Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells.
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.
Vanitharani
Ramachandran
R
International Laboratory for Tropical Agricultural Biotechnology, Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA.
Chellappan
Padmanabhan
P
Fauquet
Claude M
CM
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D010944:Plants / Q000821:virology*; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D013997:Time Factors; D014026:Nicotiana / Q000378:metabolism; D014162:Transfection
D004279:DNA, Viral; D008164:Luminescent Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins