The Arabidopsis AtFH frataxin : genetic approach with inducible RNAi to investigate its role in the iron trafficking in mitochondria

Frataxin is present in mitochondria of all eukaryotes as well as in the cytoplasm of bacteria. In humans, reduced expression of frataxin is associated with Friedreich\u2019s ataxia, a recessive inherited neurodegenerative and cardiac disorder leading to reduced life expectancy. Experimental evidence suggests that frataxin acts as an iron-chaperone protein, donating iron to the proteins involved in [Fe-S] cluster assembly and heme synthesis. It also possibly contributes to the process of iron detoxification and storage. The frataxin homolog from Arabidopsis thaliana (AtFH) is a single nuclear-encoded gene targeted to mitochondria and sharing 65% similarity with animal frataxin. We have previously shown that [1] the knocking out of AtFH gene causes arrest of Arabidopsis embryo development at the globular stage. Consistently with that, we have also shown by in situ hybridisation that AtFH is expressed, in wt Arabidopsis plants, in ovule primordia as well as in embryos at various stages of development. We are currently producing Arabidopsis transgenic lines, by inducible RNAinterference, in which microRNA can switch off AtFH frataxin transcript accumulation upon treatment with either ethanol or dexametasone. Such approach will allow to overcome experimental constraints due to embryo lethality of AtFH KO. We used the artificial microRNA web-tool designer WMD (wmd.weigelworld.org) that generates all possible amiRNAs for a gene, according to the parameters found for natural miRNA target specificity: we produced the constructs for two different amiRNAs (amiRNA1 and amiRNA2). The two frataxin amiRNAs were cloned into two different plasmids which contain the systems for either dexamethasone- or ethanol- inducible expression. The ethanol system needs also the ALCA protein inducer, whose gene is present in a second plasmid , named p425. We transformed Arabidopsis thaliana plants ecotype Columbia and we screened T1 seedlings for the presence of the following selection markers: kan resistance trait in p425 and in dexamethasone-inducible amiRNA (LOX plants) seedlings; fluorescence produced by modified GFP variants in ethanol-inducible amiRNA seeds (EtOH plants). We were able to recover 11 and 21 lines respectively for amiRNA 1 and 2 of LOX plants; 43 and 41 lines respectively for amiRNA 1 and 2 of EtOH plants; 21 lines for p425 plants. We were then able to recover, in T2 generation screening, at least 6 independent lines for each of these different 4 constructs (dexamethasone- or ethanol inducible amiRNA1 or amiRNA2). We are currently testing p425 T2 generation lines and we will cross the homozygous at single insertion with all EtOH lines since this inducing system works only when both constructs are inserted in the genome. For dexamethasone system, we are currently testing and screening LOX plants in order to find the lines with strongest AtFH silencin