A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

The fate of DNA and protein transgenic sequences in products derived from animals fed transgenic crops has recently raised public interest. Sensitive molecular tests targeting the Bt176 genetic construct and the transgenic Cry1Ab protein were developed to determine whether plant sequences, especially transgenic sequences, are present in animal products. A protocol for total DNA extraction and purification from cow whole blood samples was first drawn up and assessed by spiking with known amounts of DNA from Bt176 maize. The limit of detection for transgenic sequences (35S promoter and Bt176-specific junction sequence) was determined by both the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and the 5'-nuclease PCR assay. Four additional PCR systems were built to substantiate the results. The first detects a mono-copy maize-specific sequence (ADH promoter). Two others target multi-copy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The last one, used as a positive control, targets a mono-copy animal sequence (alpha(s1)-casein gene). Both methods detected a minimum spiking at 25 copies of Bt176 maize/mL in 10 mL whole blood samples. The sandwich ELISA kit used detected down to 1 ng transgenic Cry1Ab protein/mL spiked whole blood.

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5'junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman' chemistry.

Detection methods for genetically modified organisms (GMOs) are necessary for many applications, from seed purity assessment to compliance of food labeling in several countries. Numerous analytical methods are currently used or under development to support these needs. The currently used methods are bioassays and protein- and DNA-based detection protocols. To avoid discrepancy of results between such largely different methods and, for instance, the potential resulting legal actions, compatibility of the methods is urgently needed. Performance criteria of methods allow evaluation against a common standard. The more-common performance criteria for detection methods are precision, accuracy, sensitivity, and specificity, which together specifically address other terms used to describe the performance of a method, such as applicability, selectivity, calibration, trueness, precision, recovery, operating range, limit of quantitation, limit of detection, and ruggedness. Performance criteria should provide objective tools to accept or reject specific methods, to validate them, to ensure compatibility between validated methods, and be used on a routine basis to reject data outside an acceptable range of variability. When selecting a method of detection, it is also important to consider its applicability, its field of applications, and its limitations, by including factors such as its ability to detect the target analyte in a given matrix, the duration of the analyses, its cost effectiveness, and the necessary sample sizes for testing. Thus, the current GMO detection methods should be evaluated against a common set of performance criteria.

The phytopathogenic bacterium Xanthomonas campestris pv. pelargonii causes severe losses in vegetatively propagated Pelargonium cultivars. Symptoms are characterized by wilting of the plant, localized water-soaked lesions and stem rot (1) To overcome this problem horticulturists have to produce Xanthomonas-free planting material in insect-proof greenhouses after indexing the propagating stocks, which is expensive and time consuming. All commercial cultivars of pelargonium are susceptible to Xanthomonas and no source of resistance is known in the compatible Pelargonium germplasm. Thus, attempts were initiated to obtain Xanthomonas resistant plants by a transgenic approach.

Les récentes affaires de semences conventionnelles de colza et de maïs contaminées par des traces d’OGM ont à nouveau, s’il en était besoin, rappelé à notre bon souvenir les besoins en méthodes de détection et d’identification des OGM fiables et sensibles. Les développements se poursuivent, certes bien plus lentement qu’initialement espéré du fait du nombre important de facteurs à maîtriser et du caractère relativement inédit de ce travail. Concernant des matrices alimentaires variées, cette détection se heurte au manque de recul dans plusieurs domaines tels que celui de l’extraction et la purification d’acides nucléiques en quantité et en qualité. Bien que développées pour satisfaire aux besoins réglementaires européens concernant l’alimentation humaine, ces méthodes devraient être aisément applicables dans d’autres domaines concernés par les OGM (étude des transferts de gènes par flux pollinique, éventuels transferts horizontaux, suivi des acides nucléiques lors de l’ingestion...). Au-delà des OGM, les méthodes mises en œuvre devraient présenter un intérêt dans d’autres domaines comme la microbiologie, l’épidémiologie ou l’authentification des composants biologiques d’un produit.

Comment rester libre de choisir ce que l'on mange, OGM ou pas? L'idée d'un étiquetage obligatoire dépasse déjà largement les frontières de l'Europe. Mais construire un cadre réglementaire implique l'élaboration de solides méthodes de détection et d'identification des OGM, qui demeurent imparfaites.

The phytopathogenic fungus Botrytis cinerea can infect an extremely wide range of host plants (tomato, grapevine, strawberry, and flax) without apparent specialization. While studying genetic diversity in this fungus, we found an element which is present in multiple copies and dispersed throughout the genome of some of its isolates. DNA sequence analysis revealed that the element contained direct, long-terminal repeats (LTRs) of 596 bp whose features were characteristic of retroviral and retrotransposon LTRs. Within the element, we identified an open reading frame with sequences homologous to the reverse transcriptase and RNase H domains of retroelement pol genes. We concluded that the element we had identified was a retroelement and named it Boty. By comparing its open reading frame with sequences from other retroelements, we found that Boty is related to the gypsy family of retrotransposons. Boty was present in numerous strains isolated from grapes and tomatoes but not in isolates from lentils. We propose that Boty-containing and Boty-deficient groups represent two lineages in the population of B. cinerea.

In Erwinia chrysanthemi, conditions of iron starvation initiate production of a catechol-type siderophore and enhance production of three outer membrane polypeptides. Twenty-two mutants affected in the different stages of this iron assimilation system were isolated by mini-Mu insertion mutagenesis. All of them failed to induce systemic soft rot on axenically grown Saintpaulia plants. From the siderophore auxotrophs and the iron uptake mutants, clones having recovered the missing function(s) were isolated by using the in vivo cloning vector pULB113 (RP4::mini-Mu). An R-prime plasmid containing a ca. 35.5-kilobase-pair DNA insert was identified. Restoration of the iron functions restored partially, if not completely, the virulence of the parental strain.

Mutants in each of the pel genes (pelA, pelb, pelC, pelD and pelE) which encode the five pectate lyase isozymes (PLa, PLb, PLc, PLd or PLe) of Erwinia chrysanthemi strain 3937, a pathogen of Saintpaulia ionantha, were obtained by insertion directed mutagenesis. The five mutant strains were tested for pathogenicity by inoculating into mini-saintpaulia plants. The acidic PLa and the basic PLd and PLe isozymes are the most important for pathogenicity. The mutants which were unable to produce the neutral, PLb or PLc, isozymes behaved like the wild type strain and macerated the whole miniplant. In contrast the PelA− and PelD− mutant strains were less virulent than the wild type strain, producing fewer systemically infected plants and much more limited maceration. The PelE− mutant strain was non-invasive, with maceration and bacteria being confined to the one leaf or the part of the leaf blade which was inoculated, suggesting that plant defence reactions had taken place.

Erwinia chrysanthemi strains 3937 and B374 were shown to secrete 5 major pectate lyase (PL) isozymes with isoelectric points ranging from 4.4 to more than 9.5. To determine the number of pel genes encoding the PL and to study them, two gene libraries were constructed in Escherichia coli, using the Lambda vector L47-1 and the broad host range mobilizable cosmid pMMB33. These gene libraries provided several clones producing the PL. The pel genes of the strain B374 were also cloned by an in vivo procedure promoted by the pULB113 plasmid. Subcloning experiments have shown the existence of 5 pel genes, each with its own promoter, clustered in two regions on the chromosome of E. chrysanthemi. The restriction maps of these regions from 3937 and B374 were similar. In addition, a pectin methylesterase gene of 3937 was found closely linked to one pel gene. Different pel genes were mutagenized in E. coli using a mini-Mu phage able to promote translational fusions with lacZ. One fusion in the pelC gene was introduced by marker exchange into the chromosome of E. chrysanthemi 3937, giving rise to a strain lacking the PLc isozyme and allowing study of the regulation of pelC.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia colilacZ coding region into the hybrid plasmids were made in vivo using mini-Muduction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result on E. coli in lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47‐1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx‐positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL‐positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.

The pelC gene, which encodes one of the five major pectate lyase (PL) isoenzymes in Erwinia chrysanthemi 3937, designated PLc, was subcloned from a hybrid lambda phage into a pBR322 derivative and mutagenized with a mini-Mu-lacZ transposable element able to form fusions to the lacZ gene. One plasmid (pAD1) which had an inactivated pelC gene and a Lac+ phenotype was selected in Escherichia coli. This plasmid was introduced into Erwinia chrysanthemi, and the pelC::mini-Mu insertion was substituted for the chromosomal allele by homologous recombination. This strain lacks the PLc isoenzyme. This Erwinia chrysanthemi strain has a Lac+ phenotype that is inducible by polygalacturonate, as are the wild-type PL activities.