Two distinct RNA-dependent RNA polymerases are required for initiation and amplification of RNA silencing in the basal fungus Mucor circinelloides

Abstract

RNA-dependent RNA polymerases (RdRPs) play key roles in the RNA silencing pathway in a number of organisms. They have been involved in the production of double-stranded RNA (dsRNA) molecules that initiate the silencing mechanism as well as in the amplification of the silencing signal. The roles of RdRPs from fungi in these processes are poorly described compared with other eukaryotes. RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and an amplification process associated with two size classes of antisense small interfering RNAs (siRNAs). To investigate the function of fungal RdRP proteins in initiation and amplification of silencing we have cloned and characterized two different rdrp genes in M. circinelloides. Functional analysis of rdrp(-) disruption mutants indicates that rdrp-1 is essential for initiation of silencing by sense transgenes by producing antisense RNA transcripts derived from the transgene, but it is not necessary for amplification of the silencing signal, whereas rdrp-2 is required for efficient accumulation of the two different classes of secondary siRNAs regardless the nature of the trigger. Our results provide evidence for a functional diversification of M. circinelloides rdrp genes in different steps of the same RNA silencing pathway.