ANALYSIS OF RNA-MEDIATED GENE SILENCING USING A NEW VECTOR (PKNOCKOUT) AND AN IN PLANTA AGROBACTERIUM TRANSIENT EXPRESSION SYSTEM

Authors: Cazzonelli, Christopher; Velten, Jeffrey

Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2004
Publication Date: 12/1/2004
Citation: Cazzonelli, C.I., Velten, J.P. 2004. Analysis of rna-mediated gene silencing using a new vector (pknockout) and an in planta agrobacterium transient expression system. Plant Molecular Biology Reporter. 22:347-359.

Interpretive Summary: A new gene-silencing vector pKO has been constructed to facilitate the analysis of posttranscriptional gene silencing in an a transient expression system. The pKO binary vector was tested by cloning a firefly luciferase (Photinus pyralis) gene segment in several orientations. Expression of the silencing genetic constructs reduced expression of a firefly luciferase reporter gene by up to 95%. Gene silencing was observed at different times post-infusion in leaves from two plant species. A hairpin-RNA (double stranded transcript) producing version of the silencing plasmid, targeted to firelfly luciferase was found to simultaneously partially release silencing of an unrelated sea pansy reporter gene, resulting in reproducibly elevated sea pansy luciferase activity. This suppression effect was reduced by lowering expression from the firefly hairpin silencing construct. Viral suppressors of silencing, such as p19 and pHcPro, were found to reduce the silencing effect of hpRNA production. To our knowledge, this is the first demonstration that excess amounts of targeted dsRNA can result in nonspecific suppression of silencing of an unrelated, co-e expressed gene in plants.

Technical Abstract: A hairpin RNA (hpRNA) vector, pKNOCKOUT (pKO) has been constructed to facilitate the analysis of posttranscriptional gene silencing (PTGS) in an agrobacteria-mediated transient expression system developed for tobacco. The pKO binary vector was tested by cloning a firefly luciferase (Photinus pyralis) gene segment in the sense (sFLUC), antisense (aFLUC) and inverted repeat (ihpFLUC) orientations. The inverted repeats of the target gene are separated by the castor bean catalase intron, and when transcribed and spliced produce a self-complementary hairpin RNA. 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), indicating gene silencing, was observed in leaf tissues co-infused with pKO-sFLUC (50% reduction), pKO-aFLUC (85% reduction) or pKO-ihpFLUC (95% reduction) agrobacteria lines. Gene silencing was observed at different times post-infusion 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 co-transferred 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% eduction of FiLUC activity). Viral suppressors of PTGS such as p19 and pHcPro were found to reduce the RNAi effect of hpRNA mediated 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.