Production of transgenic pear plants expressing ferritin gene with the aim to reduce fire blight susceptibility

International audienceFire blight caused by the bacteria Erwinia amylovora is one of the most important diseases of pear. This causal agent produces a siderophore (desferrioxamine), which was identified as one of its virulence factors. This protein enables the bacteria to overcome conditions of iron limitation encountered in host tissues, and may also protect the bacteria against active oxygen species. Previous experiments indicate that the use of an iron chelator protein, encoded by the bovine lactoferrin gene, reduces fire blight symptoms in some transgenic pear clones. The aim of the present work is to test the ability of a more efficient iron chelator, plant ferritin, to reduce fire blight susceptibility in pear. In the literature, ferritin genes have been overexpressed under the control of a constitutive promoter in different transgenic plant species for various purposes. In several cases, the constitutive expression of ferritin produced negative effects such as reduced growth and chlorophyll content. Therefore, we decided to place the exogenous ferritin gene from pea under the control of a pathogen inducible promoter (sgd24) in comparison with a constitutive promoter (CaMV 35S). Two pear varieties, 'Conference' (CF) and 'Passe-Crassane' (PC) were transformed using both constructs. Transformation rates depended on variety and construct. They were respectively of 12 and 4.3% for CF and PC using the sgd24-ferritin construct. Only PC was transformed with the 35S-ferritin construct, with a transformation rate of 2%. First analyses of the transgenic clones by RT-PCR showed the expression of pea ferritin in both constructs and in all clones. The transgenic clones were acclimatized in greenhouse and exhibited normal growth. Quantification of ferritin gene expression, ferritin protein accumulation, and evaluation of fire blight resistance are underway

Generación multilingüe de boletines meteorológicos

En este artículo presentamos el software de generación de boletines meteorológicos MultiMeteo, que fue realizado en el marco del proyecto europeo del mismo nombre cuyos miembros usuarios son Météo-France, Instituto Nacional de Meteorología (España), Institut Royal Météorologique (Bélgica) y Zentralanstalt für Meteorologie und Geodynamik (Austria). Su objetivo es generar boletines en varias lenguas (inglés, francés, español, alemán y neerlandés) y varios estilos a partir de los diferentes formatos de datos de los institutos europeos. MultiMeteo utiliza AlethGen como motor de generación. Después de una panorámica de la arquitectura general, nos concentramos en la generación multilingüe.In this article we present the "MultiMeteo" software, used to generate weather forecasts. It was developed in the context of the European project of the same name for Météo-France, Instituto Nacional de Meteorología (Spain), Institut Royal Météorologique (Belgium) and Zentralanstalt für Meteorologie und Geodynamik (Austria). Its aim is to generate forecasts in several languages (English, French, Spanish, German and Dutch) and in several styles from data provided by these Met offices in various formats. The generation engine used by MultiMeteo is AlethGen. After briefly introducing the overall architecture, we describe in greater detail the multilingual generation process

Multilingual summarization by integrating linguistic resources in the MLIS-MUSI project

In this paper we will illustrate the approach to multilingual automatic abstract production adopted by the EU-sponsored project MLIS-MUSI. Although a small scale research project, MUSI has tried to tackle the challenges set by multilingual summarization by adopting an original approach based on the definition of a shared ontology and representation language, and on the reuse of existing linguistic resources. MUSI combines a linguistic-based module for relevant sentence extraction and a concept-based component to generate multilingual summaries. 1

Iron homeostasis and fire blight susceptibility in transgenic pear plants overexpressing a pea ferritin gene

UMR 1334 AGAP : Equipe AFEF ‘Architecture et Fonctionnement des Espèces fruitières’ ; Team AFFS ‘Architecture and Functioning of Fruit Species’ Contact: [email protected], [email protected] Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceThe bacterial pathogen Erwinia amylovora causes the devastating disease known as fire blight in some rosaceous plants including apple and pear. One of the pathogenicity factors affecting fire blight development is the production of a siderophore, desferrioxamine, which overcomes the limiting conditions in plant tissues and also protects bacteria against active oxygen species. In this paper we examine the effect of an iron chelator protein encoded by the pea ferritin gene on the fire blight susceptibility of pear (Pyrus communis). Transgenic pear clones expressing this gene controlled either by the constitutive promoter CaMV 355 or by the inducible promoter sgd24 promoter were produced. The transgenic clones produced were analysed by Q-RT-PCR to determine the level of expression of the pea transgene. A pathogen-inducible pattern of expression of the pea transgene was observed in sgd24-promoter transformants. Adaptation to iron deficiency in vitro was tested in some transgenic clones and different iron metabolism parameters were measured. No strong effect on iron and chlorophyll content, root reductase activity and fire blight susceptibility was detected in the transgenic lines tested. No transformants showed a significant reduction in susceptibility to fire blight in greenhouse conditions when inoculated with E. amylovor

Characterization of transgenic apples expressing the HrpN gene

International audienceFire blight is the major bacterial disease of Maloideae caused by the necrogenic bacterium Erwinia amylovora, which pathogenicity requires a functional type III protein secretion system (TTSS). The protein harpin NEa is secreted through the TTSS and targeted to the intercellular spaces of plant tissues where it acts as an elicitor of plant defense reactions. In order to create novel mechanisms for fire blight resistance in apple, we have generated transgenic apples expressing the hrpN gene from E. amylovora under the control of the constitutive CaMV35S or the pathogen-inducible tobacco Str246C promoters, with or without the tobacco PR1 gene secretion signal peptide. The characterization of these harpin NEa transgenic apples was carried out by: (1) quantification of the constitutive and induced expression of the transgene (by Q-RT-PCR); (2) in planta localization of the harpin NEa protein; (3) evaluation of fire blight resistance after greenhouse inoculation; (4) targeted transcriptomic analysis to identify defenses induced by the presence of the transgene

Elimination of the nptII marker gene in transgenic apple and pear with a chemically inducible R/Rs recombinase

The efficient production of marker-free transgenic plants is still a challenge in most fruit species even though such plants are a necessary component of many "new breeding technologies", particularly cis- and intragenesis. Marker-free plant production is also necessary for the successive stacking of genes in an elite fruit transgenic line. Here, we used a R/Rs site-specific recombinase that is post-translationally regulated by dexamethasone through fusion with a ligand-binding domain for this hormone, and a bi-functional selectable marker gene coding for a cytosine deaminase/neomycin transferase (codA-nptII) protein; this enabled a first step of positive kanamycin selection, followed by a second step of negative 5-fluorocytosine selection. The aim of our study was to optimize this system on the apple cv. Galaxy and on the pear cv. Conference by conducting a detailed study of the effects of dexamethasone and 5-fluorocytosine treatments, and by comparing an early versus a delayed selection strategy. We were able to produce marker-free transgenic pear plants for the first time, and confirm the feasibility of producing marker-free transgenic apple plants using a chemically inducible recombinase system. We recommend the use of an early selection strategy for the pear cv. Conference and a delayed selection strategy for the apple cv. Galaxy

Feasibility of alternative selection methods for transgenic apple and pear using the detoxification gene Vr-ERE

International audienceEutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. An eutypine detoxifying gene (Vr-ERE) encoding an NADPH-dependent aldehyde reductase converts eutypine into the corresponding alcohol, eutypinol, a non-toxic form of the toxin. A variety of phytotoxic compounds containing an aldehyde group can act as substrates for this enzyme, therefore opening the possibility to use Vr-ERE as an alternative selection system for plant transformation. Our preliminary experiments with apple and pear have demonstrated the following: 1) among the various substrates of VR-ERE, benzaldehyde (BD), a naturally occurring compound, is able to inhibit adventitious bud regeneration from apple as well as pear in vitro leaves, at concentrations ranging from 1 to 1.5 mM; 2) constitutive expression of Vr-ERE in transgenic ‘Greensleeves’ apple does not provoke any abnormal phenotype after one year of growth in greenhouse, but aldehyde reductase activity is reduced in all transgenic clones; 3) production of transgenic pear using BD instead of kanamycin as selection pressure is feasible, but the efficiency of the selection pressure seems low. In conclusion, the efficiency of Vr-ERE as a selective gene to produce apple and pear transgenic plants is not yet proven and further research is neede

Effect of ectopic expression of the eutypine detoxifying gene Vr-ERE in transgenic apple plants

UMR 1334 AGAP : Equipe AFEF ‘Architecture et Fonctionnement des Espèces fruitières’ ; Team AFFS ‘Architecture and Functioning of Fruit Species’ Contact: [email protected] Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceThe development of alternative selection systems without antibiotic resistance genes is a key issue to produce safer and more acceptable transgenic plants. Eutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine, which is detoxified in mung bean (Vigna radiata) by the gene Vr-ERE. Many phytotoxic compounds containing an aldehyde group can act as substrates for the Vr-ERE enzyme. The aim of the present work was to evaluate the effects of the overexpression of Vr-ERE in transgenic apple plants, as a first step towards the development of an alternative selection system. Viable transgenic apple clones expressing Vr-ERE were produced from the cultivar Greensleeves under kanamycin selection. Although the Vr-ERE transgene was normally expressed at the RNA and protein levels, the increase in aldehyde reductase activity tested on a range of potential substrates was very low in these clones. None of them revealed a significant increase in tolerance to toxic aldehydes compared to their non-transgenic control. This work with transgenic apple plants overexpressing the detoxifying gene Vr-ERE illustrates some of the difficulties in developing an alternative selection pressur

Are sheep really afraid of the wolf?

International audienceSheep are a high-olfactory sensitive species, this cue being crucial for social recognition (Hild et al. 2011) and predator avoidance (Gluesing et al. 1980). To protect themselves from predation, sheep are able to recognize cues of their predators. In a food-choice test, ewes chose to eat in the location without dog feces, this odour appeared to be repellent (Arnould et al. 1993). However, there is a trade-off between foraging and the risk of predation. We chose to test this trade-off. Our experimental design questions the motivation to eat in a context with a possible risk of predation. We hypothesize that olfactory stimuli with negative valence (wolf and cadaverine) will induce stress-like behaviors. 30 adult Ile de France ewes participated after one week of habituation to the experimental setup in the presence of four unfamiliar conspecifics. During a test, ewes entered an arena in which a plexiglas box contained the olfactory stimulus close to a bucket of pellets, for 5 minutes. The total number of vocalizations, the food intake and the food intake latency were recorded. In a first session, ewes were tested with water, 15µl orange essential oil (appetitive) and 3g of wolf feces (repulsive). In a second session, ewes were tested with water, 15g of wolf feces and 1ml of cadaverine. In this way, we tested the repulsive impact of predator feces and the putative repulsive effect of one its components. According to our hypothesis, we found a repulsive effect of wolf feces in each session by a decreased food intake (p<0.01, Friedmann test). Surprisingly, we didn't observe a repulsive effect of cadaverine even though two ewes expressed emetic behaviors. Moreover, the latency of food intake was lower for orange and wolf compared to water (p<0.05, Friedmann test) and the number of vocalizations was low and did not differ between stimuli. Taking into account all these results we are not able to conclude on the stressful impact of these olfactory stimuli of negative valence. Future work will use wireless EEG to investigate the underlying neural signatures of these stimuli

Risk Factors of Post-Large Loop Excision of the Transformation Zone Recurrent High-Grade Cervical Intraepithelial Lesion

International audienceOBJECTIVE:The aim of the study was to identify the risk factors of post-large loop excision of the transformation zone (LLETZ) recurrent disease and the impact of colposcopic guidance at the time of LLETZ on that risk.MATERIALS AND METHODS:From December 2013 to July 2014, 204 patients who had undergone LLETZ for the treatment of high-grade intraepithelial lesion with fully visible cervical squamocolumnar junction were included. The use of colposcopy during each procedure was systematically documented. The dimensions and volume of LLETZ specimens were measured at the time of the procedure before formaldehyde fixation. All participants were invited for a follow-up. The primary endpoint was the diagnosis of post-LLETZ recurrent disease defined as the histologic diagnosis of a high-grade cervical intraepithelial lesion.RESULTS:The median duration of post-LLETZ follow-up was 25.8 months. Recurrent disease was diagnosed in 8 (3.6%) patients. Older than 38 years (adjusted hazard ratio [aHR] = 11.9, 95% CI = 1.6-86.0), history of excisional therapy (aHR = 21.6, 95% CI = 3.5-135.3), and the absence of colposcopy for the guidance of LLETZ (aHR = 6.4, 95% CI = 1.1-37.7) were found to significantly increase the risk of post-LLETZ recurrent disease. The dimensions and volume of the specimen were not found to have any impact. Only positive endocervical margins were identified to significantly increase the risk of post-LLETZ recurrent disease (aHR = 14.4, 95% CI = 2.0-101.1).CONCLUSIONS:Risk factors of post-LLETZ recurrent disease are older than 38 years, history of excisional therapy, positive endocervical margins, and lack of colposcopic guidance at the time of LLETZ