Roger Frutos

Managing Insect Resistance to Plants Producing Bacillus thuringiensis Toxins

ABSTRACT: Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance...

Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner

Abstract A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype. The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein.

Comparative Genomic Analysis of Three Strains of Ehrlichia ruminantium Reveals an Active Process of Genome Size Plasticity

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

Abstract. After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the β-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.

A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype.

ABSTRACT Human respiratory syncytial virus (HRSV) is the leading cause of hospitalization of children aged <5 years due to respiratory illness in industrialized countries, and pneumonia is the leading cause of mortality among children aged <5 years worldwide. Although HRSV was first identified in 1956, a preventative vaccine has yet to be developed. Here we report the results of the first study to investigate the circulation and genetic diversity of HRSV in Cambodia among an all-ages population over 5 consecutive years. The incidences of HRSV infection among all-ages outpatient and hospitalized populations were equivalent, at 9.5% and 8.2%, respectively. Infection was most prevalent among children aged <5 years, with bronchiolitis being the most frequently observed clinical syndrome in the same age group. Circulation of HRSV was seasonal, typically coinciding with the rainy season between July and November annually. Strains belonging to HRSV groups A and B were detected with equivalent frequencies; however, we observed a potentially biennial shift in the predominant circulating HRSV genotype. The majority of HRSV group B strains belonged to the recently described BA genotype, with the exception of 10 strains classified as belonging to a novel HRSV group B genotype, SAB4, first reported here.

Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS-NRPS gene cluster for toxin production in the genus Xanthomonas.

Novel vip3-related protein from Bacillus thuringiensis

ABSTRACT A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Novel synthetic Bacillus thuringiensiscry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize

Abstract  In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins, a fully modified Bacillus thuringiensiscry1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two target insects. Molecular analyses confirmed the cry genes integration, the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations.

Distribution of cryl, cryll and cryV Genes within Bacillus thuringiensis Isolates from Spain

Summary Using a PCR-based approach, a collection of 223 isolates of Bacillus thuringiensis from Spain was screened for the presence of cry genes belonging to three families. Genes from the cryI , cryII and cryV families were found in 54%, 42%, and 66% of the isolates, respectively. Only 23% of the isolates did not show the presence of any of the genes tested. Frequencies of these genes were compared in isolates from soil samples and from samples of cereal stores and mills, being this higher in the latter. Specific primers were used to detect cryIA(a), cryIA(b), cryIA(c), cryIA(d), cryIA(e), cryIB, cryIC, cryID, cryIE, cryIF and cryIG genes. Within the cryI family, the most frequent gene was cryIA(c) (62%), followed by cryIA(a), cryID, cryIC and cryIA(b) (49, 43, 35, and 34%, respectively). A high frequency of joint occurrence was observed for cryIC and cryID ; the latter was present in 93% of the isolates containing cryIC . A random sample of 97 isolates was tested for toxicity against the insect pests Plutella xylostella and Spodoptera exigua . Among the isolates showing toxicity, the most common gene combination was cryIA-cryIC-cryID-cryII-cryV . Although in most cases toxicity could be related to gene content, in some others toxicity was unexpected according to the results obtained by PCR. We found no apparent relationship between gene content in our isolates and the serovar to which they belong.