scholarly journals A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)

2020 ◽  
Author(s):  
Ke Yang ◽  
Xin-Lin Gong ◽  
Guo-Cheng Li ◽  
Ling-Qiao Huang ◽  
Chao Ning ◽  
...  
Keyword(s):  
Plutella Xylostella ◽  
Plant Hormone ◽  
Expression System ◽  
Management Strategies ◽  
Phytophagous Insects ◽  
Larval Feeding ◽  
Specific Expression ◽  
Electrode Voltage ◽  
Oocyte Expression System ◽  
Shed Light

AbstractFeeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, we focused on PxylGr34, a bitter GR in Plutella xylostella (L.). We detected abundant PxylGr34 transcripts in the larval head and specific expression of PxylGr34 in the antennae of females. Analyses using the Xenopus oocyte expression system and two-electrode voltage-clamp recording showed that PxylGr34 is specifically tuned to the plant hormone brassinolide (BL) and its analog 24-epibrassinolide. Electrophysiological analyses revealed that the medial sensilla styloconica on the maxillary galea of the 4th instar larvae are responsive to BL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female ovipositing. Knock-down of PxylGr34 by RNAi abolished BL-induced feeding inhibition. These results shed light on gustatory coding mechanisms and deterrence of insect feeding and ovipositing, and may be useful for designing plant hormone-based pest management strategies.

scholarly journals A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ke Yang ◽  
Xin-Lin Gong ◽  
Guo-Cheng Li ◽  
Ling-Qiao Huang ◽  
Chao Ning ◽  
...  
Keyword(s):  
Plutella Xylostella ◽  
Host Plants ◽  
Plant Hormone ◽  
Expression System ◽  
Management Strategies ◽  
Phytophagous Insects ◽  
Larval Feeding ◽  
Sensilla Styloconica ◽  
Oocyte Expression System ◽  
Functional Analyses

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, the GRs in Plutella xylostella were analyzed. Through RNA sequencing and qPCR, we detected abundant PxylGr34 transcripts in the larval head and adult antennae. Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL). Electrophysiological analyses revealed that the medial sensilla styloconica of 4th instar larvae are responsive to BL and EBL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female oviposition. Knock-down of PxylGr34 by RNAi attenuates the taste responses to BL, and abolishes BL-induced feeding inhibition. These results increase our understanding of how herbivorous insects detect compounds that deter feeding and oviposition, and may be useful for designing plant hormone-based pest management strategies.

Effects of rhizobacteria on the biology and behavior of Plutella xylostella (Lepidoptera: Plutellidae)

2017 ◽  
Vol 43 (2) ◽  
pp. 195
Author(s):  
Robson Thomaz Thuler ◽  
Fernando Henrique Iost Filho ◽  
Hamilton César De Oliveira Charlo ◽  
Sergio Antônio De Bortoli
Keyword(s):  
Plutella Xylostella ◽  
Diamondback Moth ◽  
Insect Behavior ◽  
Control Treatment ◽  
Pupal Weight ◽  
Larval Feeding ◽  
Direct Effects ◽  
Plant Feeding ◽  
Kluyvera Ascorbata ◽  
And Behavior

Plant induced resistance is a tool for integrated pest management, aimed at increasing plant defense against stress, which is compatible with other techniques. Rhizobacteria act in the plant through metabolic changes and may have direct effects on plant-feeding insects. The objective of this study was to determine the effects of cabbage plants inoculated with rhizobacteria on the biology and behavior of diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Cabbage seeds inoculated with 12 rhizobacteria strains were sowed in polystyrene trays and later transplanted into the greenhouse. The cabbage plants with sufficient size to support stress were then infested with diamondback moth caterpillars. Later, healthy leaves suffering injuries were collected and taken to the laboratory to feed P. xylostella second instar caterpillars that were evaluated for larval and pupal viability and duration, pupal weight, and sex ratio. The reduction of leaf area was then calculated as a measure of the amount of larval feeding. Non-preference for feeding and oviposition assays were also performed, by comparing the control treatment and plants inoculated with different rhizobacterial strains. Plants inoculated with the strains EN4 of Kluyvera ascorbata and HPF14 of Bacillus thuringiensis negatively affected the biological characteristics of P. xylostella when such traits were evaluated together, without directly affecting the insect behavior.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Chemosensory genes in the head of Spodoptera litura larvae

2021 ◽  
pp. 1-10
Author(s):  
Lu-Lu Li ◽  
Ji-Wei Xu ◽  
Wei-Chen Yao ◽  
Hui-Hui Yang ◽  
Youssef Dewer ◽  
...  
Keyword(s):  
Spodoptera Litura ◽  
Phylogenetic Trees ◽  
Target Genes ◽  
Control Strategies ◽  
Insect Pest ◽  
Management Strategies ◽  
Food Sources ◽  
Larval Feeding ◽  
Chemosensory System ◽  
Chemosensory Genes

Abstract The tobacco cutworm Spodoptera litura (Lepidoptera: Noctuidae) is a polyphagous pest with a highly selective and sensitive chemosensory system involved in complex physiological behaviors such as searching for food sources, feeding, courtship, and oviposition. However, effective management strategies for controlling the insect pest populations under threshold levels are lacking. Therefore, there is an urgent need to formulate eco-friendly pest control strategies based on the disruption of the insect chemosensory system. In this study, we identified 158 putative chemosensory genes based on transcriptomic and genomic data for S. litura, including 45 odorant-binding proteins (OBPs, nine were new), 23 chemosensory proteins (CSPs), 60 odorant receptors (ORs, three were new), and 30 gustatory receptors (GRs, three were new), a number higher than those reported by previous transcriptome studies. Subsequently, we constructed phylogenetic trees based on these genes in moths and analyzed the dynamic expression of various genes in head capsules across larval instars using quantitative real-time polymerase chain reaction. Nine genes–SlitOBP8, SlitOBP9, SlitOBP25, SlitCSP1, SlitCSP7, SlitCSP18, SlitOR34, SlitGR240, and SlitGR242–were highly expressed in the heads of 3- to 5-day-old S. litura larvae. The genes differentially expressed in olfactory organs during larval development might play crucial roles in the chemosensory system of S. litura larvae. Our findings substantially expand the gene inventory for S. litura and present potential target genes for further studies on larval feeding in S. litura.

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

2021 ◽  
pp. 247255522110041
Author(s):  
Raffaella Cinquetti ◽  
Francesca Guia Imperiali ◽  
Salvatore Bozzaro ◽  
Daniele Zanella ◽  
Francesca Vacca ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Expression System ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Substrate Uptake ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

scholarly journals An Endogenous RNA Transcript Antisense to CNGα1 Cation Channel mRNA

2002 ◽  
Vol 13 (10) ◽  
pp. 3696-3705 ◽  
Author(s):  
Chin-Hung Cheng ◽  
David Tai-Wai Yew ◽  
Hiu-Yee Kwan ◽  
Qing Zhou ◽  
Yu Huang ◽  
...  
Keyword(s):  
Expression System ◽  
Expression Patterns ◽  
Long Term Potentiation ◽  
Information Storage ◽  
Term Potentiation ◽  
Hippocampal Ca1 ◽  
Oocyte Expression System ◽  
Neuronal Functions ◽  
Rna Transcript

CNG channels are cyclic nucleotide-gated Ca2+-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNGα1 mRNA. This transcript was capable of down-regulating the expression of sense CNGα1 in theXenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNGα1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNGα1. Treatment of human glioma cell T98 with thyroid hormone T3 caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNGα1 expression. These data suggest that the suppression of CNGα1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS.

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

scholarly journals Cellular encoding of Cy dyes for single-molecule imaging

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lilia Leisle ◽  
Rahul Chadda ◽  
John D Lueck ◽  
Daniel T Infield ◽  
Jason D Galpin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Cyanine Dye ◽  
Nonsense Suppression ◽  
Xenopus Laevis Oocyte ◽  
Cellular Environment ◽  
Oocyte Expression System ◽  
Improved Technique

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

scholarly journals A Comparison of Murine and Human Factor XI

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1055-1064 ◽  
Author(s):  
David Gailani ◽  
Mao-Fu Sun ◽  
Yuehui Sun
Keyword(s):  
Messenger Rna ◽  
Human Factor ◽  
Dextran Sulfate ◽  
Specific Activity ◽  
Expression System ◽  
Contact Activation ◽  
Factor Xi ◽  
Specific Expression ◽  
Factor Xi Deficiency ◽  
Mammalian Expression

Factor XI is a plasma glycoprotein that is required for contact activation initiated fibrin formation in vitro and for normal hemostasis in vivo. In preparation for developing a mouse model of factor XI deficiency to facilitate investigations into this protease's contributions to coagulation, we cloned the complementary DNA for murine factor XI, expressed the protein in a mammalian expression system, and compared its properties with human recombinant factor XI. The 2.8-kb murine cDNA codes for a protein of 624 amino acids with 78% homology to human factor XI. Both recombinant murine and human factor XI are 160 kD homodimers comprised of two 80 kD polypeptides connected by disulfide bonds. Murine factor XI shortens the clotting time of human factor XI deficient plasma in an activated partial thromboplastin time assay, with a specific activity 50% to 70% that of the human protein. In a purified system, murine factor XI is activated by human factor XIIa and thrombin in the presence of dextran sulfate. Murine factor XI differs from human factor XI in that it undergoes autoactivation slowly in the presence of dextran sulfate. This is due primarily to murine factor XIa preferentially cleaving a site on zymogen factor XI within the light chain, rather than the activation site between Arg371 and Val372. Northern blots of polyadenylated messenger RNA show that murine factor XI message is expressed, as expected, primarily in the liver. In contrast, messenger RNA for human factor XI was identified in liver, pancreas, and kidney. The studies show that murine and human factor XI have similar structural and enzymatic properties. However, there may be variations in tissue specific expression and subtle differences in enzyme activity across species.

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