Colette Audeon

Quasispecies in retrotransposons: a role for sequence variability in Tnt1 evolution

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 Tnt1 retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt1 is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This population structure gives to Tnt1, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt1 maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt1 evolution.

Molecular and functional characterization of Slide, an Ac-like autonomous transposable element from tobacco.

A new transposable element of tobacco, Slide, was isolated from thetl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases ofAc, Tam3 andhobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left byAc and Tam3. Tobacco lines derived from thetl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomousAc-like element characterized inSolanaceae species.

Functional analysis of the tobaccoTnt1 retrotransposon

Retroelements represent by far the largest and most widespread class of mobile genetic elements. Representatives of several classes of retrotransposons have been characterized in a broad range of plant species, but only a few of them have been shown to be active. Among these, the tobaccoTnt1 retrotransposon has been isolated after insertional mutagenesis and is one of the very few to be transcriptionally active.Tntl expression is strongly regulated in a tissue-specific and developmental manner. Moreover,Tntl expression is induced by a range of biotic or abiotic elicitors, which all have in common the ability to induce the plant defense response. Regulatory sequences involved in this elicitor-mediated induction have been located in the LTR U3 region. The link betweenTntl activation and the plant defense response might represent an example of the involvement of transposable elements in genome restructurations needed in response to environmental fluctuations such as pathogen attacks.

Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates

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.

The promoter of the Tnt1A retrotransposon is activated by ozone air pollution in tomato, but not in its natural host tobacco

The tobacco retrotransposon Tnt1A is one of the few active plant retrotransposon and is known to be transcriptionally activated in tobacco (Nicotiana tabacum) and in heterologous species by biotic and abiotic stress factors. It has been previously reported that Tnt1A expression is linked with the early steps of metabolic pathways leading to the activation of plant defence genes. As ozone is known to generate an oxidative stress in plant and activate the defence system, we have investigated, using LTR-GUS constructs, the effect of ozone on the Tnt1A promoter activation in its natural host tobacco and in an heterologous species, tomato (Lycopersicon esculentum). Plants cultivated in open top chambers (OTC) were exposed to different ozone concentrations in order to simulate either different range of a realistic chronic ozone stress or an acute ozone stress. The results show that the Tnt1A promoter is not activated by ozone in tobacco, whereas dose-dependant and cumulative effects are observed in tomato. This difference observed between tobacco and tomato is discussed. Moreover the use of such a construct to study both early response to ozone stress and regulation of the retrotransoposon is examined.