Improved plant protective efficacy of a baculovirus using an early promoter to drive insect-specific neurotoxin expression

Abstract

Genetically improved Autographa californica (Speyer) multiple nucleopolyhedrovirus (AcMNPV), the type species of the genus Nucleopolyhedrovirus, was constructed with an insect-selective toxin gene (LqhIT2) derived from the venom of the Israeli yellow scorpion, Leiurus quinquestriatus (Ehrenberg). LqhIT2 was expressed by two distinct and temporally regulated promoters. These two promoters were the early promoter p-PCm (a pu-enhanced minimal CMV promoter) and the very late p10 promoter, resulting in recombinant viruses named vAPcmIT2 and vAP10IT2, respectively. Western blot and bioassay analysis showed that the toxin gene under the control of the early p-PCm promoter was expressed 24 h earlier than under the very late p10 promoter control, and vAPcmIT2 killed faster than vAP10IT2. The EC50 (effective concentration, EC) values of wild type AcMNPV, vAP1OIT2, and vAPcmIT2 to the 3(superscript rd)-instar larvae of Trichoplusia ni (Hubner) were 1.00, 0.19, and 0.17 polyhedral inclusion bodies (PIBs)/mm^3, respectively. Compared to the very late promoter, the early promoter could shorten the effective time (ET50) by 14~17 h. Furthermore, for the 4(superscript th)-instar larvae, compared to the wild-type virus and vAP10G, a 19~23% and 30~33% reduction in ET50 were found using vAP10IT2 and vAPcmIT2, respectively. Field trials of these two viruses showed an effective paralysis of the infected larvae which then stopped feeding, resulting in a decrease in the leaf area eaten compared to those larvae infected with wild-type virus and another control virus vAP1OG, the GFP-expression recombinant AcMNPV. Scorpion toxin driven by the early p-PCm promoter shortened the time-to-paralysis of insect larvae, and thus provided an economical pest control advantage over that driven by the very late p10 promoter.