Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.
Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.
Dolja
V V
VV
Department of Biology, Texas A&M University, College Station 77843.
McBride
H J
HJ
Carrington
J C
JC
D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D016679:Genome, Viral; D005966:Glucuronidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D010942:Plant Viruses / Q000235:genetics* / Q000502:physiology*; D010944:Plants / Q000382:microbiology*; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase; D010450:Endopeptidases