Functional divergence of two ARGONAUTE 4 clade proteins on RNA-directed DNA methylation, gene expression and grain development in rice (PRJCA017578)
RNA-directed DNA methylation (RdDM) in plants triggered by 24-nt heterochromatic siRNAs (hc-siRNAs) that are loaded into ARGONAUTE 4 effectors is generally considered to control transposable elements (TEs) locally in cis, but how RdDM takes place in trans, affects gene expression and plant development remain elusive. Here, we dissect the functional diversification of AGO4a and AGO16, two rice AGO4 clade members preferentially binding hc-siRNAs with 5' adenine (A), in RdDM, gene expression and grain development in rice. We found that AGO16 has a stronger DNA methylation capacity, while a weaker influence on gene expression than AGO4a, even though they associate with similar hc-siRNA patterns. Remarkably, we found that most hc-siRNAs in rice, direct DNA methylation and affect gene expressions in trans, irrespective of being recruited into AGO4a or AGO16. In addition, we observed that the effects of DNA methylation on gene expressions by AGO4a is larger than that by AGO16, suggestive of a specific property of AGO4a in controlling gene expression via RdDM. Moreover, we find that Small Grain and Dwarf 2 (SGD2) is repressed by lower methylation at two miniature inverted-repeat TEs (MITEs) at its promoter in ago16, which results in a narrow grain of ago16 compared with wild-type plants and ago4a mutants. Finally, we demonstrate that natural variation of AGO16 haplotypes contribute to grain development in Japonica rice. Collectively, our study uncovers different features of two AGO4 proteins in rice, sheds light on more RdDM characteristics in plants and provides a potential target for breeding high-yield crops.