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Age-related macular degeneration is a major cause of vision impairment in the Western world among people of 55 years and older. Recently we have shown that autophagy is dysfunctional in the retinal pigment epithelium (RPE) of the AMD donor eyes (AMD RPE). We also showed increased reactive oxygen (ROS) production, increased cytoplasmic glycogen accumulation, mitochondrial dysfunction and disintegration, and enlarged and annular LAMP-1-positive organelles in AMD RPE. However, the underlying mechanisms inducing these abnormalities remain to be elucidated. Here, by performing a comprehensive study, we show increased PAPR2 expression, deceased NAD+, and SIRT1, increased PGC-1α acetylation (inactive form), lower AMPK activity, and overactive mTOR pathway in AMD RPE as compared to normal RPE. Metabolomics and lipidomics revealed dysregulated metabolites in AMD RPE as compared to normal RPE, including glycerophospholipid metabolism, involved in autophagy, lipid, and protein metabolisms, glutathione, guanosine, and L-glutamic acid, which are implicated in protection against oxidative stress and neurotoxicity, further supporting our observations. Our data show dysregulated metabolic pathways as important contributors to AMD pathophysiology, and facilitate the development of new treatment strategies for this debilitating disease of the visual system.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties.

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa ( Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae .

The visual cycle is an important pathway in the retinal pigment epithelium (RPE) which regenerates 11- cis retinal chromophore for the retinal photoreceptors. The central enzyme in the visual cycle is RPE65 retinol isomerase. Expression of RPE65 mRNA and protein levels are significantly lower in RPE cell culture models when compared to native RPE. This limits the use of these models to study the visual cycle. To determine the main drivers of RPE65 regulation we compared the transcriptional profiles of native and cell culture models of RPE with various levels of RPE65 expression. We also compared the levels of RPE65 expression between ARPE-19 cells grown in media supplemented with 1 mM pyruvate (PYR) or 10 mM nicotinamide (NAM). In addition, we performed experiments directed at transcriptional and translational regulation of RPE65. We show that RPE65 mRNA and protein expression is significantly higher in NAM media grown cells than PYR cells. Transfection of cells with a variety of different vectors containing RPE65 ORFs with different promoters, codon optimization, IRES, 3’ UTRs, suggest that translational effects are less important than transcriptional status. Importantly, we found that feeding with rod outer segments (ROS) decreases RPE65 expression in NAM grown cells, suggesting that certain primary functions of the RPE (here, visual cycle and phagocytosis) are not positively linked. Analysis of differentially regulated microRNAs (miRs) provides a basis for this downregulation. It appears that the regulation of RPE65 expression in ARPE-19 cells, in particular, is multifactorial, involving primarily metabolic and transcriptional status of the cells, with translation of RPE65 mRNA playing a smaller role.

Vertical transmission of plant pathogenic viruses is an important component of viral persistence, survival, and spread in agricultural production systems. This type of transmission is of considerable economic significance as it can cause major crop losses by serving as the initial focus of infection for future epidemics. Vertical transmission occurs when a virus is passed on to offspring either by direct invasion of the developing seed embryo from infected mother plants or through infected pollen grains after fertilization. We have recently demonstrated via high-throughput sequencing that mature seeds of the agriculturally important forage crop alfalfa (Medicago sativa L.) are associated with a broad range of viruses, some of which could potentially spread over long distances via seed. With the exception of the alfalfa mosaic virus, little is currently known about viral transmission through alfalfa pollen and its subsequent impact on the disease epidemiology of the crop. The objective of this study was to screen pollen from diverse alfalfa genotypes for pathogenic viruses and assess their risk of transmission. The pollen was collected from alfalfa genotypes selected for fungal disease resistance and agronomic performance in the USDA ARS pre-breeding program in Prosser, WA.

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.

Background Seed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Seed transmission largely depends on the ability of a virus to replicate in reproductive tissues and survive during the seed maturation process. It occurs through the infected embryo or mechanically through the contaminated seed coat. Alfalfa ( Medicago sativa L.) is an important legume forage crop worldwide, and except for a few individual seedborne viruses infecting the crop, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination. Methods For the detection of viruses, we used high throughput sequencing combined with bioinformatic tools and reverse transcription-polymerase chain reactions. Results Our results suggest that, in addition to common viruses, alfalfa seeds are infected by other potentially pathogenic viral species that could be vertically transmitted to offspring. Conclusions To the best of our knowledge, this is the first study of the alfalfa seed virome carried out by HTS technology. This initial screening of alfalfa germplasm accessions maintained by the NPGS showed that the crop’s mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to update germplasm distribution policies and to make decisions on the safety of distributing germplasm based on viral presence.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Background At the moment, there are a number of publications describing gene expression profiling in virus-infected plants. Most of the data are limited to specific host-pathogen interactions involving a given virus and a model host plant – usually Arabidopsis thaliana . Even though several summarizing attempts have been made, a general picture of gene expression changes in susceptible virus-host interactions is lacking. Methods To analyze transcriptome response to virus infection, we have assembled currently available microarray data on changes in gene expression levels in compatible Arabidopsis- virus interactions. We used the mean r (Pearson’s correlation coefficient) for neighboring pairs to estimate pairwise local similarity in expression in the Arabidopsis genome. Results Here we provide a functional classification of genes with altered expression levels. We also demonstrate that responsive genes may be grouped or clustered based on their co-expression pattern and chromosomal location. Conclusions In summary, we found that there is a greater variety of upregulated genes in the course of viral pathogenesis as compared to repressed genes. Distribution of the responsive genes in combined viral databases differed from that of the whole Arabidopsis genome, thus underlining a role of the specific biological processes in common mechanisms of general resistance against viruses and in physiological/cellular changes caused by infection. Using integrative platforms for the analysis of gene expression data and functional profiling, we identified overrepresented functional groups among activated and repressed genes. Each virus-host interaction is unique in terms of the genes with altered expression levels and the number of shared genes affected by all viruses is very limited. At the same time, common genes can participate in virus-, fungi- and bacteria-host interaction. According to our data, non-homologous genes that are located in close proximity to each other on the chromosomes, and whose expression profiles are modified as a result of the viral infection, occupy 12% of the genome. Among them 5% form co-expressed and co-regulated clusters.