A functional histidine-tagged replication initiator protein: implications for the study of single-stranded DNA virus replication in planta.
Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.
Vega-Arreguín
Julio C
JC
Institut des Sciences du Végétal, CNRS, Avenue de la Terrasse, Bât. 23, 91198 Gif sur Yvette Cedex, France.
Timchenko
Tatiana
T
Gronenborn
Bruno
B
Ramírez
Bertha Cecilia
BC
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D025682:Nanovirus / Q000235:genetics*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D016324:Sequence Tagged Sites; D031307:Vicia faba / Q000821:virology*; D014779:Virus Replication
D017931:DNA Primers; D004277:DNA, Single-Stranded; D010448:Peptide Initiation Factors