Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA.

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.

Characterization of the seed virome of alfalfa (Medicago sativa L).

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.

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.

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA.

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.

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.

In silico identification of transcription factors in Medicago sativa using available transcriptomic resources.

Transcription factors (TFs) are proteins that govern organismal development and response to the environment by regulating gene expression. Information on the amount and diversity of TFs within individual plant species is critical for understanding of their biological roles and evolutionary history across the plant kingdom. Currently, only scattered information on separate TFs is available for alfalfa, the most extensively cultivated forage legume in the world. In the meantime, several large transcriptomic resources that can be used to identify and characterize alfalfa TF genes are freely accessible online. In this study, we have performed an in silico analysis of transcriptome data generated in our laboratory and publicly acquirable from other sources to reveal and systematize alfalfa transcription factors. Transcriptome-wide mining enabled prediction of 983 TFs along with their sequence features and putative phylogenies of the largest families. All data were assembled into a simple open-access database named AlfalfaTFDB ( http://plantpathology.ba.ars.usda.gov/alfalfatfdb.html ). Transcriptomic analysis used in this work represents an effective approach for the identification of TF genes in plants with incomplete genomes, such as alfalfa. Integrated TF repertoires of Medicago sativa will provide an important tool for studying regulation of gene expression in other complex non-model species of agricultural significance.

Analysis of the alfalfa root transcriptome in response to salinity stress.

Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. To date, no studies have been published on a deep sequencing-based profiling of the alfalfa transcriptome in response to salinity stress. Observations gathered through research on reference genomes may not always be applicable to alfalfa. In this work, Illumina RNA-sequencing was performed in two alfalfa genotypes contrasting in salt tolerance, in order to estimate a broad spectrum of genes affected by salt stress. A total of 367,619,586 short reads were generated from cDNA libraries originated from roots of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained and, among them, 74.5% had a significant similarity to proteins in the NCBI database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496 SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that the expression of 1,165 genes, including 86 transcription factors (TFs), was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology (GO) categories using Arabidopsis GO. A random check of differentially expressed genes by quantitative real-time PCR confirmed the bioinformatic analysis of the RNA-seq data. A number of salt-responsive genes in both tested genotypes were identified and assigned to functional classes, and gene candidates with roles in the adaptation to salinity were proposed. Alfalfa-specific data on salt-responsive genes obtained in this work will be useful in understanding the molecular mechanisms of salinity tolerance in alfalfa.

Composition of the alfalfa pathobiome in commercial fields.

Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome". Pathobiome is a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), the most extensively cultivated forage legume in the world, is non-existent. Here, we approached this subject by characterizing the biodiversity of the alfalfa pathobiome using high-throughput sequencing technology. Our metagenomic study revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows us to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis.

Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses.

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.

Clustering of Pathogen-Response Genes in the Genome of Arabidopsis thaliana.

Previously, we used heterologous expressed sequence tag (EST) mapping to generate a profile of 4 935 pathogen-response genes of Arabidopsis thaliana. In this work, we performed a computer analysis of this profile, revealing 1 594 non-homologous clustered genes distributed among all A. thaliana chromosomes, whose co-regulation may be related to host responses to pathogens. To supplement computer data, we arbitrarily selected two clusters and analyzed their expression levels in A. thaliana ecotypes Col-0 and C24 during infection with the yellow strain of Cucumber mosaic virus CMV(Y). Ecotype Col-0 is susceptible to CMV(Y), whereas C24 contains the dominant resistance gene RCY1. Upon infection with CMV(Y), all clustered genes were significantly activated in the resistant ecotype C24. In addition, we demonstrated that posttranslational histone modifications associated with trimethylation of histone H3 lysine 27 are most likely involved in regulation of several cluster genes described in this study. Overall, our experiments indicated that pathogen-response genes in the genome of A. thaliana may be clustered and co-regulated.

Mapping of heterologous expressed sequence tags as an alternative to microarrays for study of defense responses in plants.

BACKGROUND: Microarray technology helped to accumulate an immense pool of data on gene expression changes in response to different environmental factors. Yet, computer- generated gene profiling using expressed sequence tags (EST) represents a valuable alternative to microarrays, which allows efficient discovery of homologous sequences in evolutionarily different species and comparison of gene sets on the whole genome scale. In this study, we used publicly available EST database derived from different plant species infected with a variety of pathogens, to generate an expression profile of homologous genes involved in defense response of a model organism, Arabidopsis thaliana. RESULTS: EST-driven prediction identified 4,935 genes (16% of the total Arabidopsis genome) which, according to the origin of EST sets, were associated with defense responses in the reference genome. Profiles of defense-related genes, obtained by mapping of heterologous EST, represent putative Arabidopsis homologs of the corresponding species. Comparison of these profiles in pairs and locating common genes allowed estimating similarity between defense-related gene sets of different plant species. To experimentally support computer data, we arbitrarily selected a number of transcription factor genes (TF) detected by EST mapping. Their expression levels were examined by real-time polymerase chain reaction during infection with yellow strain of Cucumber mosaic virus, a compatible virus systemically infecting Arabidopsis. We observed that 65% of the designated TF were upregulated in accordance with the EST-generated profile. CONCLUSION: We demonstrated that heterologous EST mapping may be efficiently used to reveal genes involved in host defense responses to pathogens. Upregulated genes identified in this study substantially overlap with those previously obtained by microarrays.

Volatile Evolution of Long Non-Coding RNA Repertoire in Retinal Pigment Epithelium: Insights from Comparison of Bovine and Human RNA Expression Profiles.

Currently, several long non-coding RNAs (lncRNAs) (TUG1, MALAT1, MEG3 and others) have been discovered to regulate normal visual function and may potentially contribute to dysfunction of the retina. We decided to extend these analyses of lncRNA genes to the retinal pigment epithelium (RPE) to determine whether there is conservation of RPE-expressed lncRNA between human and bovine genomes. We reconstructed bovine RPE lncRNAs based on genome-guided assembly. Next, we predicted homologous human transcripts based on whole genome alignment. We found a small set of conserved lncRNAs that could be involved in signature RPE functions that are conserved across mammals. However, the fraction of conserved lncRNAs in the overall pool of lncRNA found in RPE appeared to be very small (less than 5%), perhaps reflecting a fast and flexible adaptation of the mammalian eye to various environmental conditions.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae.

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.

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.

Complete Genome Sequence of the Alfalfa latent virus.

The first complete genome sequence of the Alfalfa latent carlavirus (ALV) was obtained by primer walking and Illumina RNA sequencing. The virus differs substantially from the Czech ALV isolate and the Pea streak virus isolate from Wisconsin. The absence of a clear nucleic acid-binding protein indicates ALV divergence from other carlaviruses.

Gene Expression Profiling in Viable but Nonculturable (VBNC) Cells of Pseudomonas syringae pv. syringae.

Pseudomonas syringae infects diverse crop plants and comprises at least 50 different pathovar strains with different host ranges. More information on the physiological and molecular effects of the host inhibitory environment on the pathogen is needed to develop resistant cultivars. Recently, we reported an in vitro model system that mimics the redox pulse associated with the oxidative burst in plant cells inoculated with Pseudomonas syringae pv. syringae. Using this system, we demonstrated that oxidation of acetosyringone, a major extracellular phenolic compound induced in some plants in response to bacteria, rendered Pseudomonas syringae pv. syringae to a "viable but nonculturable" (VBNC) state. Here we performed a large scale transcriptome profiling of P. s. pv. syringae in the VBNC state induced by acetosyringone treatment and identified bacterial genes and pathways presumably associated with this condition. The findings offer insight into what events occur when bacterial pathogens are first encountered and host defense responses are triggered. The acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance. We believe that this is the first work on global gene expression profiling of VBNC cells in plant pathogenic bacteria.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

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.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

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.