Barley AGO4-associated small RNAs

Barley AGO4a and AGO4b were introduced in Arabidopsis thaliana by heterologous complementation to investigate their function. Both AGO4 proteins were able to complement the expression of Arabidopsis genes regulated by RNA-derived DNA methylation pathway. In addition, HvAGO4B showed an affinity for 24-nucleotide small RNAs with A at the 5'-end nucleotide similar to AtAGO4, while also accepting 5' G, U, and C. In contrast, HvAGO4A selectively binds only 24-nt sRNAs with 5' A. This different affinity is reflected in the transposable elements-derived sRNAs, for example, AtSN1-derived sRNAs in AtAGO4 and HvAGO4B are mainly starting with G, while in HvAGO4A are only with A, inducing a reduction of TE-derived sRNAs from this locus. Heterologous complementation in Arabidopsis demonstrated the conservation of the RdDM pathway in two evolutionarily distant plants and the functional diversification that can occur in a protein belonging to the same clade.