Lost and found: Rediscovery and genomic characterization of sowthistle yellow vein virus after a 30+ year hiatus.

Beginning in the 1960's, sowthistle yellow vein virus (SYVV) was the subject of pioneering research that demonstrated propagation of a plant virus in its insect vector. Since the 1980's there has been a paucity of research on SYVV, with historic isolates no longer maintained and no genomic sequence available. Once commonly observed infecting sowthistle (Sonchus oleraceous L.) in California, SYVV incidence declined ca. 1990, likely due to displacement of the black currant aphid (Hyperomyzus lactucae L.) by an invasive non-vector aphid. In 2018, SYVV was fortuitously rediscovered infecting sowthistle in an organic citrus grove in Kern County, CA. The SYVV genome sequence (13,719 nts) obtained from the 2018 sample (designated HWY65) encoded all six expected genes: N, P, MP, M, G, and L. Nucleotide sequence (representing ∼86 % of the genome) of the SYVV Berkeley lab isolate, used by E. S. Sylvester and colleagues for the paradigm-shifting research mentioned above, was determined from an archived library of cDNA clones constructed in 1986. The two nucleotide sequences share 98.5 % identity, confirming both represent the same virus, thereby linking biology of the historic isolate with extant SYVV rediscovered in 2018. Phylogenetic analysis of the L protein indicated SYVV is positioned within a clade containing a subset of viruses currently assigned to the genus Nucleorhabdovirus. As Nucleorhabdovirus is paraphyletic, the International Committee on the Taxonomy of Viruses has proposed abolishment of the genus and establishment of three new genera. In this revised taxonomy, the clade containing SYVV constitutes a new genus designated Betanucleorhabdovirus.

Diversity and epidemiology of plant rhabdoviruses.

Plant rhabdoviruses are recognized by their large bacilliform particles and for being able to replicate in both their plant hosts and arthropod vectors. This review highlights selected, better studied examples of plant rhabdoviruses, their genetic diversity, epidemiology and interactions with plant hosts and arthropod vectors: Alfalfa dwarf virus is classified as a cytorhabdovirus, but its multifunctional phosphoprotein is localized to the plant cell nucleus. Lettuce necrotic yellows virus subtypes may differentially interact with their aphid vectors leading to changes in virus population diversity. Interactions of rhabdoviruses that infect rice, maize and other grains are tightly associated with their specific leafhopper and planthopper vectors. Future outbreaks of vector-borne nucleorhabdoviruses may be predicted based on a world distribution map of the insect vectors. The epidemiology of coffee ringspot virus and its Brevipalpus mite vector is illustrated highlighting the symptomatology and biology of a dichorhavirus and potential impacts of climate change on its epidemiology.

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.

Dichorhaviruses in their Host Plants and Mite Vectors.

A group of related bacilliform, nuclear viruses with a bisegmented negative-sense RNA genome that are transmitted by Brevipalpus mites likely in a circulative-propagative manner were recently classified in the new genus Dichorhavirus, family Rhabdoviridae. These viruses cause localized lesions on leaves, stems, and fruits of economically significant horticultural and ornamental plant species. Among its members, orchid fleck virus, citrus leprosis virus N, and coffee ringspot virus are most prominent. This chapter summarizes the current knowledge about these viruses, available detection techniques, and their interactions with their plant hosts and mite vectors.

Development of Model Systems for Plant Rhabdovirus Research.

This chapter reviews the discoveries and initial characterizations (1930-1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses.

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.

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.

The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins.

The family Rhabdoviridae consists of mostly enveloped, bullet-shaped or bacilliform viruses with a negative-sense, single-stranded RNA genome that infect vertebrates, invertebrates or plants. This ecological diversity is reflected by the diversity and complexity of their genomes. Five canonical structural protein genes are conserved in all rhabdoviruses, but may be overprinted, overlapped or interspersed with several novel and diverse accessory genes. This review gives an overview of the characteristics and diversity of rhabdoviruses, their taxonomic classification, replication mechanism, properties of classical rhabdoviruses such as rabies virus and rhabdoviruses with complex genomes, rhabdoviruses infecting aquatic species, and plant rhabdoviruses with both mono- and bipartite genomes.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

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.

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.

Dichorhavirus: a proposed new genus for Brevipalpus mite-transmitted, nuclear, bacilliform, bipartite, negative-strand RNA plant viruses.

Orchid fleck virus (OFV) is an unassigned negative-sense, single-stranded (-)ssRNA plant virus that was previously suggested to be included in the family Rhabdoviridae, order Mononegavirales. Although OFV shares some biological characteristics, including nuclear cytopathological effects, gene order, and sequence similarities, with nucleorhabdoviruses, its taxonomic status is unclear because unlike all mononegaviruses, OFV has a segmented genome and its particles are not enveloped. This article analyses the available biological, physico-chemical, and nucleotide sequence evidence that seems to indicate that OFV and several other Brevipalpus mite-transmitted short bacilliform (-)ssRNA viruses are likely related and may be classified taxonomically in novel species in a new free-floating genus Dichorhavirus.

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.

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.