Search

International audience; Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.

[EN] Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life-history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y-U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y-U isolates made it possible (i) to identify specific amino acid residues in the N-terminal of the CP protein and (ii) to assign some Y-U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY Brazilian' clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time., The authors wish to thank Claudir Lorencetti (Alliance One from Brazil) who kindly supplied tobacco PVY isolates. Transfer of materials from Brazil to France was carried out using an official letter of authorization 'LOA' delivered to L. Glais (INRA Rennes/FNPPPT Paris) by plant protection organizations. The authors gratefully acknowledge Dr Alain Palloix (INRA, Avignon) who kindly provided pepper seeds for the host range study and Maryse Guillet (FN3PT, Le Rheu) who kindly provided serum for PVY DAS-ELISA. A. W. and S. F. E. were supported by grants BFU2012-30805 from Spanish MINECO and 22371 from the John Templeton Foundation. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. J. M. C. was supported by the JAE-Doc program from CSIC. This study was supported by The Imperial Tobaccos Group and the Association for Research on Nicotianae (ARN).

International audience

International audience

National audience

National audience

National audience; Le virus Y de la pomme de terre (PVY, espèce-type du genre Potyvirus) infecte une large gamme de plantes hôtes dont des Solanacées d’intérêt agronomique tel que le tabac. Le PVY peut induire sur cet hôte des symptômes de nécroses foliaires, responsables d’importantes pertes de rendements. Toutefois, de nombreuses variétés de tabac, porteuses d’un des allèles (0, 1 ou 2) du gène récessif de résistance " va", sont capables de limiter l’expression de nécroses dues au PVY avec une efficacité variable en fonction de l’allèle en présence. Aussi, compte tenu de l’absence d’alternatives au gène "va" pour la lutte contre le PVY chez le tabac, il est important de déterminer l’impact respectif des différents allèles de ce gène sur le processus évolutif du PVY. Deux actions complémentaires visant i) à caractériser la variabilité naturelle du PVY sur tabac et ii) à étudier le processus d’adaptation du PVY aux pressions imposées par les allèles du gène "va" ont été réalisées. Ainsi, la virulence (capacité d’infection) d’une collection de 86 isolats, issus d’une prospection effectuée en France en 2007, a été caractérisée en utilisant des génotypes de tabac porteurs ou non d’un allèle du gène "va". Les isolats testés se répartissent dans huit pathotypes. Les deux-tiers des isolats, étant capables d’infecter les trois génotypes porteurs d’un allèle du gène "va", appartiennent au pathotype le plus virulent. Afin d’analyser la dynamique d’adaptation du PVY au gène "va", un clone infectieux de PVY a été inoculé à deux variétés résistantes portant respectivement l’allèle 0 et 2 du gène "va". La descendance virale obtenue à été utilisée comme inoculum pour une nouvelle série d’inoculations de ces variétés résistantes. Les données de virulence associées à ces populations virales montrent qu’un seul passage du clone infectieux sur une variété porteuse de l’allèle 2 du gène "va" peut suffire pour augmenter la virulence de ce clone au point d’être capable de surmonter la résistance des deux allèles. Ainsi, les résultats associés au pathotypage de la collection d’isolats de PVY et aux caractéristiques des populations virales issues des passages en série du clone infectieux sur des variétés résistantes contribuent à mieux caractériser l’interaction virus/plante hôte dans le cadre du pathosystème PVY/tabac.

International audience; Cultivated tobacco produces secondary alkaloids involved in the formation of nitrosamines with health concerns. The recent identification of target genes in nicotine and nornicotine biosynthetic pathways now allows biotechnological approaches for their control. We demonstrate here that mutation breeding can be used as an alternative to genetically modified (GM) plants for generating nornicotine-free tobacco. Ten alleles of the NtabCYP82E4 gene (nicotine N-demethylase) were identified by screening 1,311 M2 families of tobacco ethylmethane sulphonate (EMS) mutants. Alkaloid analysis indicated that the nornicotine contents of homozygous M2 plants carrying nonsense or missense alleles of NtabCYP82E4 were very low or near-null. Backcrossing with tobacco elite varieties yielded BC1 plants phenotypically undistinguishable from parental lines. This major objective of tobacco breeders in the last few decades could be reached in a period of less than 1.5 years, including the creation of highly mutagenised tobacco mutant collections and the detection of mutated alleles using a simple and versatile detection technology (capillary electrophoresis-single strand conformation polymorphism, CE-SSCP) accessible to most breeding companies and crop species.

International audience; LTR-retrotransposons contribute substantially to the structural diversity of plant genomes. Recent models of genome evolution suggest that retrotransposon amplification is offset by removal of retrotransposon sequences, leading to a turnover of retrotransposon populations. While bursts of amplification have been documented, it is not known whether removal of retrotransposon sequences occurs continuously, or is triggered by specific stimuli over short evolutionary periods. In this work, we have characterized the evolutionary dynamics of four populations of copia-type retrotransposons in allotetraploid tobacco (Nicotiana tabacum) and its two diploid progenitors Nicotiana sylvestris and Nicotiana tomentosiformis. We have used SSAP (Sequence-Specific Amplification Polymorphism) to evaluate the contribution retrotransposons have made to the diversity of tobacco and its diploid progenitor species, to quantify the contribution each diploid progenitor has made to tobacco's retrotransposon populations, and to estimate losses or amplifications of retrotransposon sequences subsequent to tobacco's formation. Our results show that the tobacco genome derives from a turnover of retrotransposon sequences with removals concomitant with new insertions. We have detected unique behaviour specific to each retrotransposon population, with differences likely reflecting distinct evolutionary histories and activities of particular elements. Our results indicate that the retrotransposon content of a given plant species is strongly influenced by the host evolutionary history, with periods of rapid turnover of retrotransposon sequences stimulated by allopolyploidy.

Quantitative trait loci (QTLs) were investigated in a recombinant inbred line (RIL) population descended from a cross between two flue-cured Nicotiana tabacum L. inbred lines with unrelated breeding origins. A total of 59 traits, related to diverse agronomic, leaf quality, chemical composition and smoke properties were assessed. Chemical traits and smoke mutagenicity were estimated by near infrared reflectance spectroscopy (NIRS) analyses of leaf lamina powders. Physical properties of cigarettes made from each RIL, and the total particulate matter (tar and nicotine yields), benzo[a]pyrene and CO contents of the main smoke stream generated by the cigarettes in mechanical smoking tests under a standard ISO regime were also analyzed. The RILs were screened for 184 amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR), sequence specific amplified polymorphism (SSAP), sequence characterized amplified region (SCAR) and biological markers. A partial genetic map including 18 linkage groups was constructed based on 138 of the markers. Substantial segregation distortion (47%) was observed in linkage groups throughout the genome. Seventy-five QTLs associated with 8–41.5% of the variation in the examined traits were identified on 12 linkage groups by simple and composite interval mapping. Nineteen QTLs had opposite effects to those expected from the ranking of parental means.

Phytoplasmas are sieve tube restricted, wall-less phytopathogenic bacteria belonging to the class Mollicutes that have resisted cultivation. In previous studies we have shown that single-chain Fv antibodies (scFv) directed against an immunodominant membrane protein of the culturable plant pathogenic mollicute Spiroplasma citri inhibit its growth in vitro. Based on these observations, we considered a plantibody-based strategy for controlling phytoplasmas in plants. Different genetic constructs, in which the anti-stolbur phytoplasma scFv 2A10 gene was placed either under the control of the constitutive CaMV 35S or the phloem-specific rice sucrose synthase (RSs1) promoters, were engineered for expressing antibodies in tobacco plants via the apoplastic (signal peptide) or the symplasmic (no signal peptide) route. Transformed plants were analyzed for scFv 2A10 protein expression and for their resistance to the stolbur disease both in greenhouse and field experiments. This is the first time that a plantibody strategy for pathogen resistance has been evaluated in field experiments. In the transformed plants, the apoplast-targeted scFv was functional and was produced at an average of 0.04% of soluble proteins. When the stolbur phytoplasma was graft-transmitted to tobacco plants producing secreted scFv, a short delay in symptom appearance and phytoplasma multiplication (2-weeks at maximum) was observed, together with a slight reduction in symptom severity. However, no significant resistance was observed when the phytoplasma was transmitted by its insect vector. When the single-chain molecule was targeted to the cytoplasm, the transcripts were detected with both promoters but the scFv 2A10 protein could not be detected, and the plants did not show any resistance to the disease.

National audience