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Construction of a hypervirulent and specific mycoinsecticide for locust control.

Fang W, Lu HL, King GF, St Leger RJ - Sci Rep (2014)

Bottom Line: We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption.Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption.However, specificity was retained as the recombinant strains did not cause disease in non-acridids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou. 310058, Zhejiang, China.

ABSTRACT
Locusts and grasshoppers (acridids) are among the worst pests of crops and grasslands worldwide. Metarhizium acridum, a fungal pathogen that specifically infects acridids, has been developed as a control agent but its utility is limited by slow kill time and greater expense than chemical insecticides. We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption. Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption. However, specificity was retained as the recombinant strains did not cause disease in non-acridids. Our results identify a repertoire of toxins with different modes of action that improve the utility of fungi as specific control agents of insects.

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Construction of transgenic M. acridum ARSEF324 strains expressing four arthropod toxins.(A) Schematic representations of the Metarhizium expression fragments cloned into the pFBARGFP plasmid. Pmcl1: Mcl1 promoter that targets high level production of toxins into the hemolymph; SP: signal peptide of MCL1 that ensures secretion of the expressed toxins; TER: terminator region of the TrpC gene from A. nidulans; AaIT1: NaV channel blocker from A. australis; Hv1a: CaV channel blocker from A. robustus; Hv1c: KCa channel blocker from H. versuta; Hybrid: CaV and KCa blocker from H. versuta. (B) Southern blot analysis confirming insertion of genes encoding arthropod toxins into the genome of M. acridum. Genomic DNA was digested with EcoRI and SpeI. The ORF of the herbicide resistance gene bar was used as a probe. 1: a transformant expressing Hv1a; 2: a transformant expressing Hv1c; 3: a transformant expressing hybrid-toxin; 4: a transformant expressing AaIT1. 5: the wild-type strain. (C) Western blot analysis confirming expression of arthropod toxins by M. acridum. WT: the wild-type strain; TS: transgenic strains. Polyclonal antibodies to Hv1a, Hv1c or hybrid-toxin were used to detect their respective toxins. An antiserum to A. australis venom was used to detect AaIT1. All protein samples were run on 16% SDS-PAGE (Tris-Tricine) and blotted to nitrocellulose membrane (0.2 μm). Detecting target proteins with their respective antiserum was conducted with the standard Western blotting analysis. Pictures of different toxins were equally processed and combined with the image software Photoshop CS5.
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f1: Construction of transgenic M. acridum ARSEF324 strains expressing four arthropod toxins.(A) Schematic representations of the Metarhizium expression fragments cloned into the pFBARGFP plasmid. Pmcl1: Mcl1 promoter that targets high level production of toxins into the hemolymph; SP: signal peptide of MCL1 that ensures secretion of the expressed toxins; TER: terminator region of the TrpC gene from A. nidulans; AaIT1: NaV channel blocker from A. australis; Hv1a: CaV channel blocker from A. robustus; Hv1c: KCa channel blocker from H. versuta; Hybrid: CaV and KCa blocker from H. versuta. (B) Southern blot analysis confirming insertion of genes encoding arthropod toxins into the genome of M. acridum. Genomic DNA was digested with EcoRI and SpeI. The ORF of the herbicide resistance gene bar was used as a probe. 1: a transformant expressing Hv1a; 2: a transformant expressing Hv1c; 3: a transformant expressing hybrid-toxin; 4: a transformant expressing AaIT1. 5: the wild-type strain. (C) Western blot analysis confirming expression of arthropod toxins by M. acridum. WT: the wild-type strain; TS: transgenic strains. Polyclonal antibodies to Hv1a, Hv1c or hybrid-toxin were used to detect their respective toxins. An antiserum to A. australis venom was used to detect AaIT1. All protein samples were run on 16% SDS-PAGE (Tris-Tricine) and blotted to nitrocellulose membrane (0.2 μm). Detecting target proteins with their respective antiserum was conducted with the standard Western blotting analysis. Pictures of different toxins were equally processed and combined with the image software Photoshop CS5.

Mentions: The toxin genes were chemically synthesized with the MCL1 signal peptide to ensure secretion, and they were cloned in a common transfer plasmid downstream of the Mcl1 promoter (PMcl1) (Fig. 1A) to target high-level production of each toxin into the hemolymph (Mcl1 is the most highly expressed gene in hemolymph)20. The four toxin genes were individually transformed into M. acridum strain ARSEF324. Real time RT-PCR was used to measure insecticidal toxin expression in transformants identified by Southern blot analysis as containing a single copy of the transgene (Fig. 1B). Transformants with ~1.6 ng of a specific toxin transcript in 1 μg of total RNA were subjected to Western blot analysis to identify specific products of the toxin genes. Products with sizes similar to those expected for mature, processed toxins were observed (Fig. 1C). All transformants were morphologically stable and showed wild-type levels of growth and sporulation.


Construction of a hypervirulent and specific mycoinsecticide for locust control.

Fang W, Lu HL, King GF, St Leger RJ - Sci Rep (2014)

Construction of transgenic M. acridum ARSEF324 strains expressing four arthropod toxins.(A) Schematic representations of the Metarhizium expression fragments cloned into the pFBARGFP plasmid. Pmcl1: Mcl1 promoter that targets high level production of toxins into the hemolymph; SP: signal peptide of MCL1 that ensures secretion of the expressed toxins; TER: terminator region of the TrpC gene from A. nidulans; AaIT1: NaV channel blocker from A. australis; Hv1a: CaV channel blocker from A. robustus; Hv1c: KCa channel blocker from H. versuta; Hybrid: CaV and KCa blocker from H. versuta. (B) Southern blot analysis confirming insertion of genes encoding arthropod toxins into the genome of M. acridum. Genomic DNA was digested with EcoRI and SpeI. The ORF of the herbicide resistance gene bar was used as a probe. 1: a transformant expressing Hv1a; 2: a transformant expressing Hv1c; 3: a transformant expressing hybrid-toxin; 4: a transformant expressing AaIT1. 5: the wild-type strain. (C) Western blot analysis confirming expression of arthropod toxins by M. acridum. WT: the wild-type strain; TS: transgenic strains. Polyclonal antibodies to Hv1a, Hv1c or hybrid-toxin were used to detect their respective toxins. An antiserum to A. australis venom was used to detect AaIT1. All protein samples were run on 16% SDS-PAGE (Tris-Tricine) and blotted to nitrocellulose membrane (0.2 μm). Detecting target proteins with their respective antiserum was conducted with the standard Western blotting analysis. Pictures of different toxins were equally processed and combined with the image software Photoshop CS5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256560&req=5

f1: Construction of transgenic M. acridum ARSEF324 strains expressing four arthropod toxins.(A) Schematic representations of the Metarhizium expression fragments cloned into the pFBARGFP plasmid. Pmcl1: Mcl1 promoter that targets high level production of toxins into the hemolymph; SP: signal peptide of MCL1 that ensures secretion of the expressed toxins; TER: terminator region of the TrpC gene from A. nidulans; AaIT1: NaV channel blocker from A. australis; Hv1a: CaV channel blocker from A. robustus; Hv1c: KCa channel blocker from H. versuta; Hybrid: CaV and KCa blocker from H. versuta. (B) Southern blot analysis confirming insertion of genes encoding arthropod toxins into the genome of M. acridum. Genomic DNA was digested with EcoRI and SpeI. The ORF of the herbicide resistance gene bar was used as a probe. 1: a transformant expressing Hv1a; 2: a transformant expressing Hv1c; 3: a transformant expressing hybrid-toxin; 4: a transformant expressing AaIT1. 5: the wild-type strain. (C) Western blot analysis confirming expression of arthropod toxins by M. acridum. WT: the wild-type strain; TS: transgenic strains. Polyclonal antibodies to Hv1a, Hv1c or hybrid-toxin were used to detect their respective toxins. An antiserum to A. australis venom was used to detect AaIT1. All protein samples were run on 16% SDS-PAGE (Tris-Tricine) and blotted to nitrocellulose membrane (0.2 μm). Detecting target proteins with their respective antiserum was conducted with the standard Western blotting analysis. Pictures of different toxins were equally processed and combined with the image software Photoshop CS5.
Mentions: The toxin genes were chemically synthesized with the MCL1 signal peptide to ensure secretion, and they were cloned in a common transfer plasmid downstream of the Mcl1 promoter (PMcl1) (Fig. 1A) to target high-level production of each toxin into the hemolymph (Mcl1 is the most highly expressed gene in hemolymph)20. The four toxin genes were individually transformed into M. acridum strain ARSEF324. Real time RT-PCR was used to measure insecticidal toxin expression in transformants identified by Southern blot analysis as containing a single copy of the transgene (Fig. 1B). Transformants with ~1.6 ng of a specific toxin transcript in 1 μg of total RNA were subjected to Western blot analysis to identify specific products of the toxin genes. Products with sizes similar to those expected for mature, processed toxins were observed (Fig. 1C). All transformants were morphologically stable and showed wild-type levels of growth and sporulation.

Bottom Line: We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption.Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption.However, specificity was retained as the recombinant strains did not cause disease in non-acridids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou. 310058, Zhejiang, China.

ABSTRACT
Locusts and grasshoppers (acridids) are among the worst pests of crops and grasslands worldwide. Metarhizium acridum, a fungal pathogen that specifically infects acridids, has been developed as a control agent but its utility is limited by slow kill time and greater expense than chemical insecticides. We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption. Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption. However, specificity was retained as the recombinant strains did not cause disease in non-acridids. Our results identify a repertoire of toxins with different modes of action that improve the utility of fungi as specific control agents of insects.

Show MeSH
Related in: MedlinePlus