The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins.

Background Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.Methodology/Principal FindingsBinding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that 125I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances 125I-Cry35Ab1 specific binding, and that 125I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with 125I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with 125I-Cry3Aa, or 125I-Cry8Ba.Conclusions/SignificanceCombining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.

The poliovirus RNA polymerase, 3DPOI, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C.

The ribosome-inactivating protein (RIP) from maize (Zea mays L.) is unusual in that it i s produced in the endosperm as an inactive pro-form, also known as b-32, which can be converted by limited proteolysis to a two-chain active form, cup RIP. lmmunological analysis of seed extracts from a variety of species related to maize showed that pro/cup forms of RIP are not unique to maize but are also found in other members of the Panicoideae, including Tripacum and sorghum. Ribosomes isolated from maize were quite resistant to both purified pro-and ap maize RIPs, whereas they were highly susceptible to the RIP from pokeweed. This suggests that the production of an inactive pro-RIP is nota mechanism to protect the plant's own ribosomes from deleterious action of the ap RIP. RIP derivatives with various pro-segments removed were expressed at high levels in Fscherichia coli. Measurement of their activity before and after treatment with subtilisin Carlsberg clearly identified the 25-amino acid intradomain insertion, rather than the N-or Cterminal extensions, as the major element responsible for suppression of enzymatic activity. A RIP with all three processed regions deleted had activity close to that of the native a@ form.Many plants produce RIPs, a unique class of proteins that are exceptionally potent inhibitors of eukaryotic protein synthesis (Stirpe et al., 1992;. RIPs catalytically inactivate eukaryotic, and in some cases prokaryotic, ribosomes by cleaving the N-glycosyl bond of a single specific adenine residue in the ribosomal RNA (A,,,4 in the case of rat liver ribosomes; Endo et al., 1988). Depending on the species of plant, RIPs can be expressed in leaves, roots, sap, or seeds, often at very high levels. The physiological function of RIPs is at present unclear, although evidence is accumulating that they have a role in plant defense. Leah et al. (1991) have shown that a RIP from barley seeds inhibits the growth of fungal pathogens, particularly when combined with seed chitinases and glucanases. A defensive role against the mechanical transmission of plant viruses has also been proposed Bonness et al., 1994). These results have been extended by the observation that transgenic tobacco plants expressing a RIP from barley exhibit increased tolerance to fungal infection (Logemann et al., 1992), and plants exCorresponding author; fax 1-317-337-3228. CV4 7AL, United Kingdom (M.H.)pressing a RIP from pokeweed have decreased susceptibility to vira1 infection (Lodge et al., 1993).RIPs have been classified into two types (Stirpe et al., 1992): type-1 RIPs are the most prevalent; over 40 have been described. They are typically single-chain, basic polypeptides of 25 to 32 kD with relatively low toxicity to intact cells because they do not readily cross cellular membranes. The rarer type-2 RIPs have arisen from a gene fusion between a type-1 RIP domain and a lectin-like domain. The lectin domain (or B chain) can bind to cell surfaces and mediate the delivery of the RIP (or A chain) into the cytosol of the cell. The RIP A ch...

BackgroundThe Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.ResultsThe two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.ConclusionsCry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0295-9) contains supplementary material, which is available to authorized users.