This research is designed to develop systems that can be applied to genetic dissection of plant rhabdovirus infections. Conditions suitable for molecular genetic analyses have recently been developed for several negative-strand animal virus families, and these are having an enormous impact on our understanding of these viruses. Unfortunately, virus recovery using these systems is very inefficient, and their success relies on several key elements that are not available for SYNV, including visual plaque assays in tissue cultures. Therefore the research will investigate two novel alternative strategies, one in tobacco callus tissue and one in yeast, that rely on stringent selection for recovery of replicating minigenomes suitable for genetic analyses. Objective 1 will focus on construction of autonomously replicating minigenomes containing the genes required for replication plus a herbicide resistance gene to be used for selection of plant callus cells supporting the replication of the minigenome. This will use co-transformations with the polymerase genes (N, P, and L) that are required for replication, plus a cassette for transcription of minigenome RNAs encoding the N, P, and L genes and a selectable marker. The expressed viral polymerase proteins should interact with the transcribed minigenome RNAs to "jump start" replication of the minigenome derivative. This strategy should provide selective growth of callus cultures supporting replication of SYNV minigenome replicons that expresses a herbicide resistance gene for selection in the presence of the herbicide bialaphos. These biologically active minigenomes will subsequently be used for genetic analyses to provide information on the replicative activities of SYNV, and as platforms to rescue more complex biologically active SYNV derivatives containing the five common rhabdovirus genes and a selectable antibiotic marker that can be used for discrimination of cells infected with the minigenome or the virus. Experiments will also be carried out to isolate SYNV derivatives containing all six viral genes in order to evaluate the factors necessary for whole plant infections. Objective 2 will explore use of the genetic analysis capability of yeast to study replication of SYNV and to isolate host genes required for replication. For this purpose, an existing yeast cell line expressing the N, P, and L polymerase core proteins will be transformed with a minigenome capable of expressing a selectable marker (URA3) to provide stringent recovery of yeast transformants expressing replicating minigenome derivatives. Achievement of this objective will constitute a crucial step toward creating biologically active recombinant derivatives that can be used to isolate host components required for replication of SYNV. If either of these approaches is successful, our understanding of plant rhabdoviruses replication and pathogenesis will be accelerated enormously and aspects of the yeast selection scheme could have considerable applicability to other negative-strand viruses. A large number of rhabdoviruses cause serious plant diseases, but sonchus yellow net virus (SYNV) is the only well characterized plant rhabdovirus amenable to molecular analysis. SYNV also has several unique attributes that make it attractive for comparative analyses of the infection processes of negative-strand RNA animal virus counterparts. For example, the negative-strand genome and replication strategies of SYNV share many common features with those of animal rhabdoviruses and paramyxoviruses that cause significant human, livestock and wildlife diseases. However, striking differences are evident among these agents with the nuclear phase of SYNV replication being one of the most intriguing features. SYNV is also similar in this aspect of its replication to a distantly related monopartite minus-strand virus, the neurotrophic borna disease virus. Therefore, the infection processes of SYNV have relevance to both plant and animal viruses, so, the knowledge obtained with this virus will promote better understanding of the diverse factors involved in the replication and pathology of a large order of viruses that cause diseases with enormous agricultural and human
