Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, N.; Peng, Donghai; Ruan, Lifang; Sun, Ming

doi:10.1128/aem.00340-12

Cited by 80 publications

(83 citation statements)

References 48 publications

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“…While these methods proved to be useful, they have certain limitations. Therefore, whole-genome sequencing and genome mining become reasonable alternatives for the identification of novel Cry toxins (8,35). Applying these techniques, we found that three different plasmids encode three Cry-like proteins.…”

Section: Discussionmentioning

confidence: 99%

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Iatsenko

Boichenko

Sommer

2014

Appl Environ Microbiol

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Bacillus thuringiensis has been widely used as a biopesticide, primarily for the control of insect pests, but some B. thuringiensis strains specifically target nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using Caenorhabditis elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in Escherichia coli and fed to C. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins on C. elegans are synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC 50 s) for Cry21Fa1 and Cry21Ha1 to be 13.6 g/ml and 23.9 g/ml, respectively, which are comparable to the LC 50 of nematicidal Cry5B. Finally, we found that signaling pathways which protect C. elegans against Cry5B toxin are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.

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Section: Discussionmentioning

confidence: 99%

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Iatsenko

Boichenko

Sommer

2014

Appl Environ Microbiol

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“…To investigate the virulence factors that account for this high toxicity against nematodes and to exploit additional toxin genes that may be used as biopesticides and/or in transgenic crops, we sequenced the B. thuringiensis Sbt003 genome (23). Eight potential crystal protein genes were identified via BtToxin_scanner (24), of which only four full-length toxin genes were obtained by domain structure analysis (23). Meanwhile, the genome of Sbt003 revealed a series of potential virulence factor-encoding genes based on the MvirDB database via MP3 bioinformatic prediction (see Table S1 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance

Geng

Liu

et al. 2017

Appl Environ Microbiol

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Cry proteins of Bacillus thuringiensis (Bt) have been successfully used as biopesticides and in transgenic crops throughout the world. However, resources against the most serious agricultural pathogens, plant root-knot nematodes, are limited. The genomes of several highly nematicidal virulent Bt strains from our laboratory have been sequenced, facilitating the identification of novel Cry proteins and other virulence factors. We identified two novel Cry proteins, Cry5Ca1 and Cry5Da1, that exhibit high toxicity against Meloidogyne incognita. Using the Caenorhabditis elegans model, the two Cry5 toxins were shown to negatively affect nematode life span, fertility, and survival. The 50% lethal concentrations (LC 50 s) of Cry5Ca1 and Cry5Da1 were 57.22 g/ml and 36.69 g/ml, respectively. Moreover, a synergistic effect (synergism factor, 1.61 to 2.04) was observed for nematicidal toxicity of Cry5Ca1 and Cry5Da1, which is accordant with the phylogenetic results suggesting that domain II of the two novel Cry5 toxins evolved into two independent clades. Through comparison of the depressed degree of toxicity in the ␤-methylgalactoside detoxification test, we found that the novel toxin Cry5D possesses a different galactosebinding epitope; meanwhile, the finding that Cry5D does not share a motif (GXXXE) in the corresponding loop of domain II with Cry5B could explain the different galactose binding performance. Additionally, low-level cross-resistance of C. elegans bre mutant strains was evident between Cry5B and Cry5D. These results suggest that Cry5D can be used as an alternative to delay the potential resistance of nematodes to Cry5B. IMPORTANCE Although proper gene resources for Bt crops against the most serious agricultural pathogens, plant root-knot nematodes, are limited, we have identified two novel nematicidal toxins, Cry5Ca1 and Cry5Da1, against M. incognita, which have supplied more gene candidates for Bt crops designed against nematodes. Moreover, the association of the dissimilarity between Cry5Da1 and Cry5Ba1 and their low crossresistance can be attributed not only to a low sequence similarity of domain II but also to the structural difference of the key motif and receptor-binding epitope in the loops. This association facilitates the selection of a proper candidate for the prospective design of pyramided Bt crops that can delay potential resistance.KEYWORDS Bacillus thuringiensis, Caenorhabditis elegans, Cry proteins, bre resistance, nematicidal T he agricultural economic damage caused by more than 4,100 species of plantparasitic nematodes (PPNs) has been estimated to cost US$80 billion per year (1, 2), and a large proportion of this cost is due to the serious damage caused by the plant-parasitic nematode Meloidogyne incognita (3). Although many nematophagous

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“…Cry proteins are encoded by cry genes that are frequently carried on plasmids; to date, nearly 300 cry genes have been identified and classified into 51 groups and subgroups on the basis of amino acid sequence similarity (Ye et al, 2012).…”

Section: Molecular Identification Of Cry Genementioning

confidence: 99%

Isolation and Molecular Characterization of Cry Gene for Bacillus thuringiensis Isolated from Soil of Gaza Strip

Dagga¹,

Aziz²,

Elmanama³

et al. 2016

Int.J.Curr.Microbiol.App.Sci

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Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

Cited by 80 publications

References 48 publications

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance

Isolation and Molecular Characterization of Cry Gene for Bacillus thuringiensis Isolated from Soil of Gaza Strip

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