Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.
Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.
Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada.
Andriy
A
Bilichak
Youli
Y
Yao
Igor
I
Kovalchuk
D060054:Agrobacterium / Q000502:physiology*; D017360:Arabidopsis / Q000235:genetics* / Q000382:microbiology*; D029681:Arabidopsis Proteins / Q000378:metabolism; D015139:Blotting, Southern; D053903:DNA Breaks, Double-Stranded; D019175:DNA Methylation / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015536:Down-Regulation; D044127:Epigenesis, Genetic; D017343:Genes, Plant; D056426:Genetic Loci; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D030821:Plants, Genetically Modified; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D059386:Reverse Genetics; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D021122:Protein Subunits; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C036483:T-DNA; D012321:DNA-Directed RNA Polymerases; C568761:NRPD1A protein, Arabidopsis
AGO2; Arabidopsis thaliana; NRPD1a; epigenetics; plant transformation; virus-induced gene silencing