Synthetic Biology on a Retroelement for Plant Biotechnology

Synthetic biology is an emerging field covering changes in biological processes that result in novel functionality. We are modifying a mobile genetic element of the retrotransposon class, Tto1, such that its mobility can be controlled by a chemical substance. Retrotransposons are already used as tools for insertional mutagenesis in plants. However, current experiments employ natural induction conditions for mobilization, in particular tissue culture stress. Insertion mutants can therefore be generated in those plant species for which regeneration of whole plants from tissue culture cells is straightforward. Most crop plants do unfortunately not belong to this group. The engineered Tto1 element allows insertional mutagenesis in whole plants, without tissue culture stress. It will extend the range of species for which insertional mutagenesis is possible, and thereby facilitate genetic analysis of crop plants. Our particular focus lies on barley. This crop plant is diploid, and its genome sequence determination is almost finished. The widespread use of insertion mutants currently available for the model plant Arabidopsis suggests that insertional mutants of barley can similarly contribute to understanding, and may serve as a material for crop improvement.

Retrotransposons are DNA segments that mediate their own replication within the host genome. Retrotransposon-encoded enzymes convert the element RNA into a complete DNA copy of the element. The copy is inserted into the genome at a new position, where it affects expression of neighbouring genes. The process is important for adaptation of plants to a changing environment, so that its study and manipulation promises considerable benefits. During the project, variants of the tobacco retrotransposon Tto1 were introduced into plants without additional, endogenous copies, so that properties of the element could be correlated with the DNA sequence of the construct. We could show in the model plant Arabidopsis that certain sequences of the so-called Long Terminal Repeat (LTR) region are dispensable, if Tto1 is expressed from a heterologous promoter. Shortening of LTRs shall help to subvert host defense mechanisms, and to improve control over Tto1 expression. Many tested variants of Tto1 can efficiently make DNA copies, but integration of these copies into the host genome is less efficient than desired. We have therefore investigated the integrase enzyme of Tto1. We could identify a variant integrase that is more ancient than the currently used enzyme. However, preliminary investigations suggest that both integrases have approximately equal activity. We also started to test Arabidopsis mutants in genome defense. These experiments also aimed at increasing transposition efficiency of Tto1. Furthermore, different promoters were tried for Tto1 expression. We succeeded in improving expression of Tto1. An important aspect of the project was to express Tto1 in agriculturally important plants. With the help of international collaborations, we could express Tto1 in barley and in carrot. Analysis of the ensuing plant material is ongoing. Finally, we started to analyze which sequences of Tto1 mediate enrichment of RNA in so-called virus-like particles. Knowledge of these sequences would allow to multiply any DNA sequence in a host plant. Experiments could proceed to generation of plant material, the ensuing analysis shall be carried out in the near future.