title: Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless.
abstract: Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.
authors.affiliation: John Innes Centre, Norwich Research Park, Colney, Norwich, UK.
authors.forename: Pooja
authors.initials: P
authors.lastname: Saxena
authors.forename: Yi-Cheng
authors.initials: YC
authors.lastname: Hsieh
authors.forename: Veria Y
authors.initials: VY
authors.lastname: Alvarado
authors.forename: Frank
authors.initials: F
authors.lastname: Sainsbury
authors.forename: Keith
authors.initials: K
authors.lastname: Saunders
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
authors.forename: Herman B
authors.initials: HB
authors.lastname: Scholthof
mesh_terms: D004269:DNA, Bacterial; D035264:Flowers / Q000502:physiology; D018506:Gene Expression Regulation, Plant; D016153:Genes, Suppressor; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000235:genetics*; D034622:RNA Interference; D012639:Seeds / Q000502:physiology; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development*; D017864:Tombusvirus / Q000235:genetics*; D019076:Transgenes
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D049452:Green Fluorescent Proteins