The spectrum of organisms and tissues which is susceptible to infection by a virus defines the host range of that virus. Because viruses are intimately associated with their host's cellular machinery, molecular interactions between viral and host cell components are likely to be important factors limiting viral host range. In contrast to the situation for animal-infecting viruses, little is known about the molecular interactions which determine the host range of plant viruses, and these important virus-host interactions warrant closer scrutiny. The research described in this proposal focusses on geminiviruses which are members of a large group of plant-infecting viruses that have small DNA genomes, and are therefore model systems for investigating the mechanisms of gene expression and nuclear DNA replication in higher plants. The long-term objective of this research will be to define the molecular interactions between viral and host components (both nucleic acids and proteins) in host plant species. Host range limitation will initially be investigated by using genetic and biochemical approaches to define critical stages of the geminiviral infection cycle which are blocked at the cellular level in non-host plant species. Four specific questions will be addressed in this research proposal: (1) Can specific geminiviral gene products determine host range? Recombinant DNA techniques will be used to exchange pairs of homologous genes between two geminiviruses which have no hosts in common, and the host range properties of the resulting chimeras will be evaluated. (2) What is the spectrum of mutations which can extend the viral host range? Extended host range geminivirus mutants selected in vivo after inoculation of plants with chemically mutagenized plasmid DNA will be isolated and characterized. The roles of both coding and non-coding regions of the genome will be assessed. (3) Can transcriptional trans-activation of late gene expression by the viral AL2 protein confer host specificity? In vivo transient expression assays will be used to determine whether trans-activation of viral late promoters can occur in non-host plant species. (4) What are the mechanisms underlying the host-specific cell-to-cell movement of geminiviruses in infected plants? Preliminary results show that a geminivirus can replicate in non-host protoplasts, implying that viral movement is defective in planta. Therefore, the applicability of a current model of plant virus cell-to-cell movement will be tested by evaluating the in vitro DNA-binding properties of purified viral BL1 and BR1 proteins, and a genetic approach will be used to test the hypothesis that these two proteins have distinct roles in the process of viral movement.
