Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.
Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1.
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Department of Plant Pathology, 1 Shields Avenue, University of California, Davis, California 95616, USA.
Tian
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Medina
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Falk
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D015152:Blotting, Northern; D029383:Crinivirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D003673:Defective Viruses / Q000235:genetics*; D049452:Green Fluorescent Proteins; D018545:Lactuca / Q000821:virology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014779:Virus Replication
D008164:Luminescent Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins