Molecular characterization, ecology, and epidemiology of a novel Tymovirus in Asclepias viridis from Oklahoma.

Native virus-plant interactions require more understanding and their study will provide a basis from which to identify potential sources of emerging destructive viruses in crops. A novel tymovirus sequence was detected in Asclepias viridis (green milkweed), a perennial growing in a natural setting in the Tallgrass Prairie Preserve (TGPP) of Oklahoma. It was abundant within and frequent among A. viridis plants and, to varying extents, within other dicotyledonous and one grass (Panicum virgatum) species obtained from the TGPP. Extracts from A. viridis containing the sequence were infectious to a limited number of species. The virus genome was cloned and determined to be closely related to Kennedya yellow mosaic virus. The persistence of the virus within the Oklahoma A. viridis population was monitored for five successive years. Virus was present in a high percentage of plants within representative areas of the TGPP in all years and was spreading to additional plants. Virus was present in regions adjacent to the TGPP but not in plants sampled from central and south-central Oklahoma. Virus was present in the underground caudex of the plant during the winter, suggesting overwintering in this tissue. The RNA sequence encoding the virus coat protein varied considerably between individual plants (≈3%), likely due to drift rather than selection. An infectious clone was constructed and the virus was named Asclepias asymptomatic virus (AsAV) due to the absence of obvious symptoms on A. viridis.

A host-factor interaction and localization map for a plant-adapted rhabdovirus implicates cytoplasm-tethered transcription activators in cell-to-cell movement.

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.

Genome Structure and Production of Biologically Active In Vitro Transcripts of Cucurbit-Infecting Zucchini green mottle mosaic virus.

ABSTRACT The complete nucleotide sequence of the Zucchini green mottle mosaic virus (ZGMMV), a new member of the genus Tobamovirus, has been determined. The genome of ZGMMV is 6,513 nucleotides long and contains four open reading frames coding for proteins of 131, 189, 28, and 17 kDa from the 5' to 3' end, respectively. The 5'- and 3'-non-translated regions consist of 59 and 163 residues, respectively. The sequences of the viral proteins exhibit high identity to the proteins of the members of the genus Tobamovirus and are distinct from other viruses within the subgroup of cucurbit-infecting tobamoviruses. Results from phylogenetic trees of the coding regions demonstrated that ZGMMV is a very close relative of Kyuri green mottle mosaic virus and Cucumber fruit mottle mosaic virus and is less similar to Cucumber green mottle mosaic virus. Full-length cDNA of ZGMMV was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using the 5'-end primer containing a T7 RNA promoter sequence and 3'-end primer. Capped in vitro transcript from the RT-PCR products was infectious on zucchini squash, cucumber, and Nicotiana benthamiana plants. This cell-free system to produce infectious transcripts from uncloned cDNA copies is useful for quick assessment of infectivity of transcripts from plant RNA viruses prior to cloning. Synthesized capped transcript from a full-length cDNA clone of the virus was highly infectious. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first report of a biologically active transcript from a cucurbit-infecting tobamovirus.