Characterization of cDNAs encoding three trypsin-like proteinases and mRNA quantitative analysis in Bt-resistant and -susceptible strains of Ostrinia nubilalis.
Our previous studies suggested that Bacillus thuringiensis (Bt) resistance in a Dipel-resistant strain of Ostrinia nubilalis was primarily due to reduced trypsin-like proteinase activity. In this study, we demonstrated a 254-fold resistance to Cry1Ab protoxin but only 12-fold to trypsin-activated Cry1Ab toxin in the Dipel-resistant strain. Significantly higher resistance to Cry1Ab protoxin than to trypsin-activated Cry1Ab toxin further supports the hypothesis that reduced trypsin-like proteinase activity leading to reduced activation of the Bt protoxin is a major resistance mechanism in the Dipel-resistant strain. To understand the molecular basis of reduced proteinase activity, three cDNAs, OnT2, OnT23, and OnT25, encoding full-length trypsin-like proteinases, were sequenced in Bt-resistant and -susceptible O. nubilalis larvae. Although a number of nucleotide differences were found in sequences from the Bt-resistant and -susceptible strains, the differences were not consistent with reduced trypsin-like activity in the Bt-resistant strain. However, the mRNA levels of OnT23 in the resistant strain were 2.7- and 3.8-fold lower than those of the susceptible strain as determined by northern blotting and real-time quantitative PCR, respectively. Thus, reduced trypsin-like activity may be attributed to reduced expression of OnT23 in Bt-resistant O. nubilalis. Our study provides new insights into Bt resistance management strategies, as resistance mediated by reduced Bt protoxin activation would be ineffective if resistant insects ingest a fully activated form of Cry1Ab toxin, either in spray formulations or transgenic Bt crops.
Li
Huarong
H
Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.
Oppert
Brenda
B
Higgins
Randall A
RA
Huang
Fangneng
F
Buschman
Lawrent L
LL
Gao
Jian-Rong
JR
Zhu
Kun Yan
KY
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000032:analysis; D004351:Drug Resistance; D004731:Endotoxins / Q000494:pharmacology*; D020869:Gene Expression Profiling; D006460:Hemolysin Proteins; D007300:Insect Control; D007814:Larva; D008969:Molecular Sequence Data; D009036:Moths; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018076:DNA, Complementary; D004731:Endotoxins; D006460:Hemolysin Proteins; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases