Browsing by Author "Hackland, Andrew F"

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    The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus
    (1994) Hackland, Andrew F; Thomson, Jennifer Ann; Rybicki, Edward P
    Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I.