Principal Investigator: María Rosa Ponce.
Investigators: Ana Belén Sánchez García and Rosa Micol Ponce.
Instituto de Bioingeniería. Universidad Miguel Hernández.

Identification of novel miRNA pathway elements in Arabidopsis thaliana

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are a recent addition to the inventory of mechanisms of gene expression regulation acting as developmental controls in multicellular eukaryotes. In the model plant Arabidopsis thaliana, one of the components of the miRNA mediated silencing complex (RISC) is encoded by the ARGONAUTE1 (AGO1) gene, whose ago1 mutant alleles disturb many developmental processes and often cause lethality or sterility. We isolated two viable and moderately fertile mutants, ago1-51 and ago1-52, and obtained their double mutant combinations with alleles of other miRNA machinery genes, and some miRNA target genes. All the double mutants displayed synergistic phenotypes, as expected from the functional relationship among the mutations involved. By means of a microarray analysis, we found that in the ago1-52 mutant, as well as in the other three miRNA machinery mutants already isolated in our laboratory, the mRNAs of known miRNA targets are accumulated. This prompted us to mutagenize the ago1-52 line in order to identify mutations modifying the Ago1-52 phenotype. We have screened 36,810 seeds and have identified 15 lines viable and fertile, in which the Ago1-52 phenotype is partially or almost completely supressed. Additionaly, we identified a line with an unexpected phenotype, very different to that ago1-52, which we considered a case of epistasis in which a second mutation masks the phenotype caused by ago1-52. We are obtaining the F1 progeny of the backcrosses of the above mentioned lines to Ler, with the purpose of determining the inheritance pattern of the second mutations. We have selected three lines in which the phenotype of the M2 parental reappears in a stable way in the F2 progeny in proportions that we can genetically interpret. We have initiated the positional cloning of these three supressor mutations, by crossing M3 plants to (a) Col and also (b) to a line carrying a fertile ago1 allele in a Col background.

We propose to continue this genetic and molecular approach to identify novel miRNA pathway elements acting as developmental controls in Arabidopsis thaliana. Our goals include (a) the positional cloning and functional characterization of two of the genes that supress the phenotype of the ago1-52 mutant, and (b) the low-resolution mapping of the remaining suppressor mutations identified in our screen.