Arabidopsis AGO1 and AGO10 bound sRNAs

The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the plant meristem and also give rise to all shoot organs. SAM fate is specified by class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, and here we demonstrate that AGO10 specifically interacts with miR166/165. The association is determined by a distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. Notably, the miRNA-binding ability of AGO10, but not its catalytic activity, is required for SAM development, and AGO10 has a higher binding affinity for miR166 than does AGO1, a principal contributor to miRNA-mediated silencing. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP III gene expression. Homozygous T2 progeny of complemented plants expressing ago10-3;PAGO10-HF-AGO10 and ago1;PAGO1-HF-AGO1 were used for preparation of AGO complexes. Flower samples including floral buds, open flowers, and newly set siliques (1-2 day old) were collected for protein extraction and isolation of dual-tagged AGO complexes using a two-step affinity purification. The isolated AGO complexes were divided into two parts, one aliquot was used for sRNA extraction with Trizol reagent, whereas the other part was used for monitoring protein purity by Gelcode blue staining and western blot using a monoclonal anti-Flag antibody (Sigma) as previous described.