Stephanie L. Burton

Insect resistance conferred by 283-kDa Photorhabdus luminescens protein TcdA in Arabidopsis thaliana

The tcdA gene of Photorhabdus luminescens encodes a 283-kDa protein, toxin A, that is highly toxic to a variety of insects, including some agriculturally important pests. We tested the efficacy of transgenic toxin A in Arabidopsis thaliana for control of feeding insects. Plants with toxin A expression above about 700 ng/mg of extractable protein were highly toxic to tobacco hornworm (Manduca sexta). Toxin A isolated from transgenic plants also strongly inhibited growth of the southern corn rootworm (Diabrotica undecimpunctata howardi). Addition of 5′ and 3′ untranslated regions of a tobacco osmotin gene (osm) increased toxin A production 10-fold and recovery of insect-resistant lines 12-fold. In the best line, high toxin A expression and insect resistance were maintained for at least five generations in all progeny. The intact tcdA mRNA represents the largest effective transgenic transcript produced in plants to date. These results may open a new route to transgenic pest control in agriculture.

Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus STRUCTURE AND PORE FORMATION

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ∼1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ∼30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.

Insecticidal activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) and other lepidopteran pests.

ABSTRACT Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.