Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BACKGROUND: We have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics. METHODS: This research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others. RESULTS: In this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease. CONCLUSIONS: Consistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PURPOSE: The RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter. METHODS: ARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting. RESULTS: ARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation. CONCLUSIONS: The ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated.

Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana.

In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development.