First detection of wheat streak mosaic virus in Germany: molecular and biological characteristics.

Wheat streak mosaic virus is a serious threat in wheat-producing countries. In Germany, the virus was first recorded in 2013 near Hoym. The complete sequence of isolate Hoym was obtained and compared to all other known complete WSMV sequences, including newly collected and sequenced isolates from France and Austria. Phylogenetic analysis revealed that the European isolates group together with those from the Middle East to form a separate cluster characterized by a distinct putative P1 protease cleavage site. By means of quantitative reverse transcription polymerase chain reaction, it was shown that RNA of the USA type strain PV57 accumulated to higher levels in infected wheat cv. Alcedo than did RNA of isolate Hoym.

The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids.

The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1-2 µm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla).

Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.

In vivo expression and binding activity of scFv-RWAV, which recognizes the coat protein of tomato leaf curl New Delhi virus (family Geminiviridae).

Recombinant antibodies expressed in plants have the potential to interrupt virus infections by blocking essential stages of the infection cycle. Here, we show that the expression of a recombinant single-chain variable fragment (scFv) that recognizes the coat protein of tomato leaf curl New Delhi virus (ToLCNDV) in vitro can also bind to a recombinant coat protein in vivo in the reducing environment of the plant cytosol. The scFv and its target were both expressed as fluorescent protein fusions, one incorporating green fluorescent protein (GFP) and the other DsRed. We found that the incorporation of a nuclear localization signal into the scFv construct resulted in the nuclear import of the antibody-antigen complex, as shown by colocalization of the two fluorescent signals. This demonstrates that recombinant antibodies can be targeted to the nucleus and will bind to geminivirus coat proteins therein, allowing the virus infection cycle to be interrupted during its critical replicative phase.

Complete sequence of a cryptic virus from hemp (Cannabis sativa).

Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected filamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was purified, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.

Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus.

We report the establishment of a hybridoma cell line secreting the monoclonal antibody (mAb) HAV, which recognizes the coat (AV1) protein of tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus. The cell line was obtained following immunization of mice with purified recombinant AV1 fused to glutathione S-transferase (GST). A single-chain variable fragment (scFv-SAV) was assembled from hybridoma cDNA, but sequence analysis revealed a single nucleotide deletion causing a frame shift that resulted in a 21-residue N-terminal truncation. The missing nucleotide was restored by in vitro site-directed mutagenesis to create scFv-RWAV. The binding properties of mAb HAV and the corresponding scFvs were characterized by western blot, ELISA and surface plasmon resonance spectroscopy. MAb HAV bound to AV1 with nanomolar affinity but reacted neither with the N-terminal region of the protein nor with the GST fusion partner. This suggested that the antibody recognized a linear epitope in a region of the coat protein that is conserved among begomoviruses. Both scFvs retained the antigen specificity of mAb HAV, although the dissociation rate constant of scFv-RWAV was tenfold greater than that of scFv-SAV, showing the importance of restoring the 21 N-terminal amino acids.

Comparative sequence analysis and serological and infectivity studies indicate that cocksfoot mild mosaic virus is a member of the genus Panicovirus.

The complete nucleotide sequence of the Phleum isolate of cocksfoot mild mosaic virus (CMMV-P) and the coat protein sequence of the cocksfoot isolate (CMMV-1) were determined. Comparative sequence analysis revealed a close relationship with Panicum mosaic virus (PMV; genus Panicovirus), and together with serological studies, the work supports the classification of CMMV in the family Tombusviridae, genus Panicovirus rather than, as is currently proposed, the genus Sobemovirus. A full-length cDNA clone was prepared, and RNA transcripts synthesised from cDNA were shown to be infectious when inoculated to Hordeum vulgare.

A fully recombinant ELISA using in vivo biotinylated antibody fragments for the detection of potato leafroll virus.

A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for potato leafroll virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible V3HCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 degrees C or -20 degrees C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents.

Studies of the role and function of barley stripe mosaic virus encoded proteins in replication and movement using GFP fusions.

This chapter describes techniques to investigate the localisation and function of virus-encoded proteins in plants using green fluorescent protein (GFP) transiently expressed from plasmids or infectious cDNA reporter clones of barley stripe mosaic virus. Virus movement and the localisation of GFP-tagged proteins in living cells were monitored by confocal laser scanning microscopy (CLSM). In addition, GFP expression was imaged in transgenic plants where specific organelles or subcellular structures such as endoplasmic reticulum were labelled with another fluorophore (e.g., monomeric red fluorescent protein). Using these approaches we discovered evidence for additional roles played by virus encoded movement protein TGB2 and gammab protein in virus replication. Methods are described for clone construction and mutagenesis, and for transient expression (biolistic bombardment or agrobacterium infiltration) in the epidermal cells of Nicotiana benthamiana or barley. In addition, techniques for chloroplast isolation and imaging of the different fluorescent proteins, and the avoidance of interference from autofluorescence, are described.

Unusual long-distance movement strategies of Potato mop-top virus RNAs in Nicotiana benthamiana.

The Potato mop-top virus (PMTV) genome encodes replicase, movement, and capsid proteins on three different RNA species that are encapsidated within tubular rod-shaped particles. Previously, we showed that the protein produced on translational readthrough (RT) of the coat protein (CP) gene, CP-RT, is associated with one extremity of the virus particles, and that the two RNAs encoding replicase and movement proteins can move long distance in the absence of the third RNA (RNA-CP) that encodes the capsid proteins, CP and CP-RT. Here, we examined the roles of the CP and CP-RT proteins on RNA movement using infectious clones carrying mutations in the CP and CP-RT coding domains. The results showed that, in infections established with mutant RNA-CP expressing CP together with truncated CP-RT, systemic movement of the mutant RNA-CP was inhibited but not the movement of the other two RNAs. Furthermore, RNA-CP long-distance movement was inhibited in a mutant clone expressing only CP in the absence of the CP-RT polypeptide. CP-RT was not necessary for particle assembly because virions were observed in leaf extracts infected with the CP-RT deletion mutants. RNA-CP moved long distance when protein expression was suppressed completely or when CP expression was suppressed so that only CP-RT or truncated CP-RT was expressed. CP-RT but not CP interacted with the movement protein TGB1 in the yeast two-hybrid system. CP-RT and TGB1 were detected by enzyme-linked immunosorbent assay in virus particles and the long-distance movement of RNA-CP was correlated with expression of CP-RT that interacted with TGB1; mutant RNA-CP expressing truncated CP-RT proteins that did not interact with TGB1 formed virions but did not move to upper noninoculated leaves. The results indicate that PMTV RNA-CP can move long distance in two distinct forms: either as a viral ribonucleoprotein complex or as particles that are most likely associated with CP-RT and TGB1.

Localization of viral proteins in plant cells: protein tagging.

This chapter describes techniques for in vivo imaging of fluorescent fusion proteins in living cells by confocal laser scanning microscopy (CLSM). Methods are provided for (i) producing the constructs for transient expression from plasmids or virus-based vectors, (ii) introduction of constructs to plant epidermal cells; (iii) imaging of the expressed proteins by CLSM and image processing, and (iv) studying the expression in the presence of agents that affect the integrity or function of cytoskeletal elements. Notes are provided to aid comprehension and indicate problems.

Two plant-viral movement proteins traffic in the endocytic recycling pathway.

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.

Applications of recombinant antibodies in plant pathology.

Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.