Bideshi, Dennis K.
(Department of Entomology, University of California)
,
Waldrop, Greer
(Department of Biology, Undergraduate Program in Molecular, Cellular, and Developmental Biology, University of Louisville)
,
Fernandez-Luna, Maria Teresa
(Department of Entomology, University of California)
,
Diaz-Mendoza, Mercedes
(Department of Entomology, University of California)
,
Wirth, Margaret C.
(Department of Entomology, University of California)
,
Johnson, Jeffrey J.
(Department of Entomology, University of California)
,
Park, Hyun-Woo
(Department of Entomology, University of California)
,
Federici, Brian A.
(Department of Entomology, University of California)
The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly, it enhances the larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa, and Cry4Ba) and delays the phenotypic expression of resistance to these that h...
The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly, it enhances the larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa, and Cry4Ba) and delays the phenotypic expression of resistance to these that has evolved in Culex quinquefasciatus. It is also known that Cyt1Aa, which is highly lipophilic, synergizes Cry11Aa by functioning as a surrogate membrane-bound receptor for the latter protein. Little is known, however, about whether Cyt1Aa can interact similarly with other Cry proteins not primarily mosquitocidal; for example, Cry2Aa, which is active against lepidopteran larvae, but essentially inactive or has very low toxicity to mosquito larvae. Here we demonstrate by ligand binding and enzyme-linked immunosorbent assays that Cyt1Aa and Cry2Aa form intermolecular complexes in vitro, and in addition show that Cyt1Aa facilitates binding of Cry2Aa throughout the midgut of C. quinquefasciatus larvae. As Cry2Aa and Cry11Aa share structural similarity in domain II, the interaction between Cyt1Aa and Cry2Aa could be a result of a similar mechanism previously proposed for Cry11Aa and Cyt1Aa. Finally, despite the observed interaction between Cry2Aa and Cyt1Aa, only a 2-fold enhancement in toxicity resulted against C. quinquefasciatus. Regardless, our results suggest that Cry2Aa could be a useful component of mosquitocidal endotoxin complements being developed for recombinant strains of B. thuringiensis subsp. israelensis and B. sphaericus aimed at improving the efficacy of commercial products and avoiding resistance.
The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly, it enhances the larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa, and Cry4Ba) and delays the phenotypic expression of resistance to these that has evolved in Culex quinquefasciatus. It is also known that Cyt1Aa, which is highly lipophilic, synergizes Cry11Aa by functioning as a surrogate membrane-bound receptor for the latter protein. Little is known, however, about whether Cyt1Aa can interact similarly with other Cry proteins not primarily mosquitocidal; for example, Cry2Aa, which is active against lepidopteran larvae, but essentially inactive or has very low toxicity to mosquito larvae. Here we demonstrate by ligand binding and enzyme-linked immunosorbent assays that Cyt1Aa and Cry2Aa form intermolecular complexes in vitro, and in addition show that Cyt1Aa facilitates binding of Cry2Aa throughout the midgut of C. quinquefasciatus larvae. As Cry2Aa and Cry11Aa share structural similarity in domain II, the interaction between Cyt1Aa and Cry2Aa could be a result of a similar mechanism previously proposed for Cry11Aa and Cyt1Aa. Finally, despite the observed interaction between Cry2Aa and Cyt1Aa, only a 2-fold enhancement in toxicity resulted against C. quinquefasciatus. Regardless, our results suggest that Cry2Aa could be a useful component of mosquitocidal endotoxin complements being developed for recombinant strains of B. thuringiensis subsp. israelensis and B. sphaericus aimed at improving the efficacy of commercial products and avoiding resistance.
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대상 데이터
At least 10 cells obtained from each replicate assay were selected for further microscopic analysis. Tissues and isolated cells were observed with the Nikon SM7800 and Leica DMRE fluorescent microscopes, respectively. At least three replicate assays were performed in each of these studies.
이론/모형
1 N HCl, and then centrifuged at 1,000 ×g for 5 min to pellet particulate undissolved crystals. The solution was dialyzed in PBS overnight, and total protein concentration was determined by the Bradford method[1]. Cyt1Aa in PBS (0.
성능/효과
Data for the 1:1 combination of the Cry2Aa and Cyt1Aa strains showed a statistically significant 2-fold enhancement of toxicity when the LC50 (15.2 µg/ml versus 31.2) and LC95 (127 versus 256 µg/ml) values were compared for the CqSyn mosquito strain.
4). Fourth, it is also possible that the presence of independent Cry2Aa and Cyt1Aa lesions in the midgut microvillar of CqSyn produced an additive lethal effect well below the threshold of the level required for these toxins when used independently. Finally, a combination of all of these possible mechanisms should also be considered as a viable explanation.
thuringiensis subsp. israelensis harboring plasmids pWF45, pDBF69, pWF45 and pHTC69, and pWF53 with genes encoding, respectively, Cyt1Aa (27 kDa), Cry2Aa (65 kDa), Cyt1Aa and Cry2Aa, and Cry11Aa (72 kDa) showed the presence of characteristic crystalline inclusions that formed during the sporulation phase of growth, as determined by phase contrast microscopy (data not shown). The identity of the crystal toxin proteins was confirmed by SDS-PAGE, in which each protein toxin migrated to a position corresponding to its predicted molecular mass (Fig.
후속연구
It is expected that the ratio of protoxin to spore yield in such recombinants would be higher, in contrast to when different strains producing toxins are combined. Regardless, further toxicologic studies against a wider variety of mosquito species and strains are required to clarify whether the production of a complement of diverse protoxins, including Cry2Aa, Cry4Ba, and Cyt1Aa, synthesized in a single mosquito larvicidal bacterial host produces the desired enhancement or synergistic interactions required for applied use.
To further investigate the potential for in vivo interaction between Cry2Aa and Cyt1Aa, the CqSyn and Cq4AB strains were fed purified labeled Cry2Aa alone or a combination of purified labeled Cry2Aa and Cyt1Aa toxins. Fluorescence microscopy showed that labeled Cry2Aa localized in the posterior midgut, distal from the gastric caecae in CqSyn (Fig.
참고문헌 (40)
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