Kay Scheets

Detection of members of the Tombusviridae in the Tallgrass Prairie Preserve, Osage County, Oklahoma, USA

Viruses are most frequently discovered because they cause disease in organisms of importance to humans. To expand knowledge of plant-associated viruses beyond these narrow constraints, non-cultivated plants of the Tallgrass Prairie Preserve, Osage County, Oklahoma, USA were systematically surveyed for evidence of the presence of viruses. This report discusses viruses of the family Tombusviridae putatively identified by the survey. Evidence of two carmoviruses, a tombusvirus, a panicovirus and an unclassifiable tombusvirid was found. The complete genome sequence was obtained for putative TGP carmovirus 1 from the legume Lespedeza procumbens, and the virus was detected in several other plant species including the fern Pellaea atropurpurea. Phylogenetic analysis of the sequence and partial sequence of a related virus supported strongly the placement of these viruses in the genus Carmovirus. Polymorphisms in the sequences suggested existence of two populations of TGP carmovirus 1 in the study area and year-to-year variations in infection by TGP carmovirus 3.

Pelarspovirus, a proposed new genus in the family Tombusviridae

Currently, the family Tombusviridae encompasses thirteen viral genera that contain single-stranded, positive-sense RNA genomes and isometric virions; the exception being the genus Umbravirus, whose members do not encode a coat protein (CP). A new genus, tentatively named Pelarspovirus, is proposed to be added to this family and would include five members, with Pelargonium line pattern virus recommended as the type species. Viruses assigned to this proposed genus have monopartite genomes encoding five open reading frames (ORFs) that include two 5′-proximal replication proteins, two centrally located movement proteins (MP1 and MP2) and a 3′-proximal CP that, at least for pelargonium line pattern virus (PLPV), has been shown to act also as suppressor of RNA silencing. Distinguishing characteristics of these viruses include i) production of a single, tricistronic subgenomic RNA for expression of MP and CP genes, ii) presence of a non-AUG start codon (CUG or GUG) initiating the MP2 ORF, iii) absence of AUG codons in any frame between the AUG initiation codons of MP1 and CP genes, and iv) sequence-based phylogenetic clustering of all encoded proteins in separate clades from those of other family members.

Infectious transcripts of an asymptomatic panicovirus identified from a metagenomic survey.

A new panicovirus was identified from a metagenomic survey of plant viruses in the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, USA. Thin paspalum asymptomatic virus (TPAV) was isolated from Paspalum setaceum var. muehlenbergii. The complete 4195 nucleotide sequence encodes six proteins of 44, 109, 8, 6.8, 26, and 15kDa, whose genes are similar in size and location to those of panicum mosaic virus (PMV) and cocksfoot mild mosaic virus (CMMV). Infectious transcripts made from cDNAs of the 2005 isolate were used for initial host range analyses. Of the 11 tested grasses, only Paspalum setaceum (var. stramineum), Setaria italica, and Setaria viridis were hosts, producing asymptomatic infections, and Nicotiana benthamiana was not infected. Protein and RNA alignments indicate that TPAV is more closely related to PMV than CMMV. RNA alignments and structure predictions indicate that TPAV and CMMV have conserved eight structural elements previously predicted or analyzed for PMV. The initial TPP specimen also contained RNAs related to alphacryptoviruses. Partial sequences of RNA1 (∼35%) and RNA2 (∼40%) of putative thin paspalum cryptic virus were obtained. These represent the first sequences for a putative grass-infecting alphacryptovirus.

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.