Development and characterization of the first infectious clone of alfalfa latent virus, a strain of \u3ci\u3ePea streak virus\u3c/i\u3e

Alfalfa (Medicago sativa) is a natural host plant for many plant pathogens including fungi, bacteria, nematodes and viruses. Alfalfa latent virus (ALV) is strain of Pea streak virus, a member of the carlavirus group that occurs symptomlessly in alfalfa. The first complete genomic sequence of the ALV that was recently obtained in our laboratory showed that the virus differs substantially from other members of the genus Carlavirus. Here we report generation of infectious RNA transcripts from the constructed full-length viral cDNA clone as a proof that ALV nucleotide sequence is correct and as an initial step toward development of the ALV-based vector for gene silencing and expression of foreign proteins in alfalfa. This is the first report describing the development of a complete cDNA clone of the ALV strain of Pea streak virus and its infectivity in the diagnostic pea (Pisum sativum) and natural alfalfa hosts

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

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode

Development and characterization of the first infectious clone of alfalfa latent virus, a strain of \u3ci\u3ePea streak virus\u3c/i\u3e

Alfalfa (Medicago sativa) is a natural host plant for many plant pathogens including fungi, bacteria, nematodes and viruses. Alfalfa latent virus (ALV) is strain of Pea streak virus, a member of the carlavirus group that occurs symptomlessly in alfalfa. The first complete genomic sequence of the ALV that was recently obtained in our laboratory showed that the virus differs substantially from other members of the genus Carlavirus. Here we report generation of infectious RNA transcripts from the constructed full-length viral cDNA clone as a proof that ALV nucleotide sequence is correct and as an initial step toward development of the ALV-based vector for gene silencing and expression of foreign proteins in alfalfa. This is the first report describing the development of a complete cDNA clone of the ALV strain of Pea streak virus and its infectivity in the diagnostic pea (Pisum sativum) and natural alfalfa hosts

Transient expression of the ectodomain of matrix protein 2 (M2e) of avian influenza A virus in plants

We have previously reported an expression system based on the capsid protein gene (CP) of cucumber mosaic virus (CMV) placed under transcriptional control of a potato virus X (PVX)-based vector. PVX-expressed CMV CP formed virus-like particles, which served as carriers for heterologous antigens of the Newcastle disease virus (NDV). In this work, we applied our expression tool toward the development of plant-derived vaccine candidate against avian influenza A virus. Twenty-three amino acid-long extracellular domain of the viral M2 protein (M2e) was engineered into the internal motif 5 of CMV CP and the recombinant gene then was transiently expressed in plants through a PVX vector. Chimeric CMV capsids reacted with specific antibodies produced to synthetic M2e epitope of the H5N1 strain of the virus. In addition, CMV CP-M2e protein was expressed to high levels in Escherichia coli bacterial cells and was recognized by antibodies to both CMV and M2e. This initial study demonstrates the feasibility of using plant virus-based vectors for expression of antigenic epitopes of H5N1 avian influenza in plants

Improvement of PVX/CMV CP expression tool for display of short foreign antigens

We have previously reported that Potato virus X-expressed coat protein of Cucumber mosaic virus (CMV) formed virus-like particles (VLPs), which served as carriers for display of different neutralizing epitopes of Newcastle disease virus (NDV). In this work, we further modified the purification protocol of recombinant VLPs carrying short neutralizing epitopes of the NDV proteins and demonstrated that self-contained capsid protein subunits of CMV transiently expressed from heterologous virus packaged into individual virions morphologically resembling and/or indistinguishable from wild type CMV particles. Homogeneity of the final preparation represents an advance over our previous study, where VLPs were found to be of variable size. Chickens immunized with purified VLPs developed antigen-specific response

Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3' poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3' proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659

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

Abstract 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