UBC Theses and Dissertations
Molecular characterization of the cucumber necrosis virus coat protein gene McLean, Morven A.
A cucumber necrosis tombusvirus (CNV) mutant which lacks the coding sequence for the coat protein protruding domain, PD(-), was constructed by site-directed mutagenesis of an infectious CNV cDNA clone, pK2/M5 (wild-type, 4701 nt). Transcripts of PD(-) were infectious in Nicotiana clevelandii, however, the resulting local lesions were significantly smaller than those on the corresponding leaves of plants inoculated with wild-type transcript. In addition, systemic symptoms took 8 to 12 days longer to develop than in wild type-inoculated plants. N. clevelandii inoculated with sap from systemically infected leaves of PD(-) transcript-inoculated plants showed local lesions the same as wild-type andthe delay in systemic symptom development was either reduced or, occasionally, lost. Systemic symptoms in both PD(-) transcript-and sap-inoculated N. clevelandii were either similar to thesystemic necrosis of wild-type or an oak-leaf necrosis was observed. High molecular weight RNA from mutant- and wild-type-infected plants was extracted and analyzed by Northern blotting. Full-length PD(-) RNA could be detected only rarely in RNA preparations from transcript-inoculated leaves; a further deleted, stable RNA species of approximately 3800 nucleotides was found in preparations from systemically infected leaves of PD(-) transcript-and sap-inoculated plants. CNV coat protein could not be detected by ELISA or ISEM in PD(-)-infected leaf material. The ca. 3.8 kbRNA, when cloned and sequenced, was found to have lost all but 74of the 1140 nucleotides of the CNV coat protein open reading frame. Transcripts from this coat-proteinless CNV cDNA clone produced symptoms on N. clevelandii that were similar to those induced inPD(-)-sap-inoculated plants; wild-type and oak-leaf necroses were observed and there was a trend for systemic symptoms to be delayed when compared with wild-type. It would appear that CNV is able to replicate and move systemically, in both transcript-inoculated and sap-inoculated N. clevelandii, in the absence of a functional coat protein. Additionally, mechanical transmission of this virus occurs in the absence of the coat protein, however, such transmission is less efficient when compared with wild-type infections. CNV and tomato bushy stunt tombus virus (TBSV) have the same genome organization and share high levels of nucleotide and amino acid sequence similarity throughout their genomes, yet differ biologically. CNV, for which the natural vector is the fungus Olpidium radicale, has been found only in greenhouse-grown cucumbers. TBSV, by contrast, has a relatively diverse natural host range and has no known vector. The role of the CNV coat protein in determining transmission by 0. radicale was examined by exchanging the coat protein genes contained in full-length cDNA clones of CNV and TBSV. Transcripts from the resulting chimeric clones (CNV with TBSV coat protein and vice versa) were infectious in N. clevelandii. Virus particles, produced in infected plants, were purified and tested for in vitro acquisition and transmission to roots of cucumber seedlings by zoospores of a cucumber strain of O. radicale. Particles produced by the TBSV chimeric clone encoding the CNV coat protein were transmitted, while those produced by the CNV chimeric clone encoding the TBSV coat protein were not. These results demonstrate that the coat protein gene of the CNV genome is specifically involved in fungus transmissibility.
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