Analysis of the interaction of plant retrotransposon Tto1 with host organisms: mechanistic aspects and potential applications

Retrotransposons comprise over 50% of the DNA of higher eukaryotes such as maize or humans. They can be viewed either as intrinsic parts of the organism where they occur, or, alternatively, as molecular parasites with the major goal to replicate element sequences more often than host DNA replication allows multiplication of the rest of the genome. This goal is achieved by reverse transcription of element mRNA, followed by integration of the reverse transcript into the host genome. Although retrotransposons have the potential to cause damage by insertion into important sequences (e.g., structural genes), defects due to retrotransposon insertion are apparently rare under "natural" conditions. One possible explanation for this apparent paradox is that the host organism exerts significant control over retrotransposon activity. Analysis of host factors involved in retrotransposition, or in its suppression, is inherently difficult and was, so far, only possible in exceptional cases. The project shall allow the investigation of a plant element from the Ty1/copia family of long terminal repeat containing (LTR-) retrotransposons, Tto1 from tobacco. The focus of interest is the interaction between the element and the host organism. We have discovered that Arabidopsis thaliana Col-0 can destroy newly introduced copies of Tto1, and want to study the molecular basis of this transposon defense mechanism. Furthermore, we want to investigate the finding that Tto1 is most active in plant callus tissue, even though transcription of the element can also be found under a number of other conditions. Tto1 shall be expressed in the two model plant species, Arabidopsis thaliana and Lotus japonicus, and in the microbial host, Saccharomyces cerevisiae. The first two hosts shall allow to study Tto1 in a "natural", yet background-free environment, whereas the latter host allows a biochemical disection of Tto1 replication, and a comparison with the well-known yeast retrotransposon Ty1. The knowledge gained from the proposed work might also help to use Tto1-derived constructs for efficient insertional mutagenesis. This latter method of molecular analysis should be particularly helpful in Lotus japonicus, a model plant for the investigation of symbiosis with soil bacteria to convert air into organic nitrogen.

The majority of the DNA of multi-cellular organisms consists of sequence repeats. A considerable fraction of this repetitive DNA are so-called retroelements. Retroelements encode enzymes that catalyze the deposition of element copies at new positions in the genome. Our interest in retroelements stems from their ability to change the host genome through mutations caused by such integration events. This activity is usually restricted to stressful conditions. Because it is potentially damaging to the host organism, it might be tightly regulated. Understanding whether such a regulation actually exists, and if so, how it works, can help to activate retroelements at will and thus to use them for mutational analysis. Investigations focused on the plant retrotransposon Tto1. This element, and variants of the element produced in the course of the project, were transformed into Arabidopsis plants to correlate the changes present in the variants with their activity. Usually, so-called RNA transcripts produced by cellular enzymes from genes are translated to give rise to proteins. We found, however, that most transcripts produced from Tto1 in plant cells are not translated. However, if the natural transcript is extended by choosing a start site that lies further upstream, the ensuing RNA can be translated to produce Tto1 proteins. We concluded that the plant host has a critical influence on Tto1 activity, by choice of the transcription start site. Identification of a biologically active Tto1 RNA allowed us to replace the normal, stress-inducible transcriptional control elements of Tto1 by those regulated by other stimuli. This allowed to observe intermediates of Tto1 multiplication in absence of stress conditions. Attempts were made to transfer this knowledge to the crop plant barley (Hordeum vulgare). Constructs that allowed observation of Tto1 activity in Arabidopsis were transferred into barley. First results will be used to improve Tto1 performance in barley. Once successful, Tto1 expression in barley shall allow to induce mutations in this crop plant, and to study the nature of these mutations in a way similar to research that is currently only possible with model plant species.