Increasing Efficiency of Bioassays: Evaluating Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae)

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera. Bacillus thuringiensis, which is a Gram-positive bacterium, is known for its specific toxicity toward insect pests (1). This toxicity is largely attributed to the insecticidal crystal proteins encoded by the cry genes (2-4). The cry1-type genes of B. thuringiensis are highly toxic to lepidopteran pests, and some genes have been used to develop plants with resistance to insect pests (5-7). Because of their potential application and commercial value, much research has focused on the discovery of novel cry1 genes; to date, approximately 258 cry1 genes have been cloned and named, and 50 distinct holotypes have been classified (http://www.lifesci .sussex.ac.uk/home/Neil_Crickmore/Bt/). Previous research has revealed that cry1 genes are typically found in clusters; for example, B. thuringiensis strains HD12 and HD525 contain at least four different cry1-type genes (8).PCR is a simple and convenient investigative method and has been widely used to identify the vast variety of cry genes by the use of different primers (9-12). PCR-restriction fragment length polymorphism (PCR-RFLP) is a modified PCR technique that is generally used for the identification of known and unknown cry1-type genes (except for cry1I-type genes), and of parts of the cry7-type and cry9-type genes, according to the fragment lengths of digested PCR-amplified products described by Kuo and Chak (8). Some primers have been designed for the identification of cry1-type genes (13) and cry8-type genes (14) based on the PCR-RFLP method. However, it is becoming difficult to identify novel cry1-type genes using the PCR-RFLP method (8) because of the increase in the numbers of cry1-type genes.To resolve this problem, an improved PCR-RFLP method was designed to directly identify the fourth class of cry1-type genes by dividing...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

Shu

Liu

Zhou

et al. 2013

“…Cry1 proteins that are active against lepidopteran insects are produced as crystalline parasporal inclusions during sporulation. Generally, the crystals are composed of protoxins of approximately 130 kDa, but cry1I-type genes are usually silent genes capable of encoding a protein of about 81 kDa in B. thuringiensis strains (9,13,21,24). We decided to screen B. thuringiensis isolates for cry1I genes with the aim of finding novel cry1I genes, which could encode insecticidal proteins toxic to insensitive or resistant insect pests.…”

mentioning

confidence: 99%

Identification of cry1I- Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry1I- Type Gene

Song

Zhang

et al. 2003

108

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis ␦-endotoxin nomenclature committee.Crystal proteins from the gram-positive spore-forming bacterium Bacillus thuringiensis are toxic to a wide variety of insects that are economically important as pests. Many different genes encoding the B. thuringiensis endotoxin have been isolated and characterized. The genes have been classified as cry1 to cry40, cyt1, and cyt2 and are ranked according to their homology (10, 21; see also the B. thuringiensis toxin nomenclature website at http://www.biols.susx.ac.uk/home/Neil_Crickmore /Bt/). Cry1 proteins that are active against lepidopteran insects are produced as crystalline parasporal inclusions during sporulation. Generally, the crystals are composed of protoxins of approximately 130 kDa, but cry1I-type genes are usually silent genes capable of encoding a protein of about 81 kDa in B. thuringiensis strains (9,13,21,24). We decided to screen B. thuringiensis isolates for cry1I genes with the aim of finding novel cry1I genes, which could encode insecticidal proteins toxic to insensitive or resistant insect pests.This screening approach included the development of an analysis protocol based on PCR-restriction fragment length polymorphism (RFLP). PCR-based methods have been developed to detect different cry genes from B. thuringiensis strains (1-8, 11, 13, 15, 17, 23). More than 80 primer pairs have been designed to identify entire groups and individual cry genes (19). Several specific primers and probes for Southern blotting were designed to detect cry1I-type genes. A wide distribution of cry1I-type genes among many different B. thuringiensis strains has been reported (13,22,25). However, the list of cry genes is increasing, and novel PCR primers are needed in order to identify some of the recently described genes (5).The present study establishes a PCR-RFLP method for identify...

“…Each concentration was tested with 24 insects that were individually placed in each well, and the amount of the surviving insects was recorded after 7 days. Toxicity against B. mori neonate larvae was conducted on mulberry leaves using the leaf-dip bioassay (45), and the number of surviving larvae was recorded after 2 days. The bioassays were repeated three times using five different concentrations of the proteins.…”

Section: Methodsmentioning

confidence: 99%

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Zhou

Liu

Liang

et al. 2017

Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.