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

Authors:
Dr Orlando Borrás-Hidalgo, PhD
Dr Orlando Borrás-Hidalgo, PhD
CIGB
Senior Scientist
Molecular Biology
Havana | Cuba
Denis J Wright
Denis J Wright
Imperial College London
United Kingdom
Dr. Camilo Ayra-Pardo, PhD
Dr. Camilo Ayra-Pardo, PhD
Henan Provincial Engineering Laboratory of Insect Bio-reactor / Nanyang Normal University
Associate Professor
Plant Biotechnology
Nanyang, Henan | China

Environ Microbiol 2010 Nov;12(11):2894-903

Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.

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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November 2010
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