Practicing virology: making and knowing a mid-twentieth century experiment with Tobacco mosaic virus.

Tobacco mosaic virus (TMV) has served as a model organism for pathbreaking work in plant pathology, virology, biochemistry and applied genetics for more than a century. We were intrigued by a photograph published in Phytopathology in 1934 showing that Tabasco pepper plants responded to TMV infection with localized necrotic lesions, followed by abscission of the inoculated leaves. This dramatic outcome of a biological response to infection observed by Francis O. Holmes, a virologist at the Rockefeller Institute for Medical Research, was used to score plants for resistance to TMV infection. Our objective was to gain a better understanding of early to mid-twentieth century ideas of genetic resistance to viruses in crop plants. We investigated Holmes' observation as a practical exercise in reworking an experiment, having been inspired by Pamela Smith's innovative Making and Knowing Project. We had a great deal of difficulty replicating Holmes' experiment, finding that biological materials and experimental customs change over time, in ways that ideas do not. Using complementary tools plus careful study and interpretation of the original text and figures, we were able to rework, yet only partially replicate, this experiment. Reading peer-reviewed manuscripts that cited Holmes' 1934 report provided an additional level of insight into the interpretation and replication of this work in the decades that followed. From this, we touch on how experimental reworking can inform our strategies to address the reproducibility "crisis" in twenty-first century science.

Brachypodium: A Monocot Grass Model Genus for Plant Biology.

The genus Brachypodium represents a model system that is advancing our knowledge of the biology of grasses, including small grains, in the postgenomics era. The most widely used species, Brachypodium distachyon, is a C3 plant that is distributed worldwide. B. distachyon has a small genome, short life cycle, and small stature and is amenable to genetic transformation. Due to the intensive and thoughtful development of this grass as a model organism, it is well-suited for laboratory and field experimentation. The intent of this review is to introduce this model system genus and describe some key outcomes of nearly a decade of research since the first draft genome sequence of the flagship species, B. distachyon, was completed. We discuss characteristics and features of B. distachyon and its congeners that make the genus a valuable model system for studies in ecology, evolution, genetics, and genomics in the grasses, review current hot topics in Brachypodium research, and highlight the potential for future analysis using this system in the coming years.

Brachypodium and plant viruses: entwined tools for discovery.

In just a decade, Brachypodium distachyon (Brachypodium) has fulfilled its initial promise as a key tool for realizing new strategies for understanding host and pathogen biology during virus infections of the Poaceae. For this Tansley Insight, I have identified four areas - from the laboratory to the field - that may be particularly fruitful to explore, with a particular focus on Brachypodium-virus infections. These focus areas include: mechanisms of RNA modification of host plants and viruses; coevolution of virus-host interactions; viruses as tools of discovery; and how to explicate the complex outcomes during multivirus infections. Here, I broadly frame our current knowledge of Brachypodium-virus interactions and how these findings may inform virus studies of grasses in the laboratory, field and natural settings.

Genome-wide analysis of alternative splicing landscapes modulated during plant-virus interactions in Brachypodium distachyon.

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes.

Spicing Up the N Gene: F. O. Holmes and Tobacco mosaic virus Resistance in Capsicum and Nicotiana Plants.

One of the seminal events in plant pathology was the discovery by Francis O. Holmes that necrotic local lesions induced on certain species of Nicotiana following rub-inoculation of Tobacco mosaic virus (TMV) was due to a specific interaction involving a dominant host gene (N). From this, Holmes had an idea that if the N gene from N. glutinosa was introgressed into susceptible tobacco, the greatly reduced titer of TMV would, by extension, prevent subsequent infection of tomato and pepper plants by field workers whose hands were contaminated with TMV from their use of chewing and smoking tobacco. The ultimate outcome has many surprising twists and turns, including Holmes' failure to obtain fertile crosses of N. glutinosa × N. tabacum after 3 years of intensive work. Progress was made with N. digluta, a rare amphidiploid that was readily crossed with N. tabacum. And, importantly, the first demonstration by Holmes of the utility of interspecies hybridization for virus resistance was made with Capsicum (pepper) species with the identification of the L gene in Tabasco pepper, that he introgressed into commercial bell pepper varieties. Holmes' findings are important as they predate Flor's gene-for-gene hypothesis, show the use of interspecies hybridization for control of plant pathogens, and the use of the local lesion as a bioassay to monitor resistance events in crop plants.

A Beacon for Applied Plant Pathology: The Origins of Plant Disease.

This year marks a full century since the founding of the journal Plant Disease. The story of how the journal developed, from its origins as a service publication of the USDA in 1917 to the leading applied journal in the field today, reflects on major historical themes in plant pathology. Central to this narrative is the delicate balancing act in plant pathology between fundamental and applied science. During the 1960s and 1970s, substantial numbers of plant pathologists in the U.S. expressed concerns through the American Phytopathological Society (APS) over what they viewed as an alarming and increasing scarcity of applied papers in the flagship journal, Phytopathology. These concerns led increasingly to calls for a second APS journal devoted to applied research. After a period of uncertainty and indecision, the dissolution of the USDA Plant Disease Reporter (PDR) in 1979 offered APS leadership an unusual opportunity to assume publication of a journal with a 63-year legacy of publishing practical plant pathology. In a bold move, APS Council, with the decision in 1979 to take on the publication of PDR under the new title, Plant Disease, provided plant pathologists and the larger agricultural science community with an innovative vehicle to communicate applied plant pathology.

Plant immune responses against viruses: how does a virus cause disease?

Plants respond to pathogens using elaborate networks of genetic interactions. Recently, significant progress has been made in understanding RNA silencing and how viruses counter this apparently ubiquitous antiviral defense. In addition, plants also induce hypersensitive and systemic acquired resistance responses, which together limit the virus to infected cells and impart resistance to the noninfected tissues. Molecular processes such as the ubiquitin proteasome system and DNA methylation are also critical to antiviral defenses. Here, we provide a summary and update of advances in plant antiviral immune responses, beyond RNA silencing mechanisms-advances that went relatively unnoticed in the realm of RNA silencing and nonviral immune responses. We also document the rise of Brachypodium and Setaria species as model grasses to study antiviral responses in Poaceae, aspects that have been relatively understudied, despite grasses being the primary source of our calories, as well as animal feed, forage, recreation, and biofuel needs in the 21st century. Finally, we outline critical gaps, future prospects, and considerations central to studying plant antiviral immunity. To promote an integrated model of plant immunity, we discuss analogous viral and nonviral immune concepts and propose working definitions of viral effectors, effector-triggered immunity, and viral pathogen-triggered immunity.

Multi-Year Pathogen Survey of Biofuel Switchgrass Breeding Plots Reveals High Prevalence of Infections by Panicum mosaic virus and Its Satellite Virus.

Switchgrass (Panicum virgatum) cultivars are currently under development as lignocellulosic feedstock. Here we present a survey of three established switchgrass experimental nurseries in Nebraska in which we identified Panicum mosaic virus (PMV) as the most prevalent virus. In 2012, 72% of 139 symptomatic plants tested positive for PMV. Of the PMV-positive samples, 19% were coinfected with its satellite virus (SPMV). Less than 14% of all sampled plants in 2012 were positive for four additional viruses known to infect switchgrass. In 2013, randomized sampling of switchgrass individuals from the same 2012 breeding plots revealed that infection by PMV or PMV+SPMV was both more prevalent and associated with more severe symptoms in the cultivar Summer, and experimental lines with Summer parentage, than populations derived from the cultivar Kanlow. A 3-year analysis, from 2012 to 2014, showed that previously uninfected switchgrass plants acquire PMV or PMV+SPMV between harvest cycles. In contrast, some plants apparently did not maintain PMV infections at detectable levels from year-to-year. These findings suggest that PMV and SPMV should be considered important pathogens of switchgrass and serious potential threats to biofuel crop production efficiency.

Comparative analysis of antiviral responses in Brachypodium distachyon and Setaria viridis reveals conserved and unique outcomes among C3 and C4 plant defenses.

Viral diseases cause significant losses in global agricultural production, yet little is known about grass antiviral defense mechanisms. We previously reported on host immune responses triggered by Panicum mosaic virus (PMV) and its satellite virus (SPMV) in the model C3 grass Brachypodium distachyon. To aid comparative analyses of C3 and C4 grass antiviral defenses, here, we establish B. distachyon and Setaria viridis (a C4 grass) as compatible hosts for seven grass-infecting viruses, including PMV and SPMV, Brome mosaic virus, Barley stripe mosaic virus, Maize mild mottle virus, Sorghum yellow banding virus, Wheat streak mosaic virus (WSMV), and Foxtail mosaic virus (FoMV). Etiological and molecular characterization of the fourteen grass-virus pathosystems showed evidence for conserved crosstalk among salicylic acid (SA), jasmonic acid, and ethylene pathways in B. distachyon and S. viridis. Strikingly, expression of PHYTOALEXIN DEFICIENT4, an upstream modulator of SA signaling, was consistently suppressed during most virus infections in B. distachyon and S. viridis. Hierarchical clustering analyses further identified unique antiviral responses triggered by two morphologically similar viruses, FoMV and WSMV, and uncovered other host-dependent effects. Together, the results of this study establish B. distachyon and S. viridis as models for the analysis of plant-virus interactions and provide the first framework for conserved and unique features of C3 and C4 grass antiviral defenses.

The Greening of One Health: Plants, Pathogens, and the Environment.

One Health has an aspirational goal of ensuring the health of humans, animals, plants, and the environment through transdisciplinary, collaborative research. At its essence, One Health addresses the human clash with Nature by formulating strategies to repair and restore a (globally) perturbed ecosystem. A more nuanced evaluation of humankind's impact on the environment (Nature, Earth, Gaia) would fully intercalate plants, plant pathogens, and beneficial plant microbes into One Health. Here, several examples point out how plants and plant microbes are keystones of One Health. Meaningful cross-pollination between plant, animal, and human health practitioners can drive discovery and application of innovative tools to address the many complex problems within the One Health framework.

Characterization of a viral synergism in the monocot Brachypodium distachyon reveals distinctly altered host molecular processes associated with disease.

Panicum mosaic virus (PMV) and its satellite virus (SPMV) together infect several small grain crops, biofuel, and forage and turf grasses. Here, we establish the emerging monocot model Brachypodium (Brachypodium distachyon) as an alternate host to study PMV- and SPMV-host interactions and viral synergism. Infection of Brachypodium with PMV+SPMV induced chlorosis and necrosis of leaves, reduced seed set, caused stunting, and lowered biomass, more than PMV alone. Toward gaining a molecular understanding of PMV- and SPMV-affected host processes, we used a custom-designed microarray and analyzed global changes in gene expression of PMV- and PMV+SPMV-infected plants. PMV infection by itself modulated expression of putative genes functioning in carbon metabolism, photosynthesis, metabolite transport, protein modification, cell wall remodeling, and cell death. Many of these genes were additively altered in a coinfection with PMV+SPMV and correlated to the exacerbated symptoms of PMV+SPMV coinfected plants. PMV+SPMV coinfection also uniquely altered expression of certain genes, including transcription and splicing factors. Among the host defenses commonly affected in PMV and PMV+SPMV coinfections, expression of an antiviral RNA silencing component, SILENCING DEFECTIVE3, was suppressed. Several salicylic acid signaling components, such as pathogenesis-related genes and WRKY transcription factors, were up-regulated. By contrast, several genes in jasmonic acid and ethylene responses were down-regulated. Strikingly, numerous protein kinases, including several classes of receptor-like kinases, were misexpressed. Taken together, our results identified distinctly altered immune responses in monocot antiviral defenses and provide insights into monocot viral synergism.

The Past Is Present: Coevolution of Viruses and Host Resistance Within Geographic Centers of Plant Diversity.

Understanding the coevolutionary history of plants, pathogens, and disease resistance is vital for plant pathology. Here, I review Francis O. Holmes's work with tobacco mosaic virus (TMV) framed by the foundational work of Nikolai Vavilov on the geographic centers of origin of plants and crop wild relatives (CWRs) and T. Harper Goodspeed's taxonomy of the genus Nicotiana. Holmes developed a hypothesis that the origin of host resistance to viruses was due to coevolution of both at a geographic center. In the 1950s, Holmes proved that genetic resistance to TMV, especially dominant R-genes, was centered in South America for Nicotiana and other solanaceous plants, including Capsicum, potato, and tomato. One seeming exception was eggplant (Solanum melongena). Not until the acceptance of plate tectonics in the 1960s and recent advances in evolutionary taxonomy did it become evident that northeast Africa was the home of eggplant CWRs, far from Holmes's geographic center for TMV-R-gene coevolution. Unbeknownst to most plant pathologists, Holmes's ideas predated those of H.H. Flor, including experimental proof of the gene-for-gene interaction, identification of R-genes, and deployment of dominant host genes to protect crop plants from virus-associated yield losses.

Plant virology: an RNA treasure trove.

Key principles pertaining to RNA biology not infrequently have their origins in plant virology. Examples have arisen from studies on viral RNA-intrinsic properties and the infection process from gene expression, replication, movement, and defense evasion to biotechnological applications. Since RNA is at the core of the central dogma in molecular biology, how plant virology assisted in the reinforcement or adaptations of this concept, while at other instances shook up elements of the doctrine, is discussed. Moreover, despite the negative effects of viral diseases in agriculture worldwide, plant viruses can be considered a scientific treasure trove. Today they remain tools of discovery for biotechnology, studying evolution, cell biology, and host-microbe interactions.

Induction and suppression of RNA silencing: insights from plant viral infections--a BARD workshop report.

An international workshop on ''Induction and Suppression of RNA Silencing: Insights from Plant Viral Infections'' was sponsored by the United States-Israel Binational Agricultural Research and Development Fund (BARD) and organized in Eilat, Israel in March 2010. The focus of this workshop was on molecular mechanisms employed by viruses or their hosts, and their interactions, for the regulation of virus-induced silencing and suppression. Several of the talks also served as potent reminders of scientific hubris and the need to be attentive to earlier results, both for analyses and perspective regarding new findings.

The society that almost wasn't: issues of professional identity and the creation of the American Phytopathological Society in 1908.

The creation of The American Phytopathological Society (APS) in 1908 was a response to the developing professionalism in the biological and agricultural sciences in the United States between 1880 and 1920. During this period, a new generation of plant pathologists emerged in the United States Department of Agriculture, agricultural colleges, and state agricultural experiment stations with a methodological and theoretical framework to determine the cause and nature of disease and make control recommendations based on experimental evidence. These plant pathologists, in turn, became eager to establish a professional identity, for some an identity separate from traditional botany and mycology. For these scientists, the goal would be facilitated by establishing a new society for plant pathologists. The story of the creation of APS is best understood within the nature of the ensuing debates over identity and the merits of forming a new society among its first generation of scientists.

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation.

Positive-sense RNA viruses in the Tombusviridae family have genomes lacking a 5' cap structure and prototypical 3' polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3' ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon (herein called Brachypodium) and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3' untranslated region-a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.IMPORTANCE The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5' cap structure and 3' polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3' untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents (i.e., satellite virus and noncoding satellite RNAs), all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3' untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.

Multiple activities associated with the capsid protein of satellite panicum mosaic virus are controlled separately by the N- and C-terminal regions.

The 17-kDa capsid protein (CP) of satellite panicum mosaic virus (SPMV) contains a distinct N-terminal arginine-rich motif (N-ARM) which is required for SPMV virion assembly and the activity of SPMV CP to promote systemic accumulation of its cognate RNA. The present study indicates that SPMV CP also is involved in SPMV RNA accumulation in inoculated leaves and that this activity is also dependent on a functional N-ARM. In addition, deletions of a C-terminal region abolish virion assembly and impair SPMV RNA accumulation in both inoculated and systemic leaves. Unlike the N-ARM mutations, substantial deletions of the SPMV CP C-terminus do not affect SPMV RNA binding activity. Interestingly, SPMV CP also binds Panicum mosaic virus genomic RNA via N-ARM-mediated CP:RNA interactions. Mutations of the N-ARM and the C-terminal regions significantly reduce SPMV CP titers and result in symptom attenuation. In contrast, virions were not associated per se with symptom exacerbation or successful SPMV RNA accumulation. The results show the existence of a correlation between N- and C-termini-mediated contributions for CP accumulation, symptom induction, defective-interfering RNA accumulation, and temperature sensitivity of SPMV RNA maintenance. The data provide further evidence that SPMV CP has multiple roles during infection, which might involve the formation of nonvirion CP:RNA complexes whose stability is controlled in a biologically relevant manner by the N- and C-termini of the CP.

A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis.

A novel primer design method is described for site-directed fragment deletion, insertion, and substitution by PCR that is based on inverse PCR using a single pair of partially complementary primers. This method allowed insertion or substitution of fragments up to 27 bp and deletion of fragments up to 105 bp with screening of candidate colonies complete within 24h. To demonstrate the principle behind this new mutagenesis strategy, a series of deletions, insertions, and substitutions were introduced into the capsid protein gene of satellite panicum mosaic virus (SPMV). This method can potentially facilitate high-throughput gene engineering, structure-function analyses, and library construction to study virus-host interactions.

De novo generation of helper virus-satellite chimera RNAs results in disease attenuation and satellite sequence acquisition in a host-dependent manner.

Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions.