2008 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research project is to develop a transient expression vector based on the negative stranded RNA genomes of the nucleorhabdovirus Maize mosaic virus (MMV). 1b.Approach (from AD-416) 1. Construction a of transient expression vector based on MMV genome in yeast; 2. Analysis of yeast cells and cell lines from diverse organisms for susceptibility to MMV invasion and replication; and, 3. Optimization of the vascular puncture inoculation (VPI) for introduction of wild type and recombinant MMV into maize plants. 3.Progress Report Demonstrated that two viral proteins of Maize fine streak virus (MFSV) use host cell systems for protein transport to move into the plant and insect cell nucleus. MFSV is a nucleorhabdovirus that is transmitted to maize by the black-faced leafhopper Graminella nigrifons. The virus replicates in both its plant host and in its insect vector. Nucleorhabdoviruses replicate in the nucleus of their plant and insect hosts, so the nuclear import of viral proteins is critical for virus assembly. In cellular organisms, two proteins called importin alpha and beta recognize and transport proteins carrying nuclear localization signals into the nucleus. To test the role of the importin proteins in MFSV protein tranport into the nucleus, plasmids carrying fluorescent-protein fusions of the MFSV N and P proteins were transfected into Drosophila S2 cells in which the synthesis of importin alpha was knocked down by RNA interference. In importin alpha-depleted cells the MFSV N protein was distributed throughout the cells, whereas the MFSV N protein was found only in nuclei of control cells. When MFSV N and P genes were co-expressed in importin alpha-depleted cells, both N and P proteins were found throughout cells in contrast to the nuclear localization observed in control cells. ADODR contacts Ohio State University PI personally or by email. Ohio State University researchers working on project meet weekly with ADODR.