A modified viral satellite DNA-based gene silencing vector is effective in association with heterologous begomoviruses

We have previously reported effective gene silencing of a transgene and endogenous plant genes in tobacco and tomato plants using a modified viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we constructed a similar gene silencing vector (DNADeltaC12beta) based on the satellite DNAbeta associated with Tobacco curly shoot virus (TbCSV) by replacing its betaC1 gene with a multiple cloning site. Strong and stable silencing of cognate genes was achieved when this vector, carrying a fragment of the green fluorescent protein (GFP) transgene or a sulfur (Su) endogenous gene encoding one unit of the chloroplast enzyme magnesium chelatase required for chlorophyll II production, was co-agroinoculated with TbCSV used as a helper virus. GFP silenced transgenic Nicotiana benthamiana plants appear red under UV illumination due to loss of green fluorescence, while the Su silenced plants appear white as a result of failure to synthesize chlorophyll. Our results show that the efficiency of Su silencing is independent of the insert orientation in both N. benthamiana and N. glutinosa plants. Most significant however, is the observation that in association with heterologous begomoviruses, such as TYLCCNV or Malvastrum yellow vein virus, the DNADeltaC12beta vector could still effectively induce transgene and endogenous gene silencing in tobacco plants. These observations suggest that the modified viral satellite DNA vector can be applied as a reverse genetics tool for the study, analysis and discovery of gene function in more plants.