One of the most important achievements of the past decade has been the development of strategies for reverse genetic analyses of negative strand RNA viruses infecting animals. Reverse genetics methods have now been applied to members of most families of these viruses, but technical obstacles have prevented applications of similar technologies to the plant negative strand viruses. The goal of this application is to develop methods for genetic analysis of a plant rhabdovirus, sonchus yellow net virus (SYNV) and to devise strategies that can be adapted to other plant negative strand viruses. SYNV is a particularly appropriate subject for these studies because of the extensive background information we have accumulated about the virus and the large repertoire of available recombinant materials that can be applied to the proposed study. We have recently made important advances in my laboratory by constructing vectors that permit routine expression of large amounts of proteins in nearly every cell of leaves infiltrated with bacteria harboring the vectors. The approach should be easily modified to provide simultaneous expression of the core polymerase proteins and cloned SYNV RNA derivatives for de novo generation of recombinant derivatives of the virus. Achievement of these goals will lead to a better understanding of the replication and pathology of the negative strand viruses as a whole, and will also provide a basis for engineering of protein expression vehicles suitable for stable and large scale plant-based production of proteins that are difficult to produce by other means. This application thus represents a new approach to recovery of recombinant plant negative strand viruses, and provides opportunities to explore applications that may have implications for biotechnology and medicine. Specific Aim 1 is to generate a recombinant viroplasm capable of replicating SYNV RNA derivatives and expressing reporter genes encoded within the derivatives. Specific Aim 2 is to construct a minigenome that can replicate autonomously and express encoded reporter genes when incorporated into biologically active nucleocapsids. A bipartite virus consisting of the autonomously replicating minigenome and a minigenome containing the matrix and envelope proteins will also be tested for the ability to infect plants. Genetic analyses using this system could provide valuable information about the requirements of each of the viral genes for aspects of replication, morphogenesis and cell-to-cell movement. Specific Aim 3 focuses on construction of biologically active SYNV derivatives that can be used to evaluate the conditions necessary for whole plant infections, the roles of the viral genes in disease development, and the feasibility of using SYNV as a vehicle to invade host plants and express abundant amounts of foreign proteins suitable for vaccines and other biomedical uses. [unreadable] [unreadable]