title: Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR.
abstract: RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.
authors.affiliation: Plant Biotechnology Research Group, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Taylor
authors.forename: Johan
authors.initials: J
authors.lastname: Harmse
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.forename: Fiona
authors.initials: F
authors.lastname: Van Den Berg
authors.forename: Marco S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: Marie E C
authors.initials: ME
authors.lastname: Rey
mesh_terms: D020137:Base Pair Mismatch / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics*; D003641:Deamination / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D055029:Inverted Repeat Sequences / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000187:drug effects / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics / Q000821:virology; D014170:Transformation, Genetic / Q000187:drug effects
chemical_list: D004279:DNA, Viral; D013447:Sulfites; C009279:sodium bisulfite