MicroRNA – ARGONAUTE associations and their roles in plant development

MicroRNAs are small RNAs which associate with ARGONAUTE (AGO) proteins and regulate target mRNAs in various ways with the most common being cleavage. In Arabidopsis, AtAGO10 interacts exclusively with miR166 and is required for shoot apical meristem maintenance. In maize, ZmAGO10 can bind both miR166 and miR394, but its role in maize development is unknown. MiR394 is adaxially expressed, while its F-box target genes accumulate abaxially. Moreover, a downward-curled leaf phenotype is observed in adult ZmAGO10 double mutant plants, suggesting a role of the pathway in leaf patterning and development. The aim of this project was to characterize maize mutants in this pathway, and to identify the basis for the differential miRNA associations between AtAGO10 and ZmAGO10 using tobacco infiltration-mediated competitive binding assays. Western blots were used to confirm the absence of the ZmAGO10 proteins in ago10 double mutants, and quantitative RT-PCR (qPCR) was used to analyze F-box target gene expression in wild-type (WT) and mutant plants. Regarding a possible role of the pathway in leaf polarity, the expression of the ARF family genes was analyzed using qPCR. Finally, a cloning strategy was designed in order to generate transgenic maize plants harboring resistant versions of the F-box genes. As a preliminary step for the binding assays, tobacco leaves were infiltrated with Agrobacteria containing either 3xFLAG-AGO proteins or microRNA hairpins from both Arabidopsis and maize. Protein or microRNA accumulation was analyzed after one, two and three days. The Western blots showed that the mutants were severely compromised in AGO10 protein production. However, no significant change was observed in F-box target gene expression between WT and mutant samples. It could be that a different AGO protein takes over AGO10 activity, or that target gene regulation occurs at the protein rather than the transcript level. No significant difference in ARF3 gene expression was observed between mutants and WT samples, indicating that this polarity pathway is not affected by mutations in AGO10 genes. The designed strategy for the resistant version of the F-box genes involves several primer combinations to amplify the genes, the addition of the YFP reporter gene, and a megaprimer to mutate the miR394 binding site. Results from the tobacco infiltration experiments need to be repeated as they were inconclusive. This study has been a useful first step toward the molecular characterization of the miR394 pathway in maize and its findings can be used as a guide for future investigation.