Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants.
Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.
Haviv
Sabrina
S
The S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel.
Galiakparov
Nurbol
N
Goszczynski
Dariusz E
DE
Batuman
Ozgur
O
Czosnek
Henryk
H
Mawassi
Munir
M
D001665:Binding Sites; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D016679:Genome, Viral; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D053830:Plant Viral Movement Proteins; D010942:Plant Viruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012328:RNA Viruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics
D036022:Capsid Proteins; D053830:Plant Viral Movement Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D004262:DNA Restriction Enzymes; D005966:Glucuronidase