Stabilization of GFP Expression in a Transient Expression System using Viral Suppressors of Silencing
Advisor:Finer, John J.
Contributors:Chiera, Joseph M.
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Series/Report no.:Horticulture and Crop Science. Graduate student poster competition, 2007
Using transient expression systems, introduction of the green fluorescent protein (gfp) gene results in peak GFP expression 24 hrs post-bombardment (hpb), followed by a rapid decline within 72 hpb. The decline in GFP expression during transient expression probably results from the silencing of the gfp gene. Gene silencing or RNA silencing were developed by plants as a mechanism to combat viruses. As a counter measure, viruses encode certain proteins that suppress the silencing mechanism of plants. We evaluated six viral “suppressors of silencing” for their abilities to stabilize GFP expression using a transient expression system. Suppressor constructs were generated to allow introduction of the suppressor as a fusion with the gfp gene or on a separate plasmid from the gfp gene. Various suppressor constructs were introduced into lima bean (Phaseolus lunatus L.) cotyledons from germinating seedlings, using particle bombardment. Post-introduction, GFP expression was tracked over time using an automated image collection and analysis system. The silencing suppressors HC-Pro and p19 stabilized GFP expression when introduced as 3΄ gfp translational fusions, while p21 and γb stabilized GFP expression when co-introduced with the gfp gene on separate plasmids. The last two suppressors, AL2 and replicase, did not stabilize GFP expression in lima bean system. Introduction of the 3΄ gfp fusion of p19 (35S-gfp::p19-nos) into soybean (Glycine max (L.) Merrill) resulted in recovery of plants with an abnormal downward-leaf curling phenotype, suggesting that p19 not only affects the expression of the gene it is fused with, but also expression of other genes in-trans.
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