SUMMARYHere, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells. INTRODUCTIONPlant-adapted rhabdoviruses are classified into two genera. Members of the genus Nucleorhabdovirus differ from members of the genus Cytorhabdovirus and their mammal-, fishand insect-infecting relatives in that they replicate and undergo morphogenesis in nuclei of infected cells Reed et al., 2005;Revill et al., 2005;Tsai et al., 2005;Dietzgen et al., 2006).Nucleorhabdoviruses share many of the structural features of animal rhabdoviruses such as vesicular stomatitis virus (VSV; Jackson et al., 2005). They are consequently composed of an infectious nucleocapsid 'core' surrounded by a phospholipid membrane. The core can be purified by density-gradient centrifugation of non-ionic detergenttreated virions (Wagner et al., 1996). In the case of Sonchus yellow net virus (SYNV), the core is a ribonucleoprotein (RNP) complex that consists of the negative-strand genomic RNA (Jackson & Christie, 1977) encapsidated by three associated proteins, namely the nucleocapsid (N), phospho-(P) and polymerase (L) proteins Zuidema et al., 1987;Choi et al., 1992). The membrane fraction of mature virions contains a glycoprotein (G) that protrudes from the surface of the virion (Goldberg et al., 1...

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