Engineering of alfalfa mosaic virus RNA 3 into an expression vector.
RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protein (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.
Sanchez-Navarro
J
J
Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, The Netherlands.
Miglino
R
R
Ragozzino
A
A
Bol
J F
JF
D020413:3' Untranslated Regions; D017794:Alfalfa mosaic virus / Q000235:genetics* / Q000472:pathogenicity; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D018076:DNA, Complementary / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018519:Meristem / Q000821:virology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000378:metabolism; D053830:Plant Viral Movement Proteins; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011523:Protoplasts / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics; D014774:Virulence
D020413:3' Untranslated Regions; D036022:Capsid Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C095691:coat protein, Alfalfa mosaic virus; D049452:Green Fluorescent Proteins