Specific binding between the coat protein (CP) and the helper component (HC) of the tobacco vein mottling potyvirus (TVMV) was characterized using a protein blotting-overlay protocol. In this in vitro assay, HC interacted with either virions or CP monomers originating from the aphid-transmissible TVMV-AT but not from the non-aphid-transmissible TVMV-NAT. There was a strong correlation between the aphid transmissibility of a series of TVMV variants having mutations in the DAG motif of the CP and their ability to bind HC. Expression of TVMV CP derivatives in bacteria allowed a precise determination of the minimum domain mediating HC binding. This domain is composed of seven amino acids, including the DAG motif (DTVDAGK), located in the N-terminus of the TVMV CP at amino acid positions 2 to 8.

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

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Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N-P and M-M interactions are common to all three viruses. Associations unique to only one virus include P-M for LNYV, G-Mv for SYNV and M-Mv, M-G and N-M for PYDV. The cognate N-P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed. INTRODUCTIONRhabdoviruses are pathogens of vertebrates, invertebrates and plants that can have serious effects on human health, agriculture and wildlife. Taxonomically, the family Rhabdoviridae is composed of six genera, four of which infect animals and humans and replicate in the cytoplasm of infected cells. Rhabdoviruses that infect plants are assigned to two genera, Nucleorhabdovirus and Cytorhabdovirus . Nucleorhabdoviruses replicate and assemble in the nucleus, whereas this occurs in the cytoplasm for the cytorhabdoviruses (Jackson et al., 2005). Lettuce necrotic yellows virus (LNYV) is the type species of the genus Cytorhabdovirus. In 1954, LNYV was first recognized as a destructive pathogen of lettuce (Lactuca sativa L.) causing a chlorotic and flattened appearance in the mature leaves with varying degrees of necrosis (Stubbs & Grogan, 1963). The virus forms viroplasms in the cytoplasm of infected cells and is sap transmissible from lettuce or sowthistle (Sonchus sp.) to several indicator species including Nicotiana glutinosa and petunia (Petunia 6 hybrida), but not to lettuce or Nicotiana benthamiana. LNYV has been reported from Australia and New Zealand, where it is transmitted in a propagative and persistent manner by the aphid vector Hyperomyzus lactucae (Dietzgen et al., 2006;Stubbs & Grogan, 1963). LNYV biological, physicochemical and genomic properties have been reviewed (Dietzgen et al., 2007). Phylogenetically, LNYV is most closely related to two other cytorhabdoviruses, Strawberry crinkle ...

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