Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing.
Using particle bombardment, a DNA expression vector containing the green fluorescent protein (GFP) reporter gene was introduced into plant cells. Expression of the GFP gene was transient; resulting in peak GFP Expression about 24 h post introduction and a rapid decline thereafter. This well known decline in gene expression has previously been attributed to pre-integrative DNA events that involved the loss of introduced DNA or cell death. Here, we show that post-transcriptional gene silencing (PTGS) is also involved. Introduction of a GFP expression vector alone resulted in a rapid decline in transient expression after 30 h. However, if GFP was expressed as a translational fusion to the RNA silencing suppressor protein HCPro from tobacco etch potyvirus, transgene expression was extended to well over 100 h. Mutant analyses of HCPro showed that a functional HCPro protein was required for this extension of transient expression. Various deletion and translational fusion analyses confirmed that the C-terminal region of the protein was important for suppressor activity and the entire protein was required for optimal suppression of host silencing. The transient nature of gene expression during particle bombardment appears to result from induction of PTGS, which can be mitigated by the presence of a suppressor of silencing. The use of RNA silencing suppressor proteins may make particle bombardment-mediated transient expression assays more useful for evaluating factors that effect gene expression.
Chiera
Joseph M
JM
Department of Horticulture and Crop Science, OARDC/The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA.
Lindbo
John A
JA
Finer
John J
JJ
D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D027805:Phaseolus / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D010957:Plasmids; D017800:Potyvirus / Q000235:genetics; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D034741:RNA, Small Interfering; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins