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Molecular characterization of the interaction between cucumber necrosis virus and zoospores of the fungal vector olpidium bornovanus Robbins, Marjorie Ann

Abstract

In this thesis, the molecular biology of the interaction between Cucumber necrosis tombusvirus (CNV) and zoospores of its fungal vector, Olpidium bornovanus, was examined. Serial passage in the absence of O. bornovanus produced a pool of C NV variants deficient in fungus transmission. Variants of CNV had mutations and/or deletions in genomic RNA that affected various stages of the virus multiplication-cycle, thereby decreasing transmissibility. One transmission mutant, LL5, was selected for further characterization since this mutant retained stable, intact particles and was as infectious as wild-type CNV. Sequencing of the LL5 coat protein (CP) and subsequent site-directed mutagenesis of the CNV CP indicated that a single nucleotide substitution resulting in a Glu to Lys substitution in the CNV CP shell domain was responsible for the reduced transmission of LL5. An in vitro binding assay was developed to determine if LL5 was deficient at the level of attachment to fungal zoospores. LL5 bound zoospores at approximately 50% the level of wild-type CNV indicating that the reduced ability to bind zoospores was at least partially responsible for the reduced transmission. Further examination of the binding of several O. bornovanus-transmitted viruses to O. bornovanus zoospores indicated that the ability to bind zoospores in vitro correlates with the known biological specificity of transmission. Additionally, it was found that binding of CNV to O. bornovanus was saturable, with a maximum of 1.75 X 10[superscript 4] virus particles bound per zoospore. It was also determined that CNV can competitively inhibit binding of another O. bornovanus-transmitted virus, Melon necrotic spot carmovirus. Taken together, these results suggest that O. bornovanus zoospores contain specific receptor molecule(s) for attachment of C N V particles. Amino acid sequence relationships among the coat proteins of several small spherical plant RNA viruses including four known to be transmitted by O. bornovanus were examined. Interestingly, the coat protein sequences of the four O. bornovanus transmitted viruses were closely related, despite the affinities of these viruses to distinct taxonomic groups. Two regions within the otherwise non-conserved coat protein protruding domain were particularly conserved. These data further suggest the involvement of specific coat protein sequences in the fungus transmission process.

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