RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin.
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.
Rodríguez-Cabrera
Lianet
L
Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
Trujillo-Bacallao
Damian
D
Borrás-Hidalgo
Orlando
O
Wright
Denis J
DJ
Ayra-Pardo
Camilo
C
D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D004064:Digestive System / Q000378:metabolism; D004731:Endotoxins / Q000633:toxicity*; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D006460:Hemolysin Proteins / Q000633:toxicity*; D054884:Host-Pathogen Interactions; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000633:toxicity*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology
D000083722:Bacillus thuringiensis Toxins; C029103:Bacillus thuringiensis protoxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011498:Protein Precursors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases