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A novel procedure for the localization of viral RNAs in protoplasts and whole plants
Author(s) -
Zhang Fengli,
Simon Anne E.
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.01837.x
Subject(s) - green fluorescent protein , biology , fusion protein , protoplast , cytoplasm , virus , nuclear localization sequence , microbiology and biotechnology , movement protein , virology , plant virus , rna , coat protein , gene , recombinant dna , genetics
Summary Analysis of virus spread using co‐expressed reporter proteins has provided important details on cell‐to‐cell and long‐distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non‐viral segments or loss of any open‐reading frames, procedures required to detect viruses non‐evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CP MS2 ) to a 19 base hairpin (hp). A fusion protein, consisting of the CP MS2 , green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear‐localized upon transient expression in protoplasts. However, addition of the hp to the 3′ untranslated region of Turnip crinkle virus (TCV‐hp) and co‐transfection of the virus and fusion protein construct into protoplasts resulted in the re‐location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP‐NLS‐CP MS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV‐hp‐inoculated leaves at 2 days post‐inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.