Karen-Beth G. Scholthof

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.

The disease triangle: pathogens, the environment and society.

The primary means to define any disease is by naming a pathogen or agent that negatively affects the health of the host organism. Another assumed, but often overlooked, determinant of disease is the environment, which includes deleterious physical and social effects on mankind. The disease triangle is a conceptual model that shows the interactions between the environment, the host and an infectious (or abiotic) agent. This model can be used to predict epidemiological outcomes in plant health and public health, both in local and global communities. Here, the Irish potato famine of the mid-nineteenth century is used as an example to show how the disease triangle, originally devised to interpret plant disease outcomes, can be applied to public health. In parallel, malaria is used to discuss the role of the environment in disease transmission and control. In both examples, the disease triangle is used as a tool to discuss parameters that influence socioeconomic outcomes as a result of host–pathogen interactions involving plants and humans.

Tobacco Mosaic Virus: Pioneering Research for a Century

One century ago, M.W. Beijerinck contended that the filterable agent of tobacco mosaic disease was neither a bacterium nor any corpuscular body, but rather that it was a contagium vivum fluidum ([Beijerinck, 1898][1]). Beijerincks contribution followed A. Mayers path-breaking work on tobacco

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.

A Synergism Induced by Satellite Panicum Mosaic Virus

Panicum mosaic virus (PMV) induces a mild mottle on millet plants, but the addition of its satellite virus (SPMV) causes a severe chlorosis and stunting. Immunoblots, RNA blots, and sucrose density gradient analyses revealed increases of PMV RNA and its p8 and capsid proteins when plants were co-infected with SPMV. A unique feature associated with this satellite virus interaction was an increased rate of systemic infection of PMV.

Control of Plant Virus Diseases by Pathogen-Derived Resistance in Transgenic Plants.

Plant viruses have an enormous negative impact on agricultural crop production throughout the world, and, consequently, agronomists and plant pathologists have devoted considerable effort toward controlling virus diseases during this century. Prior to the advent of genetic engineering, traditional plant breeding methodology was sometimes successfully applied to develop resistance to viruses of agronomically important crops. In addition, standard techniques of plant pathology, including quarantine, eradication, crop rotation, and certified virus-free stock, have been important tools to control virus diseases, although each has disadvantages, such as expense, questionable effectiveness, and lack of reliability on a yearly basis. The prospects for pathogen-mediated intervention in virus disease development were first realized in 1929 when H.H. McKinney demonstrated that tobacco could be protected from infection by a severe strain of TMV by prior inoculation with

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

Virus infection altered alternative splicing in >100 Brachypodium distachyon defense-related genes, with little effect on the genome-wide ratios of different splicing types. 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.

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.

A translational enhancer element on the 3′‐proximal end of the Panicum mosaic virus genome

Panicum mosaic virus (PMV) is a single‐stranded positive‐sense RNA virus in the family Tombusviridae. PMV genomic RNA (gRNA) and subgenomic RNA (sgRNA) are not capped or polyadenylated. We have determined that PMV uses a cap‐independent mechanism of translation. A 116‐nucleotide translational enhancer (TE) region on the 3′‐untranslated region of both the gRNA and sgRNA has been identified. The TE is required for efficient translation of viral proteins in vitro. For mutants with a compromised TE, addition of cap analog, or transposition of the cis‐active TE to another location, both restored translational competence of the 5′‐proximal sgRNA genes in vitro.

The Capsid Protein of Satellite Panicum Mosaic Virus Contributes to Systemic Invasion and Interacts with Its Helper Virus

ABSTRACT Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5′-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5′-untranslated region (5′-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5′-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5′-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.