Genetics‐based methods for agricultural insect pest management

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Insecticidal effects of selected biological control agents on the larvae of Plutella xylostella (Lepidoptera: Plutellidae)

To identify a more effective and safe biological control agent against a common cabbage pest, Plutella xylostella (L.) (Lepidoptera: Plutellidae), the insecticidal effects of selected biological agents were evaluated. The highest insecticidal effects determined were 100, 73.5, 45.5, 47 and 55.3% using toxin HD‐1 (isolated from the Harry Dumagae strain of Bacillus thuringiensis), toxin BTS‐1 (isolated from the tenebrionis strain of B. thuringiensis), B. thuringiensis Berliner, B. thuringiensis israelensis and B. thuringiensis kurstaki, respectively.     

Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes

Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight 'toxin complexes' (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of approximately 40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants.     

CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.

     

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis

Insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are widely used as environmentally friendly insecticides. As the only known Cry protein with insecticidal activity against Locusta migratoria manilensis, a locust subspecies that causes extensive destruction of crops, the Cry7Ca1 protein from Bt strain BTH‐13 identified in our previous study is of particular interest to locust prevention and control. However, the three‐dimensional structure of Cry7Ca1 toxin (the active form of the Cry7Ca1 protein) and the mechanisms of the Cry7Ca1 insecticidal specificity remain largely elusive. Here, we report a 2.3 Å crystal structure of the Cry7Ca1 toxin and carry out a systematic comparison of all available Cry toxins structures. A cluster of six loops in Cry toxin domain II, named Apex here, are the most variable structural elements and were documented to contribute in insecticidal specificity. The Cry7Ca1 toxin Apex loops are different from those of other Cry toxins in length, conformation, and sequence. Electrostatic potential analysis further revealed that Cry7Ca1 is the only structure‐available Cry toxin that does not have a high contrast of surface electrostatic potentials in the Apex. We further suggest that the L1/L2 loops in the center of the Cry7Ca1 Apex may be worthy of attention in future efforts to unravel the Cry7Ca1 insecticidal specificity as they exhibit unique features not found in the corresponding regions of other Cry toxins. Our work highlights the uniqueness of the Apex in the Cry7Ca1 toxin and may assist exploration of the insecticidal mechanism of the Cry7Ca1 against Locusta migratoria manilensis.     

Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp

Aims: Some Cry proteins produced by the soil bacterium Bacillus thuringiensis (Bt) or by transgenic Bt plants persist in agricultural soils for an extended period of time, which may pose a hazard for nontarget soil organisms. The aims of our study were to screen for soil fungi capable of degrading the Cry1Ac toxin and to identify the mechanisms that lead to the inactivation of this protein. Methods and Results: Of the eight fungal strains screened, only one, Chrysosporium sp., was found to produce extracellular proteases capable of degrading the 66‐kDa Cry1Ac at the N‐terminal end of amino acid 125 (alanine). The proteolytic products of the Cry1Ac toxin did not exhibit any insecticidal activity against Helicoverpa armigera, in contrast to its high toxicity exhibited in the native form. Conclusions: Proteases elaborated by the Chrysosporium sp. degrade the Cry1Ac toxin in a way that it looses its insecticidal activity against H. armigera. Significance and Impact of the Study:  Chrysosporium sp., a specific soil micro‐organism capable of producing proteases that degrade the Cry1Ac toxin into inactive products under controlled conditions is being reported for the first time. Application of this observation needs to be further tested in field conditions.     

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells

Photorhabdus luminescens Tc toxins consist of the cell‐binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP‐ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP‐ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP‐ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP‐ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.     

Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

The tc genes of Photorhabdus: a growing family

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.     

Elicitin genes in <Emphasis Type="Italic">Phytophthora infestans</Emphasis> are clustered and interspersed with various transposon-like elements

Sequencing and annotation of a contiguous stretch of genomic DNA (112.3 kb) from the oomycete plant pathogen Phytophthora infestans revealed the order, spacing and genomic context of four members of the elicitin (inf) gene family. Analysis of the GC content at the third codon position (GC3) of six genes encoded in the region, and a set of randomly selected coding regions as well as random genomic regions, showed that a high GC3 value is a general feature of Phytophthora genes that can be exploited to optimize gene prediction programs for Phytophthora species. At least one-third of the annotated 112.3-kb P. infestans sequence consisted of transposons or transposon-like elements. The most prominent were four Tc3/gypsy and Tc1/copia type retrotransposons and three DNA transposons that belong to the Tc1/mariner, Pogo and PiggyBac groups, respectively. Comparative analysis of other available genomic sequences suggests that transposable elements are highly heterogeneous and ubiquitous in the P. infestans genome.Electronic Supplementary Material Supplementary material is available for this article at     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

A Cry1Ac Toxin Variant Generated by Directed Evolution has Enhanced Toxicity against Lepidopteran Insects

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.     

Isolation,cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region ‘2.3 kbp’ of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC₅₀ was found to be 142.4 µ/mL while the LC₅₀ was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.     

The crystal δ-endotoxins of Bacillus thuringiensis: Models for their mechanism of action on the insect gut

The crystal δ-endotoxins of Bacillus thuringiensis (Bt) are a family of insecticidal proteins which have been known for some time to kill insects by lysing their gut epithelial cells, but the precise molecular mechanism of toxicity has remained elusive. The recent publication of the crystal structure of a Bt δ-endotoxin has made it possible for us to model the molecular events that occur as the toxin binds to its receptor and inserts into the membrane to form a pore. Using our knowledge of insect gut physiology, we can also predict the effect on the insect of the formation of a toxic pore. We present a new model to explain the events that occur in the insect gut during toxin action.