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A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

Chen W, Liu C, Xiao Y, Zhang D, Zhang Y, Li X, Tabashnik BE, Wu K - PLoS ONE (2015)

Bottom Line: Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains.Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase.The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, West Yuanmingyuan Road, Beijing, 100193, China.

ABSTRACT
Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

No MeSH data available.


Related in: MedlinePlus

Detection of HaALP1f expressed in E. coli.(A) SDS-PAGE separation of the protein extracts from HaALP1f-expressing E. coli cells. M: Molecular weight markers; Lane 1: total extract from non-induced HaALP1f-expressing E. coli cells; Lane 2: total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 3: the supernatant (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 4: The pelleted inclusion body (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 5: Purified HaALP1f from the pelleted inclusion body from IPTG-induced HaALP1f-expressing E. coli cells by using Ni-affinity column. (B) Detection of purified HaALP1f by Western blot.
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pone.0126288.g004: Detection of HaALP1f expressed in E. coli.(A) SDS-PAGE separation of the protein extracts from HaALP1f-expressing E. coli cells. M: Molecular weight markers; Lane 1: total extract from non-induced HaALP1f-expressing E. coli cells; Lane 2: total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 3: the supernatant (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 4: The pelleted inclusion body (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 5: Purified HaALP1f from the pelleted inclusion body from IPTG-induced HaALP1f-expressing E. coli cells by using Ni-affinity column. (B) Detection of purified HaALP1f by Western blot.

Mentions: Using PCR amplification, we cloned a previously described 780 bp cDNA fragment of the gene from H. armigera encoding HaALP1 [56]. The peptide encoded by this cDNA fragment, referred to here as HaALP1f, is predicted to have 260 amino acid residues (192A-T451) and a molecular weight of 30 kDa. SDS-PAGE analysis of the total extracts of Escherichia coli cells transformed with the his-tagged HaALP1f-pET28a+ construct revealed a protein band of the expected size (about 35 kDa, indicated by a black arrowhead in Fig 4A. This band was not only the strongest protein band, but also the only inducible band by IPTG (compared lane 1 and 2 in Fig 4A). This band was present in the supernatant (lane 3 in Fig 4A) and the pelleted inclusion bodies (lane 4 in Fig 4A). After purification with Ni-affinity column that captures his-tagged proteins, the 35–37 kDa band was the only visible band on the gel (lane 5 in Fig 4A). Western blot hybridization of the Ni column-purified proteins from IPTG-induced HaALP1f-expressing E. coli cells with the anti-his antibody showed that the visible 35–37 kDa band was strongly hybridized with the anti-his antibody, confirming that this band represented the his-tagged HaALP1f (Fig 4B). Western blot also revealed a weak positive band of around 74 kD (Fig 4B), suggesting that a small portion of the heterologously expressed HaALP1f existed as a homodimmer.


A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

Chen W, Liu C, Xiao Y, Zhang D, Zhang Y, Li X, Tabashnik BE, Wu K - PLoS ONE (2015)

Detection of HaALP1f expressed in E. coli.(A) SDS-PAGE separation of the protein extracts from HaALP1f-expressing E. coli cells. M: Molecular weight markers; Lane 1: total extract from non-induced HaALP1f-expressing E. coli cells; Lane 2: total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 3: the supernatant (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 4: The pelleted inclusion body (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 5: Purified HaALP1f from the pelleted inclusion body from IPTG-induced HaALP1f-expressing E. coli cells by using Ni-affinity column. (B) Detection of purified HaALP1f by Western blot.
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pone.0126288.g004: Detection of HaALP1f expressed in E. coli.(A) SDS-PAGE separation of the protein extracts from HaALP1f-expressing E. coli cells. M: Molecular weight markers; Lane 1: total extract from non-induced HaALP1f-expressing E. coli cells; Lane 2: total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 3: the supernatant (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 4: The pelleted inclusion body (from 20 min centrifugation at 25,000×g) of total extract from IPTG-induced HaALP1f-expressing E. coli cells; Lane 5: Purified HaALP1f from the pelleted inclusion body from IPTG-induced HaALP1f-expressing E. coli cells by using Ni-affinity column. (B) Detection of purified HaALP1f by Western blot.
Mentions: Using PCR amplification, we cloned a previously described 780 bp cDNA fragment of the gene from H. armigera encoding HaALP1 [56]. The peptide encoded by this cDNA fragment, referred to here as HaALP1f, is predicted to have 260 amino acid residues (192A-T451) and a molecular weight of 30 kDa. SDS-PAGE analysis of the total extracts of Escherichia coli cells transformed with the his-tagged HaALP1f-pET28a+ construct revealed a protein band of the expected size (about 35 kDa, indicated by a black arrowhead in Fig 4A. This band was not only the strongest protein band, but also the only inducible band by IPTG (compared lane 1 and 2 in Fig 4A). This band was present in the supernatant (lane 3 in Fig 4A) and the pelleted inclusion bodies (lane 4 in Fig 4A). After purification with Ni-affinity column that captures his-tagged proteins, the 35–37 kDa band was the only visible band on the gel (lane 5 in Fig 4A). Western blot hybridization of the Ni column-purified proteins from IPTG-induced HaALP1f-expressing E. coli cells with the anti-his antibody showed that the visible 35–37 kDa band was strongly hybridized with the anti-his antibody, confirming that this band represented the his-tagged HaALP1f (Fig 4B). Western blot also revealed a weak positive band of around 74 kD (Fig 4B), suggesting that a small portion of the heterologously expressed HaALP1f existed as a homodimmer.

Bottom Line: Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains.Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase.The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, West Yuanmingyuan Road, Beijing, 100193, China.

ABSTRACT
Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

No MeSH data available.


Related in: MedlinePlus