During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

Insecticidal crystal proteins of Bacillus thuringiensis.

characterized by its ability to produce crystalline inclusions during spor-ulation. TIhese inclusions consist of proteins exhibiting a highly specific insecticidal activity (reviewed in references 4 and 97). Many B. thuriuigiensis strains with different insect host spectra have been identified (9). TIhey ar-e classified into different serotypes or subspecies based on their flagellar antigens. Most strains are active against larvae of certain members of the Lepidoptera, but some show toxicity against dipterian (reviewed in refer-ence 22) or coleopteran (53) species. For several cryst.al-producing strains.

Insecticidal crystal proteins of Bacilllus thuringiensis

In recent years, the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), has become the most destructive insect of cruciferous plants throughout the world, and the annual cost for managing it is estimated to be US $I billion (168) Members of the plant family Cruciferae occur temperate and tropical climates and represent a diverse, widespread, and important plant group that includes cabbage, broccoli, cauliflower, collards, rapeseed, mustard, and Chinese cabbage, the most important vegetable crop grown in China (90), the most populous country in the world Although the diamondback moth is believed to have originated in the Mediterranean area (64), the source of some of our most important crucifers (185), diamondback moths now occur wherever cmcifers are grown, and this insect is believed to be the most universally distributed of all Lepidoptera (107) Absence of effective natural enemies, especially parasitoids, is believed to be a major cause of the diamondback moth’s pest status in most parts of the world (92) Lack of parasitoids in a particular area may have occurred because the diamondback moth is better able than its natural-enemy complex to become established in newly planted cmcifers Reports on the ability of diamondback moths to migrate long distances are numerous (19, 40, 54, 58, 108, 120,

http://web.entomology.cornell.edu/shelton/publications/pdf/Talekar-Shelton_1993_Ann_Rev_Ent_DBM.pdf

Biology, Ecology, and Management of the Diamondback Moth

▪ Abstract This review examines potential impacts of transgenic cultivars on insect population dynamics and evolution. Experience with classically bred, insecticidal cultivars has demonstrated that a solid understanding of both the target insect's ecology and the cultivar's performance under varied field conditions will be essential for predicting area-wide effects of transgenic cultivars on pest and natural enemy dynamics. This experience has also demonstrated the evolutionary capacity of pests for adaptive response to insecticidal traits in crops. Biochemical and genetic studies of insect adaptation to the Bacillus thuringiensis (Bt) toxins expressed by currently marketed transgenic cultivars indicate a high risk for rapid adaptation if these cultivars are misused. Theoretical and practical issues involved in implementing strategies to delay pest adaptation to insecticidal cultivars are reviewed. Emphasis is placed on examining the “high dose”/refuge strategy that has become the goal of industry and reg...

Sustainability of Transgenic Insecticidal Cultivars: Integrating Pest Genetics and Ecology

Binding studies were performed with two 125I-labeled Bacillus thuringiensis delta-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One delta-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other delta-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis delta-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

The biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryIC] were compared between the field population and a laboratory strain. The field population proved resistant (greater than 200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brush-border membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.

https://www.pnas.org/content/pnas/88/12/5119.full.pdf

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor.

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.

https://aem.asm.org/content/aem/62/2/564.full.pdf

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella).

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin.

Aims: To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis.



Methods and Results:  Following a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48·9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains.



Conclusions:  Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study.



Significance and Impact of the Study:  This is the first time that vip1 and vip2 genes have been identified by PCR-RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2009.04199.x

J. Van Rie

Papers

Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor.

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella).

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin.

Screening and identification of vip genes in Bacillus thuringiensis strains