahadur Shastri Building, Pusa Campus, New Delhi, 110012, India.

Key Message: Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.
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http://dx.doi.org/10.1007/s00299-014-1732-2DOI Listing
May 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

Effects of deficiency and excess of zinc on morphophysiological traits and spatiotemporal regulation of zinc-responsive genes reveal incidence of cross talk between micro- and macronutrients.

Environ Sci Technol 2013 May 3;47(10):5327-35. Epub 2013 May 3.

National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012, India.

Zinc (Zn) is an essential micronutrient which affects plant growth and development in deficiency and can be toxic when present in excess. In Arabidopsis thaliana , different families of cation transporters play pivotal roles in Zn homeostasis. In the present study, we evaluated the effects of Zn in its deficiency (0 μM; Zn-) and excess (75 μM; Zn++) on various morphophysiological and molecular traits. Primary root length was reduced in Zn- seedlings, whereas there were significant increases in the number and length of lateral roots under Zn- and Zn++ conditions, respectively. Concentration of various macro- and microelements showed variations under different Zn regimes and notable among them was the reduced level of iron (Fe) in Zn++ seedlings compared to Zn+. Certain members of the ZIP family (ZIP4, ZIP9, and ZIP12) showed significant induction in roots and shoots of the Zn- seedlings. Their suppression under Zn++ condition indicated their transcriptional regulation by Zn and their roles in the maintenance of its homeostasis. Zn-deficiency-mediated induction of HMA2 in roots and shoots suggested its role in effluxing Zn into xylem for long-distance transport. Attenuation in the expression of Fe-responsive FRO2 and IRT1 in Zn- roots and their induction in Zn++ roots provided empirical evidence toward the prevalence of a cross talk between Zn and Fe homeostasis. Variable effects of Zn- and Zn++ on the expression of subset of genes involved in the homeostasis of phosphate (Pi), potassium (K), and sulfur (S) further highlighted the prevalence of cross talk between the sensing and signaling cascades of Zn and macronutrients. Further, the inducibility of ZIP4 and ZIP12 in response to cadmium (cd) treatment could be harnessed by tailoring them in homologous or heterologous plant system for removing pollutant toxic heavy metals from the environment.
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http://dx.doi.org/10.1021/es400113yDOI Listing
May 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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