Strategies to enhance Agrobacterium-mediated plant transformation systems based on suppression of gene silencing

Plant genetic transformation involves inserting at least one novel (foreign) gene into plants to produce 'Genetically Modified' (GM) plants. GM plants are mostly used for basic research to understand how genes work in plants. To study the effect of all genes tens of thousands of different GM plants have to be produced. Plants are generally transformed using a bacterium, called Agrobacterium, which naturally transfers its own bacterial genes into plants. When the bacterial genes are replaced by a novel gene, Agrobacterium can transfer it into the plant DNA. In the last ten years, these transformation technologies have also been used to produce improved GM crop varieties, such as maize, soybean, cotton and oilseed rape. However, for both basic and applied research, current transformation technologies still have limitations related to how often and where the novel gene is inserted into the plant DNA. This short project aims at testing a new idea to improve plant transformation technologies. The idea is based on new findings on how Agrobacterium interacts with plants in nature. In February 2006, a group of French scientists showed that Agrobacterium naturally triggers a response system in plants that recognises and inactivates foreign DNA. This response system, called 'gene silencing', has already been described when plants respond to viruses but never in response to bacteria. We intend to alter this gene silencing mechanism in plants to improve the relationship between plants and Agrobacterium and therefore increase the effectiveness of plant transformation when Agrobacterium is used to transfer novel genes. In order to do so, we will test the effects of 'suppressors of gene silencing' during transformation experiments. Preliminary experiments conducted in our laboratory suggested that this approach could improve transformation efficiency. We propose to investigate different ways to use these suppressors of gene silencing in different plant species such as tobacco and rice. We are using rice because it is a relatively simple crop and a cereal, so results in rice could be quickly tested and used in wheat and barley, both important cereal crops in the UK.

Plant transgenic technologies are key to state-of-the-art plant molecular genetics and GM crop improvement. The development of facile and high-throughput Agrobacterium-mediated transformation systems in Arabidopsis thaliana and, to a lesser extend, in rice have been critical to the development of functional genomic programmes in both species. However, current Agrobacterium-mediated transformation systems remain inefficient in many important plant species (especially in crops). A study published in February 2006 demonstrated for the first time, that virulent Agrobacterium tumefaciens infections induce a RNA interference (RNAi) response in A. thaliana. RNAi-deficient plants were shown to be hyper-susceptible to the virulent bacterium. This pilot project aims at testing if these recent findings could be used to improve existing plant transformation technologies through novel modifications based on suppression of the plant gene silencing pathways. The major approach taken is to over-express genes known as suppressors of gene silencing during plant transformation. Preliminary experiments conducted in our laboratory showed great potential for this approach. Novel dual-binary vectors will be developed and tested for the stable transformation of rice, tobacco and A. thaliana. We will assess the best strategy to deliver these suppressors of gene silencing, to maximise their effect on transformation efficiency and to minimise their integration into the plant genome. The P38 gene from Turnip Crinkle Virus and the P25 gene from Potato Virus X will be tested as they are known to specifically inhibit the production of siRNAs with otherwise little or no effect on plant development in A. thaliana. We will use the dual binary vector system pGreen/pSoup to undertake multiple T-DNA delivery and unlinked T-DNA integration in plants. Generic means to enhanced plant transformation technologies would have a major impact in both basic and applied plant transgenic science.