Characterization of Soybean yellow shoot virus, a New Member of the Family Potyviridae Infecting Soybean Plants in Brazil.

A new virus species, belonging to the family Potyviridae and capable of infecting most of the soybean cultivars grown in Brazil, was collected in Lavras, Minas Gerais, Brazil, and named Soybean yellow shoot virus (SoyYSV). In this study, the complete 9,052-nucleotide genome of SoyYSV was determined and the structural, biological, and molecular properties of the virus were investigated. The SoyYSV genome encoded a single polyprotein that could be subsequently cleaved, generating 11 proteins. The SoyYSV genome shared 49% nucleotide and 36% amino acid sequence identity with Blackberry virus Y. However, the P1 protein of SoyYSV was much smaller and lacked the ALK1 domain characteristic of the genus Brambyvirus. Electron microscopy revealed flexuous filamentous virus particles, 760 to 780 nm in length, and cytoplasmic inclusions typical of those found in plant cells infected with Potyviridae species. In addition to soybean, SoyYSV infected species in the Amaranthaceae, Caricaceae, Fabaceae, and Solanaceae families. Among the most common potyviruses present in Brazil, only SoyYSV induced local necrotic lesions in Carica papaya L. SoyYSV was transmissible by Myzus persicae and Aphis gossypii but lacked the HC-Pro domain required for aphid transmission in other potyviruses. No seed transmission in soybean was observed.

Sonchus yellow net virus core particles form on ring-like nuclear structure enriched in viral phosphoprotein.

The phosphoprotein (P) of the nucleorhabdovirus sonchus yellow net virus has been shown to accumulate in ring-shaped structures in virus-infected nuclei. Further examination by live-cell imaging, in combination with structural examination by transmission electron microscopy and immunolocalization demonstrated that P-rings do not form in association with nucleoli. Furthermore, viral cores were shown to condense on the nucleoplasm-contacting surface of the rings. The data presented here offer evidence for the site of nucleocapsid assembly in SYNV-infected nuclei.

The Rise and Rise of Nicotiana benthamiana: A Plant for All Reasons.

A decade ago, the value of Nicotiana benthamiana as a tool for plant molecular biologists was beginning to be appreciated. Scientists were using it to study plant-microbe and protein-protein interactions, and it was the species of choice with which to activate plasmid-encoded viruses, screen for gene functions with virus-induced gene silencing (VIGS), and transiently express genes by leaf agroinfiltration. However, little information about the species' origin, diversity, genetics, and genomics was available, and biologists were asking the question of whether N. benthamiana is a second fiddle or virtuoso. In this review, we look at the increased knowledge about the species and its applications over the past decade. Although N. benthamiana may still be the sidekick to Arabidopsis, it shines ever more brightly with realized and yet-to-be-exploited potential.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Protein Localization and Interaction Studies in Plants: Toward Defining Complete Proteomes by Visualization.

Protein interaction and localization studies in plants are a fundamental component of achieving mechanistic understanding of virus:plant interactions at the systems level. Many such studies are conducted using transient expression assays in leaves of Nicotiana benthamiana, the most widely used experimental plant host in virology, examined by laser-scanning confocal microscopy. This chapter provides a workflow for protein interaction and localization experiments, with particular attention to the many control and supporting assays that may also need to be performed. Basic principles of microscopy are introduced to aid researchers in the early stages of adding imaging techniques to their experimental repertoire. Three major types of imaging-based experiments are discussed in detail: (i) protein localization using autofluorescent proteins, (ii) colocalization studies, and (iii) bimolecular fluorescence complementation, with emphasis on judicious interpretation of the data obtained from these approaches. In addition to establishing a general framework for protein localization experiments in plants, the need for proteome-scale localization projects is discussed, with emphasis on nuclear-localized proteins.

Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus.

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12 792 nt long and organized into seven ORFs with the gene order 3'-N-X-P-Y-M-G-L-5', which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. Cloned ORFs for each gene, except L, were used to construct a protein interaction and localization map (PILM) for this virus, which shares greater than 80 % amino acid similarity in all ORFs except X and P with the sanguinolenta strain of this species (SYDV). Protein localization patterns and interactions unique to each viral strain were identified, resulting in strain-specific PILMs. Localization of CYDV and SYDV proteins in virus-infected cells mapped subcellular loci likely to be sites of replication, morphogenesis and movement.

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins.

BACKGROUND: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and ß-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. CONCLUSION/SIGNIFICANCE: Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.

Clinical phenotypes associated with circulating tumor cell enumeration in metastatic castration-resistant prostate cancer.

BACKGROUND: The presence of ≥5 circulating tumor cells (CTCs) is prognostic for shorter survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, some men have low CTCs despite widespread disease, suggesting heterogeneity in CTC phenotype or detection. The aim of this study was to evaluate the association of CTC enumeration with clinical disease characteristics and overall survival in men with mCRPC at our institution. DESIGN: CTCs were enumerated using the CellSearch method in a prospective correlative study in men with mCRPC starting a new systemic therapy. The primary objective was to determine the clinical phenotype of the subset of men with mCRPC who have a poor prognosis and low CTCs. Secondary end points included associations of CTCs with survival and known prognostic biomarkers, before therapy and at progression. RESULTS: At baseline, median CTC count was 16 cells and prostate-specific antigen (PSA) level was 178 ng/ml. At progression, median CTC count was 42, PSA level was 245 ng/ml, levels of lactate dehydrogenase and alkaline phosphatase rose, and level of hemoglobin dropped. The median overall survival for this heavily pretreated population was 11.2 months, and the multivariable hazard ratio for death of men with CTCs<5 vs.≥5 was 0.43 (95% CI: 0.24-0.77). Median progression-free survival was 4.4 months. CTC enumeration modestly correlated with lactate dehydrogenase and alkaline phosphatase levels but only weakly correlated with PSA and hemoglobin levels. We were unable to identify a consistent subgroup of poor prognosis men with a low number of CTCs. CONCLUSION: CTC enumeration appears to be prognostic in men with mCRPC and describes a phenotype of hematogenous dissemination that cannot be predicted based on standard clinical and laboratory assessments.

Nicotiana benthamiana: Its History and Future as a Model for Plant-Pathogen Interactions.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Addi- tionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus- induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, inter- action, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions cur- rently used by the research community. In addition to ad- dressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco.

KEY MESSAGE: Phylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen. ABSTRACT: Tobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene--as a fusion with the GFP gene--targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.

Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses.

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins.

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RT-PCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Lettuce necrotic yellows cytorhabdovirus protein localization and interaction map, and comparison with nucleorhabdoviruses.

Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N-P and M-M interactions are common to all three viruses. Associations unique to only one virus include P-M for LNYV, G-Mv for SYNV and M-Mv, M-G and N-M for PYDV. The cognate N-P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed.

A host-factor interaction and localization map for a plant-adapted rhabdovirus implicates cytoplasm-tethered transcription activators in cell-to-cell movement.

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus.

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine the subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.

Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta.

Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.

Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

Cloning and subcellular localization of the phosphoprotein and nucleocapsid proteins of Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus.

We have cloned and characterized mRNAs corresponding to the phosphoprotein (P) and nucleocapsid (N) genes of the sanguinolenta strain of Potato yellow dwarf virus (PYDV). The P and N messenger RNAs both begin with a common AAACA pentanucleotide and are 1546nt and 962nt in length, and capable of encoding 52kDa and 31kDa proteins, respectively. The N mRNA contains a 12nt 5' non-translated sequence (NTS) and a 83nt 3'-NTS. Similarly, the P mRNA has a 19nt 5'-NTS and a 125nt 3'-NTS. Primary structure analyses revealed three potential phosphorylation sites in the P protein and six in the N protein. Despite a lack of predictable nuclear localization signals (NLSs) in either protein, transient expression of the P and N proteins in N. benthamiana showed that both proteins are targeted exclusively to nuclei. Phylogenetic analyses showed that PYDV is most closely related to Maize mosaic virus and Taro vein chlorosis virus, which also lack predictable NLSs in their N proteins. The present data further distinguish PYDV from SYNV and suggest that, together, these viruses serve to provide a more comprehensive view of rhabdovirus cell biology, which can be studied in a common host plant.

Membrane and protein dynamics in virus-infected plant cells.

In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells.