Functionality of a plant dicer-like protein in generating small rnas in a heterologous saccharomyces cerevisiae system

SiRNAs are the central players of RNAi in plants, fungi, insects, nematodes and mammals. Plant DCLs are encoded by multigenic families and fall into four clades containing orthologs of DCL1, DCL2, DCL3 and DCL4 genes originally discovered in Arabidopsis thaliana (At) and conserved in all plants, including Nicotiana benthamiana (Nb). In At, DCL1 primarily generates 21-22 nt miRNAs, while DCL4, DCL2 and DCL3 mainly generate 21, 22 and 24 nt endogenous siRNAs, respectively. Upon viral infection, all four DCLs generate viral siRNAs: DCL4 and DCL2 produce 21 and 22 nt siRNAs in defense against cytoplasmic RNA viruses, while DCL3 and, less efficiently, DCL1 produce 24 and 21 nt siRNAs in defense against nuclear DNA viruses. The application of siRNAs for crop protection is still limited, mainly because of poor knowledge about the biogenesis and function of distinct siRNA size-classes. A HA-tagged version of the Nb DCL4 (HANbDCL4) was cloned under the control of the cupper inducible promoter CUP1, transformed into Saccharomyces cerevisiae YPH499 cells and successfully expressed. The protein expression was detected by Western blot analysis and varied along with the growth temperature. Differential centrifugation of protein extracts was used to separate a membrane-enriched and a soluble cytoplasmic fractions. Western blot analysis showed that HANbDCL4 was mostly associated to the cell membrane fraction. In addition, it was only partially removed after 0.1 M Na2CO3 or 1 M KCl treatments. The partial association of HANbDCL4 to cell membranes was confirmed by immunofluorescence analysis of yeast cells, in which the expressed HANbDCL4 was mostly detected close to the nuclear membrane. HANbDCL4 was expressed in yeast cells together with the cymbidium ringspot virus defective interfering (DI) RNA and RNA dependent RNA polymerase (p92). Robust DI RNA replication was not affected by HANbDCL4 expression. By small RNA-seq preliminar evidences were obtained that HANbDCL4 is functional in generating sRNAs of viral origin when expressed in the tombusvirus-S. cerevisiae heterologous system.