The quasi-completion of the Arabidopsis genome sequence has provided an extremely important resource for plant biologists and scientists interested in comparative genomics. However, experimental proof of the function of less than 10% of the predicted 28000 genes has been obtained so far. High-throughput methods for determining gene function are therefore crucial to rapid progress. Reverse genetics approaches based on targeted disruption of gene expression are powerful tools towards this goal. Several labs have demonstrated that it is possible to artificially trigger post-transcriptional gene-silencing (PTGS) by "RNA interference" (RNAi), the production of double strand RNAs by a carefully designed transgene, the most efficient RNAs being the "hairpin" type (hpRNA).
To create a gene encoding such a hairpin RNA, one first has to identify a short region of the target transcript that does not contain significant sequence similarity to other transcripts in the cell (to avoid cross-silencing). The first objective of AGRIKOLA is therefore to clone gene-specific tags (GSTs, 150-600bp long) for as many of the 25000+ Arabidopsis genes as possible. The second necessary step for constructing a gene capable of expressing hairpin RNA is to clone the GSTs on both sides of the intron spacer in inverted orientation. By conventional methods, this is tricky, time-consuming and difficult to adapt to high-throughput protocols. Therefore the second objective of this project is to use high-throughput recombinational cloning techniques to transfer each of the c. 25000 GSTs to an hpRNA vector. Once these first two objectives have been reached, we shall have constructed a set of plasmids each carrying a gene construct capable of specifically silencing one particular Arabidopsis gene.
Some genes are essential for cell survival or are required for growth. Such genes are difficult to analyse by insertion mutagenesis or silencing as loss of expression is lethal. Hence the next objective is to create a second-generation hpRNA vector whose expression can be controlled such that gene-silencing is only triggered when required. This vector will greatly facilitate the analysis of genes whose mutation is lethal or leads to a very severe phenotype. The c. 25000 GSTs will be transferred to this inducible vector. Again, once this second pair of objectives has been achieved, we shall have constructed a set of plasmids each capable of specifically silencing one particular Arabidopsis gene, but on this occasion the silencing can be controlled at will. The two sets of plasmids will be made available to the scientific community via the Nottingham Arabidopsis Stock Centre.
Although sufficient is known about RNAi and gene-silencing to be sure that the approach described here should be generally effective, no large-scale systematic attempts to carry out gene-silencing have yet been undertaken in plants. Therefore we intend to examine the effects of expressing RNAi in Arabidopsis on a large scale. We will transform 4000 constitutive hpRNA clones covering chosen genes for which GSTs can be designed into Arabidopsis and examine the resulting phenotypes at a basic visual level. This effort should reveal useful information on the efficacy of this approach and the types of phenotypes that can be obtained, including the proportion of clones which generate severe or lethal phenotypes. More detailed analysis would be useful, but is inconceivable on a systematic scale within the project given the number of lines that we propose to generate. We will however analyse in detail 150-200 selected transformed lines silenced for genes of particular interest. We will analyse the efficacy, stability and specificity of the gene-silencing produced. We will compare the level of the silencing triggered by constitutive and inducible hpRNA constructs carrying the same GSTs and introduced in independent transformed lines. These experiments will help identify the function of important Arabidopsis genes for which mutants are not currently available.