Tnt1 elements are a superfamily of LTR-retrotransposons distributed in the Solanaceae plant family and represent good model systems for studying regulatory and evolutionary controls established between hosts and transposable elements. Tnt1 retrotransposons tightly control their activation, by restricting expression to specific conditions. The Tnt1A element, originally discovered in tobacco, is expressed in response to stress, and its activation by microbial factors is followed by amplification, demonstrating that factors of pathogen origin can generate genetic diversity in plants. The Tnt1A promoter has the potential to be activated by various biotic and abiotic stimuli but a number of these are specifically repressed in tobacco and are revealed only when the LTR promoter is placed in a heterologous context. We propose that a tobacco- and stimulus-specific repression has been established in order to minimize activation in conditions that might generate germinal transposition. In addition to tight transcriptional controls, Tnt1A retrotransposons self-regulate their activity through gradual generation of defective copies that have reduced transcriptional activity. Tnt1 retrotransposons found in various Solanaceae species are characterized by a high level of variability in the LTR sequences involved in transcription, and have evolved by gaining new expression patterns, mostly associated with responses to diverse stress conditions. Tnt1A insertions associated with genic regions are initially favored but seem subsequently counter-selected, while insertions in repetitive DNA are maintained. On the other hand, amplification and loss of insertions may result from more brutal occurrences, as suggested by the large restructuring of Tnt1 populations observed in tobacco compared to each of its parental species. The distribution of Tnt1 elements thus appears as a dynamic flux, with amplification counterbalanced by loss of insertions. Tnt1 insertion polymorphisms are too high to reveal species relationships in the Nicotiana genus, but can be used to evaluate species relationships in the Lycopersicon and Capsicum genera. This also demonstrates that the behavior of Tnt1 retrotransposons differs between host species, most probably in correlation to differences in expression conditions and in the evolutionary and environmental history of each host.

Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross-resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51(I381V Y461H) or CYP51(I381V ΔY459/) (G460) , respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.

Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici, have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides’ efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici. The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici.

SummaryWe have analyzed the stress-induced ampli®cation of the tobacco Tnt1 element, one of the rare active plant retrotransposons. Tnt1 mobility was monitored using the retrotransposon-anchored SSAP strategy that allows the screening of multiple insertion sites of high copy number elements. We have screened for Tnt1 insertion polymorphisms in plants regenerated from mesophyll leaf cells, either via explant culture or via protoplast isolation. The second procedure includes an overnight exposure to fungal extracts known to induce high levels of Tnt1 transcription. Newly transposed Tnt1 copies were detected in nearly 25% of the plants regenerated via protoplast isolation, and in less than 3% of the plants derived from explant culture. These results show that Tnt1 transcription is followed by transposition, and that fungal extracts ef®ciently activate Tnt1 mobility. Transcription appears to be the key step to controlling Tnt1 ampli®cation, as newly transposed Tnt1 copies show high sequence similarities to the subpopulations of transcribed Tnt1 elements. Our results provide direct evidence that factors of microbial origin are able to induce retrotransposon ampli®cation in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.

Transcriptional regulation of the tobacco retrotransposon Tnt1 was monitored in Arabidopsis thaliana and Lycopersicon esculentum. In these two heterologous host species, Tnt1 expression is developmentally regulated, and its tissue‐specific pattern of transcription is similar to that observed in tobacco, with the exception that Tnt1 is expressed in their flowers. Induction of Tnt1 by microbial elicitors, previously found in tobacco, is maintained in Arabidopsis and tomato. Tnt1 activation is not strictly dependent upon a Hypersensitive Response‐like necrotic response, but also occurs in a plant‐pathogen compatible interaction triggered by viral or bacterial infections. The putative role of Tnt1 in generating genetic variability in its host plant is discussed.

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Retroviral replication is a very error-prone process. Replication of retroviruses gives rise to populations of closely related but different genomes referred to as 'quasispecies'. This huge swarm of different sequences constitutes a reservoir of potentially useful genomes in case of an environmental change, endowing retroviruses with extreme adaptability. Retrotransposons are mobile genetic elements closely related to retroviruses, and retrotransposition is as error prone as retroviral replication. The Tnt! retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt! is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This popUlation structure gives to Tntl, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt! maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt! evolution.

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip Ò GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the S. Leimanis Á S. Hamels (&) Á J. Remacle Eppendorf Array Technologies, 20 Rue du séminaire, 5000 Namur, Belgium e-mail: hamels.s@eppendorf.be DOI 10.1007/s00217-008-0886-y study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra-and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip Ò GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.123 Eur Food Res Technol