Ourmia melon virusOurmia melon virus (OuMV) is the type member of the genus Ourmiavirus. OuMV was first isolated in melon crops in Ourmia in Iran (35,53), the only country where its presence has been documented. Since its early discovery, OuMV has raised curiosity because of its unique virion morphology compared to other plant viruses: bacilliform particles of three different lengths with pointed ends and a tubular body containing discontinuities marked by fissures (35). Evidence was shown that each genomic RNA is encapsidated independently (35). Within the same genus are two other viruses: Epirus cherry virus (EpCV) (7) and Cassava virus C (CaVC) (3). No biological vector has been implicated in plant-to-plant transmission in the field (1, 42). Recent nucleotide sequence and phylogenetic analyses have shown that the three Ourmiavirus species have a conserved genome organization (53). All have a trisegmented plus-strand RNA genome. Each segment potentially encodes only one protein (Fig. 1). The largest RNA segment contains open reading frame 1 (ORF1) which encodes a putative RNA-dependent RNA polymerase (RdRP). The RdRP is phylogenetically related to the RdRPs of viruses in the Narnaviridae, a heterogeneous group comprised chiefly of viruses which infect fungi, including the yeast Saccharomyces cerevisiae. The RNA2 ORF2 encodes a putative movement protein (MP) (circa 30 kDa). In contrast to the phylogenetic relationships for RNA1, the OuMV MP shares similarities with MPs of plant-infecting viruses of the genus Tombusvirus. Finally, RNA3 encodes the 22-kDa coat protein (CP) which has no obvious phylogenetic relationships to any protein in the database (53). The different phylogenetic origins of RNA1 and RNA2 (from a fungal virus and a plant virus, respectively) point to interkingdom virus reassortment as a possible new mechanism of viral evolution apt to induce the emergence of a novel viral genus (32, 53). To better understand the molecular mechanisms behind the Ourmiavirus life cycle, a reverse genetic system is required.Here, we describe the development of a reverse genetic system for OuMV launched by a mix of three Agrobacterium tumefaciens clones harboring binary plasmids expressing each of the genomic RNAs under the control of the 35S promoter. Using this system, we provide new genetic evidence for the role of each virus-encoded protein in the infection cycle in N. benthamiana plants. Furthermore, we identified unexpected genetic requirements for virion assembly. MATERIALS AND METHODSConstruction of full-length agroinfectious clones. The full-length cDNAs of each of the three genomic segments was obtained through reverse transcription-PCR (RT-PCR) using as a template RNA obtained from plants infected with the OuMV isolate VE9. RNA was extracted with a Spectrum Plant Total RNA kit and buffer A (Sigma, St. Louis, MO), following the manufacturer's protocol. Reverse transcription was carried out as previously described (65). PCR was done using Phusion (Finnzymes, Espoo, Finland) as previously described and with ...

Cpkk1 and Cpkk2 are two previously characterized Mitogen-activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search.