Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus.
To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome component RNA 3 of the virus was modified and split into two sub-components, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (MP) was replaced by a reporter gene encoding the green fluorescent protein (GFP), to monitor virus replication and movement. In RNA 3B, the coat protein (CP) gene was eliminated and a multiple cloning site (MCS) was created for foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector system was evaluated for its ability to deliver and express the bacterial beta-glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat protein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated leaf and enter the minor veins of the inoculated leaf. Foreign gene expression was detected in the inoculated leaves. However, intermolecular recombination between RNA 3A and 3B occurred frequently, preventing efficient systemic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.
Zhao
Y
Y
Molecular Plant Pathology Laboratory, Plant Sciences Institute, USDA-ARS, Beltsville, Maryland, USA.
Hammond
J
J
Tousignant
M E
ME
Hammond
R W
RW
D002213:Capsid / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D003001:Cloning, Molecular; D017799:Cucumovirus / Q000235:genetics* / Q000502:physiology*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000821:virology; D053830:Plant Viral Movement Proteins; D010947:Plants, Toxic; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D036022:Capsid Proteins; D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D014764:Viral Proteins; C065651:coat protein, Bean Yellow Mosaic Virus; D049452:Green Fluorescent Proteins; C087151:RNA 3 coat protein, prune dwarf ilarvirus; D005966:Glucuronidase