Huarong Li

Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris)

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.

Virus-derived genes for insect-resistant transgenic plants.

Insect viruses have evolved to counter physiological barriers to infection presented by the host insect. For the Lepidoptera (butterflies and moths), these barriers include (1) the peritrophic membrane (PM) lining the gut, which presents a physical barrier to virus infection of the midgut epithelial cells, (2) the basement membrane (BM) that overlies the gut thereby restricting secondary infection of other tissues, and (3) the immune system of the host insect. Hence, insect viruses provide a resource for genes that disrupt host physiology in a specific manner, and these genes in turn serve as a resource both for the study of physiological processes, and for disruption of these processes for pest management purposes. There are several examples of the application of genes used by an insect virus to overcome the PM barrier for production of insect-resistant transgenic plants. There are other examples of intrahemocoelic effectors, such as BM-degrading proteases that can only be used with an appropriate system for delivery of the agent from the gut into the hemocoel (body cavity) of the insect pest. In this chapter, we describe (1) baculovirus- and entomopoxvirus-derived genes that alter the physiology of the host insect, (2) use of these and homologous genes for production of insect-resistant transgenic plants, (3) other viral genes that have potential for use in development of insect-resistant transgenic plants, and (4) the use of plant lectins for delivery of intrahemocoelic toxins from transgenic plants. Plant expression of polydnavirus-derived genes is described by Gill et al. (this volume, pp. 393-426).

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.

Protocols for microapplicator-assisted infection of lepidopteran larvae with baculovirus.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be infected with microapplicator techniques in the gut with baculovirus polyhedra and in the hemolymph with budded virus. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents. Formulation and application of baculoviruses for pest control purposes are described elsewhere.

Histopathological Effects of Bt and TcdA Insecticidal Proteins on the Midgut Epithelium of Western Corn Rootworm Larvae (Diabrotica virgifera virgifera)

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is a major corn pest in the United States, causing annual losses of over

Impact of a basement membrane-degrading protease on dissemination and secondary infection of Autographa californica multiple nucleopolyhedrovirus in Heliothis virescens (Fabricus).

1 billion. One approach to protect against crop loss by this insect is the use of transgenic corn hybrids expressing one or more crystal (Cry) proteins derived from Bacillus thuringiensis. Cry34Ab1 and Cry35Ab1 together comprise a binary insecticidal toxin with specific activity against WCR. These proteins have been developed as insect resistance traits in commercialized corn hybrids resistant to WCR feeding damage. Cry34/35Ab1 is a pore forming toxin, but the specific effects of Cry34/35Ab1 on WCR cells and tissues have not been well characterized microscopically, and the overall histopathology is poorly understood. Using high-resolution resin-based histopathology methods, the effects of Cry34/35Ab1 as well as Cry3Aa1, Cry6Aa1, and the Photorhabdus toxin complex protein TcdA have been directly visualized and documented. Clear symptoms of intoxication were observed for all insecticidal proteins tested, including swelling and sloughing of enterocytes, constriction of midgut circular muscles, stem cell activation, and obstruction of the midgut lumen. These data demonstrate the effects of these insecticidal proteins on WCR midgut cells, and the collective response of the midgut to intoxication. Taken together, these results advance our understanding of the insect cell biology and pathology of these insecticidal proteins, which should further the field of insect resistance traits and corn rootworm management.