Use of virus-induced gene silencing in Arabidopsis thaliana to identify trans-acting factors involved in mitochondrial editing

RNA editing is any post-transcriptional modification to the primary transcript. In the organelles of flowering plants, some C’s undergo C-U editing. Depending on the location of the C that undergoes editing, the amino acid that the codon encodes for can be changed and ultimately affect the final protein product. As of now, only two trans-acting factors involved in mitochondrial C-U editing have been identified and both contain a pentratricopeptide-repeat motif and a DYW domain (PPR-DYW), suggesting that other editing factors are likely to be PPR-DYW proteins. For the most part, editing in the mitochondria is not 100% and the extent of editing also varies between different ascensions ofArabidopsis thaliana. These natural variations were used for quantitative trait loci (QTL) mapping and previous work in the lab identified QTLs that are responsible for the editing variations at several sites. My project was to silence in total expression of ten PPR-DYW nuclear-encoded genes within several QTLs that controlled editing at eight different C sites in mitochondrial transcripts. Virus-induced gene silencing (VIGS) was used to silence the PPR-DYW candidate gene and the level of RNA editing was assayed using poisoned primer extension (PPE) reaction. The PPE reaction yields two products of different lengths with the longer corresponding to the edited transcript and the shorter to the unedited and a ratio of the products tells the level of editing of the transcripts. I expected the silencing of an important PPR-DYW gene involved in editing to reduce the level of editing in VIGS treated plants compared to controls. Silencing of genes not involved in editing are not expected to have an affect on editing extent. Results of my project did not show a significant difference between control and VIGS treated plants for most sites that I looked at, suggesting that these candidate PPR-DYW genes are not involved in RNA editing. However, compared to controls, two mitochondrial editing sites showed slightly reduced editing when a particular PPR-DYW gene was silenced. This suggests that the PPR-DYW gene may regulate editing at these two sites.