Analysis of RNA-mediated gene silencing using a new vector (pKNOCKOUT) and an in planta Agrobacterium transient expression system

A hairpin RNA (hpRNA) vector, pKNOCKOUT (pKO) has been constructed to facilitate the analysis of posttranscriptional gene silencing (PTGS) in an Agrobacterium-mediated transient expression system developed for tobacco. The pKO binary vector was tested by cloning a firefly luciferase (Photinus pyralis) gene segment in sense (sFLUC), antisense (aFLUC), and inverted repeat (ihpFLUC) orientations. The inverted repeats of the target gene are separated by the castor bean catalase intron (CbCi) and, when transcribed and spliced, produce a self-complementary hpRNA. hpRNA-mediated gene silencing exploits a cellular mechanism that recognizes double-stranded RNA (dsRNA) and subjects it, and homologous mRNA molecules, to sequence-specific degradation. Agrobacteria harboring compatible plasmids, pE1778-RiLUC (Renilla luciferase normalization construct) and pTCaMV35S-FiLUC (functional firefly luciferase test construct), were co-infused with different variants of pKO-FLUC plasmids into Nicotiana benthamiana and Nicotiana tobaccum leaf tissues. Reduced firefly luciferase reporter gene activity (from pTCaMV35S-FiLUC) indicated gene silencing and was observed in leaf tissues co-infused with pKO-sFLUC (50% reduction), pKO-aFLUC (85% reduction), or pKO-ihpFLUC (96% reduction) agrobacteria lines. Gene silencing was observed at different times postinfusion in leaves from both Nicotiana species. The hpRNA-mediated interference with FiLUC reporter gene expression, as measured by reduced firefly luciferase activity, was found to also suppress silencing of the cotransferred Renilla reniformis luciferase normalization reporter gene, resulting in reproducibly elevated RiLUC activity. This suppression effect was reduced by lowering the percentage of infused pKO-ihpFLUC agrobacteria without markedly effecting hpRNA-mediated gene silencing of FiLUC (all pKO-ihpFLUC dilutions produced >90% reduction of FiLUC activity). Viral suppressors of PTGS, such as p19 and HcPro, were found to reduce the RNAi effect of hpRNAmediated gene silencing. To our knowledge, this is the first demonstration that excess amounts of targeted dsRNA can result in nonspecific suppression of PTGS of an unrelated, co-infused reporter gene in plants.