scholarly journals Fishing for New Bt Receptors in Diamondback Moth

2017 ◽  
Author(s):  
Yazhou Chen ◽  
Yuping Huang ◽  
Qun Liu ◽  
Jun Xu ◽  
Saskia Hogenhout ◽  
...  
Keyword(s):  
Brush Border ◽  
Molecular Mechanisms ◽  
Brush Border Membrane ◽  
Insect Pests ◽  
Diamondback Moth ◽  
Genetically Modified Crops ◽  
Ongoing Research ◽  
Bt Toxins ◽  
Insect Gut ◽  
Immunoblotting Technique

ABSTRACTBt toxins bind to receptors in the brush border membrane of the insect gut and create pores, leading to insect death. Bt-resistant insects demonstrate reduced binding of the Bt toxins to gut membranes. However, our understanding of the gut receptors involved in Bt toxin binding, and which receptors confer resistance to these toxins is incomplete, especially in diamondback moth (Plutella xylostella), a major agricultural pest. Identifying receptors has remained challenging because we lack sufficiently sensitive methods to detect Bt receptor interactions. Here, we report a modified far-immunoblotting technique, which revealed a broad spectrum of binding targets for the Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd in diamondback moth. We confirm the role of the glucosinolate sulfatases GSS1 and GSS2 in Cry1Bd toxicity. GSS1 and GSS2 bind directly to Cry1Bd, and their expression is crucial for Cry1Bd toxicity. These results improve our understanding of the molecular mechanisms of Bt toxicity.AUTHOR SUMMARYThe Bt toxins, from the soil bacteriumBacillus thuringiensis, have wide applications in agriculture as insecticides applied to plants or expressed in genetically modified crops. Bt toxins bind to receptors in the brush border membrane of the insect gut and create pores leading to insect death. The success of the Bt toxins in controlling insect pests has been hindered by the emergence of resistant insects, which show reduced binding of Bt to their gut membranes. Although ongoing research has identified a few receptors, many remain unknown and the mechanisms by which these receptors cause resistance remain unclear. Here, we used a modified far-immunoblotting technique to identify proteins that bind to the toxins Cry1Ac, Cry1Ab, and Cry1Bd in the diamondback moth. This identified two glucosinolate sulfatases that bind directly to Cry1Bd; also, the toxicity of Cry1Bd requires expression of these glucosinolate sulfatases. Therefore, identification of these candidate receptors improves our understanding of Bt function and resistance.

Proximal Tubular Phosphate Reabsorption: Molecular Mechanisms

2000 ◽  
Vol 80 (4) ◽  
pp. 1373-1409 ◽  
Author(s):  
Heini Murer ◽  
Nati Hernando ◽  
Ian Forster ◽  
Jürg Biber
Keyword(s):  
Brush Border ◽  
Molecular Mechanisms ◽  
Brush Border Membrane ◽  
Cell Model ◽  
Expression System ◽  
Cellular Mechanisms ◽  
Membrane Expression ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Proximal Tubular

Renal proximal tubular reabsorption of Pi is a key element in overall Pi homeostasis, and it involves a secondary active Pi transport mechanism. Among the molecularly identified sodium-phosphate (Na/Pi) cotransport systems a brush-border membrane type IIa Na-Pi cotransporter is the key player in proximal tubular Pi reabsorption. Physiological and pathophysiological alterations in renal Pi reabsorption are related to altered brush-border membrane expression/content of the type IIa Na-Picotransporter. Complex membrane retrieval/insertion mechanisms are involved in modulating transporter content in the brush-border membrane. In a tissue culture model (OK cells) expressing intrinsically the type IIa Na-Pi cotransporter, the cellular cascades involved in “physiological/pathophysiological” control of Pi reabsorption have been explored. As this cell model offers a “proximal tubular” environment, it is useful for characterization (in heterologous expression studies) of the cellular/molecular requirements for transport regulation. Finally, the oocyte expression system has permitted a thorough characterization of the transport characteristics and of structure/function relationships. Thus the cloning of the type IIa Na-Pi cotransporter (in 1993) provided the tools to study renal brush-border membrane Na-Pi cotransport function/regulation at the cellular/molecular level as well as at the organ level and led to an understanding of cellular mechanisms involved in control of proximal tubular Pi handling and, thus, of overall Pihomeostasis.

scholarly journals Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

2011 ◽  
Vol 77 (10) ◽  
pp. 3182-3188 ◽  
Author(s):  
C. Gouffon ◽  
A. Van Vliet ◽  
J. Van Rie ◽  
S. Jansens ◽  
J. L. Jurat-Fuentes
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Target Range ◽  
Content Type ◽  
Bt Toxins

ABSTRACTThe use of combinations ofBacillus thuringiensis(Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species,Heliothis virescens,Helicoverpa zea, andHelicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

scholarly journals Independent and Synergistic Effects of Knocking out Two ABC Transporter Genes on Resistance to Bacillus thuringiensis Toxins Cry1Ac and Cry1Fa in Diamondback Moth

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 9
Author(s):  
Shan Zhao ◽  
Dong Jiang ◽  
Falong Wang ◽  
Yihua Yang ◽  
Bruce E. Tabashnik ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Abc Transporter ◽  
Insect Pests ◽  
Synergistic Effects ◽  
Diamondback Moth ◽  
Cross Resistance ◽  
Vegetable Crops ◽  
Double Knockout ◽  
Bt Toxins ◽  
Abc Transporter Genes

Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world’s most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.

Sodium-phosphate transporter adaptation to dietary phosphate deprivation in normal and hypophosphatemic mice

1995 ◽  
Vol 268 (6) ◽  
pp. G917-G924 ◽  
Author(s):  
J. F. Collins ◽  
N. Bulus ◽  
F. K. Ghishan
Keyword(s):  
Brush Border ◽  
Molecular Mechanisms ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Xenopus Laevis Oocytes ◽  
Protein Levels ◽  
Renal Brush Border Membrane ◽  
Renal Tubular Reabsorption ◽  
Renal Tubular

The X-linked hypophosphatemic (Hyp) mouse is a model for hypophosphatemic vitamin D-resistant rickets and is a homologue of human X-linked hypophosphatemia. The defect in the Hyp mouse appears to be related to decreased renal tubular reabsorption of P(i) via the renal brush-border membrane (Na(+)-P(i)) transporter. Dietary P(i) deprivation upregulates Na(+)-P(i) transport activity in brush-border membrane vesicles (BBMV) isolated from both normal and Hyp mice; however, the molecular mechanisms underlying this phenomenon are not known. The current studies were designed to investigate the effect of P(i) deprivation on the renal Na(+)-P(i) transporter. Low P(i) diet upregulated Na(+)-P(i) transporter activity in isolated BBMV by 2.1-fold in normal and Hyp mice (n = 3, P = 0.01). Low P(i) diet also induced a 1.9 +/- 0.3-fold increase in normal mice and 2.9 +/- 0.4-fold increase in Hyp mice in Na(+)-P(i) transporter message levels (n = 3, P = 0.028). The increase in message level encoding the Na(+)-P(i) transporter stimulated increased Na(+)-dependent P(i) uptake by Xenopus laevis oocytes when poly(A)+ RNA was injected into them from mice on low P(i) diet (approximately 1.67-fold in normal mice and 1.33-fold in Hyp mice). Immunoreactive protein levels increased 2.3 +/- 0.4-fold in normal mice and 8.2 +/- 0.5 in the Hyp mouse kidney cortexes (n = 3, P = 0.0001) in response to dietary P(i) deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Peran Brush Border Membrane Vesicle (BBMV) dalam proses infeksi Baculovirus

2017 ◽  
Vol 2 (2) ◽  
pp. 8
Author(s):  
Arman Wijonarko
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Insect Pests ◽  
Membrane Vesicle ◽  
Apical Part ◽  
Brush Border Membrane Vesicle ◽  
Control Programs ◽  
Pseudaletia Separata ◽  
Baculovirus Infection ◽  
Columnar Cells

The Role of Brush Border Membrane Vesicle (BBMV) in The Baculovirus Infection. Although baculovirus becoming recognized as potential important agents for control pests, there are several important obstacles for commercial use insecticide. One important obstacle is their narrow host. The other obstacle is due to its development resistance, the phenomenon whereby host larvae become progressively more resistant to fatal infection as they age within and among instars. From practical stand point, this phenomenon impacts the effectiveness of baculovirus control programs against agricultural and forest insect pests that is necessary to adjust application levels in the response to the demography of the target insect population. Purified columnar cells derived from midgut of armyworm Pseudaletia separata was used for fusion assay and observed by means of fluorescence assay based on self-quenching of octadecylrhodamine B chloride (R18). Confocal microscopy of the columnar cells that had been combined with labeled virus showed that the fluorescence appeared on the apical part in the brush border membrane, this indicated that there was fusion between virus and the cells. Two dimensional SDS-PAGE and immunobloting assay confirmed that the receptor exist within the columnar cells.

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles

1989 ◽  
Vol 257 (5) ◽  
pp. C971-C975 ◽  
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
G. Flouret ◽  
D. R. Peterson
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Luminal Membrane ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Brush Border

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

Sign in / Sign up

Export Citation Format

Share Document