RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin

Authors

  • Lianet Rodríguez–Cabrera,

    1. Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
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  • Damian Trujillo–Bacallao,

    1. Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
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  • Orlando Borrás–Hidalgo,

    1. Plant Functional Genomic Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
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  • Denis J. Wright,

    1. Division of Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, Silwood Park campus, Ascot, Berkshire SL5 7PY, UK.
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  • Camilo Ayra–Pardo

    Corresponding author
    1. Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
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E-mail camilo.ayra@cigb.edu.cu; Tel. (+53) 7 2716022; Fax (+53) 7 2731779.

Summary

SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.

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