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

Discovery of the first pectin methylesterase gene in a plant-parasitic nematode

The root lesion nematode (RLN) Pratylenchus penetrans is considered one of the most damaging plant-parasitic nematode (PPN) species worldwide affecting important agronomic and horticultural crops (e.g. potato, corn). The successful invasion of roots by RLNs is related to their ability to overcome the barrier imposed by the plant cell wall. Like other PPNs, RLNs are equipped with a protrusible stylet that mechanically disrupts the cell wall and through which cell wall-degrading enzymes are secreted to facilitate penetration and migration of the nematode through host roots. Herein, we identified and characterized a pectin methylesterase gene for P. penetrans. Sequence analysis confirm the eukaryotic gene structure of Pp-pme. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato Virus X-based vector. PMEs have been so far described for plants, fungus, bacteria, and to a restrict number of insects. To our knowledge, this is the first report a PME within the phylum Nematoda

The root lesion nematode effector Ppen10370 is essential for parasitism of Pratylenchus penetrans

The root lesion nematode, Pratylenchus penetrans, is a migratory species that attacks a broad range of crops. Like other plant pathogens, P. penetrans deploys a battery of secreted protein effectors to manipulate plant hosts and induce disease. Although several candidate effectors of P. penetrans have been identified, detailed mechanisms of their functions and particularly their host targets remain largely unexplored. In this study, a repertoire of candidate genes encoding pioneer effectors of P. penetrans was amplified from mixed life stages of the nematode, cloned and subjected to transient expression in a heterologous host Nicotiana benthamiana using potato virus X-based gene vector. Among seven analyzed genes, the candidate effector designated as Ppen10370 triggered pleiotropic phenotypes substantially different from those produced by wild type infection. Transcriptome analysis of plants expressing Ppen10370 demonstrated that observed phenotypic changes were likely related to disruption of core biological processes in the plant due to effector-originated activities. Cross-species comparative analysis of Ppen10370 identified homologue gene sequences in five other Pratylenchus species and their transcripts were found to be localized specifically in the nematode esophageal glands by in situ hybridization. RNA silencing of the Ppen10370 resulted in a significant reduction of nematode reproduction and development, demonstrating an important role of the esophageal gland effector for parasitism

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

A new esophageal gland transcriptome reveals signatures of large scale de novo effector birth in the root lesion nematode Pratylenchus penetrans.

BACKGROUND: The root lesion nematode Pratylenchus penetrans is a migratory plant-parasitic nematode responsible for economically important losses in a wide number of crops. Despite the importance of P. penetrans, the molecular mechanisms employed by this nematode to promote virulence remain largely unknown. RESULTS: Here we generated a new and comprehensive esophageal glands-specific transcriptome library for P. penetrans. In-depth analysis of this transcriptome enabled a robust identification of a catalogue of 30 new candidate effector genes, which were experimentally validated in the esophageal glands by in situ hybridization. We further validated the expression of a multifaceted network of candidate effectors during the interaction with different plants. To advance our understanding of the "effectorome" of P. penetrans, we adopted a phylogenetic approach and compared the expanded effector repertoire of P. penetrans to the genome/transcriptome of other nematode species with similar or contrasting parasitism strategies. Our data allowed us to infer plausible evolutionary histories that shaped the effector repertoire of P. penetrans, as well as other close and distant plant-parasitic nematodes. Two remarkable trends were apparent: 1) large scale effector birth in the Pratylenchidae in general and P. penetrans in particular, and 2) large scale effector death in sedentary (endo) plant-parasitic nematodes. CONCLUSIONS: Our study doubles the number of validated Pratylenchus penetrans effectors reported in the literature. The dramatic effector gene gain in P. penetrans could be related to the remarkable ability of this nematode to parasitize a large number of plants. Our data provide valuable insights into nematode parasitism and contribute towards basic understating of the adaptation of P. penetrans and other root lesion nematodes to specific host plants

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