Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells

Abstract

Gene silencing mediated by double-stranded RNA is a sequence-specific RNA degradation mechanism highly conserved in eukaryotes that serves as an antiviral defense pathway in both plants and Drosophila. Short interfering RNAs (siRNAs), the 21- to 23-nt double-stranded intermediates of this natural defense mechanism, are becoming powerful tools for reducing gene expression and countering viral infection in a variety of mammalian cells. Here we report the use of siRNAs to target reporter gene expression and viral DNA accumulation in cultured plant cells. Transient expression of reporter genes encoding either GFP or red fluorescent protein from Discosoma was specifically reduced by 58% and 47%, respectively, at 24 h after codelivery of cognate siRNAs in BY2 protoplasts. In contrast to mammalian systems, the siRNA-induced silencing of GFP expression was transitive as indicated by the presence of siRNAs representing parts of the target RNA outside the region homologous to the triggering siRNA. Codelivery of an siRNA designed to target the mRNA encoding the replication-associated protein (AC1) of the geminivirus African cassava mosaic virus (ACMV) from Cameroon blocked AC1 mRNA accumulation by approximately 91% and inhibited accumulation of the ACMV genomic DNA by approximately 66% at 36 and 48 h after transfection. As with siRNA-induced reporter gene silencing, the siRNA targeting ACMV AC1 was specific and did not affect the replication of East African cassava mosaic Cameroon virus. This report demonstrates the occurrence of siRNA-mediated suppression of gene expression in cultured plant cells and that siRNA can interfere with and suppress accumulation of a nuclear-replicated DNA virus.

Fig. 1.

(A) Genome organization of DNA-A and DNA-B of ACMV-[CM]. DNA-A contains six ORFs: AC1–AC4, AV1, and AV2. DNA-B contains the ORFs BV1 and BC1. V represents virion sense genes, and C represents complementary sense genes. AC1 encodes replication-associated protein (Rep). (B) Schematic representation of the AC1-coding sequence of ACMV DNA-A. The siAC1 sequence (nucleotides 161–181) is as indicated. CRA, common region in DNA-A; CRB, common region in DNA-B; CP, coat protein; TrAP, transcriptional activator protein; REn, replication enhancer protein; MP, movement protein; NSP, nuclear-shuttle protein.

Fig. 2.

Effect of siRNA on EGFP and DsRed2 expression in BY2 tobacco protoplasts. Both the reporter plasmids were driven by the CaMV35S promoter. (A) Schematic representation of the EGFP-coding region and the siRNA sequence (nucleotides 124–144) designed to target EGFP. The slashed area represents the sequence used for the probe in D. NOS-T, nopaline synthase terminator. (B) Schematic representation of DsRed2-coding region and the corresponding siRNA sequence (nucleotides 191–211) to target DsRed2. (C) Protoplasts were transfected either with the EGFP and DsRed2 plasmid DNAs together or in combination with either siGFP or siRNA cognate to red fluorescent protein. Triplicate electroporation experiments were performed in every case. Columns and bars indicate mean and SD values, respectively. GFP and DsRed fluorescence are normalized with nontransfected control cells. (D) RNA gel blot showing siRNA accumulation in protoplasts. Lane 1, protoplasts transfected with pCaMV35S-GFP plasmid DNA with siGFP; lane 2, protoplasts transfected with pCaMV35S-GFP plasmid DNA; lane M, 21-, 30-, and 43-nt end-labeled oligos are shown as molecular size markers.

Fig. 3.

Effect of siRNA targeted to the AC1 of ACMV on ACMV and EACMCV DNA accumulation. Southern blots showing relative levels of viral DNA accumulation 36–48 h posttransfection in BY2 tobacco protoplasts. For cotransfection, either infectious DNA-A and DNA-B clones of ACMV or EACMCV with (+) or without (-) siAC1 were used. Five micrograms of total DNA isolated from protoplasts was loaded in each lane. The blots were probed with [α-³²P]dCTP-labeled ACMV-specific (Left) or EACMV-specific (Right) DNA. Ethidium bromide-stained gel to show loading control. h.p.i., Hours postinoculation; OC, open circular; SS, single strand; SC, super-coiled, viral DNA forms.

Fig. 4.

Northern blot analysis of AC1-specific mRNA in transfected BY2 tobacco protoplasts. Ten micrograms of total RNA isolated from protoplasts inoculated with the infectious clones of DNA-A and DNA-B of ACMV-[CM] with (+) or without (-) siRNA specific for AC1 of ACMV-[CM] was loaded in each lane. AC1-specific [α-³²P]dCTP-labeled DNA was used as the probe. Ethidium bromide staining of ribosomal RNA (rRNA) shows equal loading of the samples. h.p.i., Hours postinoculation.