RESUMO
Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.
Assuntos
Arabidopsis , Epitopos , Solanum lycopersicum , Epitopos/imunologia , Solanum lycopersicum/imunologia , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Arabidopsis/imunologia , Arabidopsis/genética , Genoma Bacteriano , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Bactérias/imunologia , Bactérias/genética , Proteínas e Peptídeos de Choque Frio/genética , Proteínas e Peptídeos de Choque Frio/imunologia , Proteínas e Peptídeos de Choque Frio/metabolismoRESUMO
Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.
RESUMO
Plants rely on innate immune systems to defend against a wide variety of biotic attackers. Key components of innate immunity include cell-surface pattern-recognition receptors (PRRs), which recognize pest- and pathogen-associated molecular patterns (PAMPs). Unlike other classes of receptors that often have visible cell-death immune outputs upon activation, PRRs generally lack rapid methods for assessing function. Here, we describe a genetically encoded bioluminescent reporter of immune activation by heterologously expressed PRRs in the model organism Nicotiana benthamiana. We characterized N. benthamiana transcriptome changes in response to Agrobacterium tumefaciens and subsequent PAMP treatment to identify pattern-triggered immunity (PTI)-associated marker genes, which were then used to generate promoter-luciferase fusion fungal bioluminescence pathway (FBP) constructs. A reporter construct termed pFBP_2xNbLYS1::LUZ allows for robust detection of PTI activation by heterologously expressed PRRs. Consistent with known PTI signaling pathways, reporter activation by receptor-like protein (RLP) PRRs is dependent on the known adaptor of RLP PRRs, i.e., SOBIR1. The FBP reporter minimizes the amount of labor, reagents, and time needed to assay function of PRRs and displays robust sensitivity at biologically relevant PAMP concentrations, making it ideal for high throughput screens. The tools described in this paper will be powerful for investigations of PRR function and characterization of the structure-function of plant cell-surface receptors. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.
Assuntos
Reconhecimento da Imunidade Inata , Nicotiana , Nicotiana/microbiologia , Plantas/microbiologia , Imunidade Inata , Receptores de Reconhecimento de Padrão/metabolismo , Imunidade Vegetal/genética , Doenças das Plantas/microbiologiaRESUMO
As a first step in innate immunity, pattern recognition receptors (PRRs) recognize the distinct pathogen and herbivore-associated molecular patterns and mediate activation of immune responses, but specific steps in the evolution of new PRR sensing functions are not well understood. We employed comparative genomic and functional analyses to define evolutionary events leading to the sensing of the herbivore-associated peptide inceptin (In11) by the PRR inceptin receptor (INR) in legume plant species. Existing and de novo genome assemblies revealed that the presence of a functional INR gene corresponded with ability to respond to In11 across ~53 million years (my) of evolution. In11 recognition is unique to the clade of Phaseoloid legumes, and only a single clade of INR homologs from Phaseoloids was functional in a heterologous model. The syntenic loci of several non-Phaseoloid outgroup species nonetheless contain non-functional INR-like homologs, suggesting that an ancestral gene insertion event and diversification preceded the evolution of a specific INR receptor function ~28 my ago. Chimeric and ancestrally reconstructed receptors indicated that 16 amino acid differences in the C1 leucine-rich repeat domain and C2 intervening motif mediate gain of In11 recognition. Thus, high PRR diversity was likely followed by a small number of mutations to expand innate immune recognition to a novel peptide elicitor. Analysis of INR evolution provides a model for functional diversification of other germline-encoded PRRs.
The health status of a plant depends on the immune system it inherits from its parents. Plants have many receptor proteins that can recognize distinct molecules from insects and microbes, and trigger an immune response. Inheriting the right set of receptors allows plants to detect certain threats and to cope with diseases and pests. Soybeans, chickpeas and other closely-related crop plants belong to a family of plants known as the legumes. Previous studies have found that, unlike other plants, some legumes are able to respond to oral secretions from caterpillars. These plants have a receptor known as INR that binds to a molecule called inceptin in the secretions. However, it remained unclear how or when INR evolved. To address this gap, Snoeck et al. tested immune responses to inceptin in the leaves of 22 species of legume. The experiments revealed that only members of a subgroup of legumes called the Phaseoloids were able to recognize the molecule. Analyzing the genomes of several legume species revealed that the gene encoding INR first emerged around 28 million years ago. Among the descendants of the legumes that first evolved this receptor, only the crop plant soybean and a few other species were unable to respond to inceptin. The genomic data indicated that these species had in fact lost the gene encoding INR over evolutionary time. Snoeck et al. then combined data from genes encoding modern-day receptors to reconstruct the sequence of building blocks that make up the 28-million-year-old version of INR. This ancestral receptor was able to respond to inceptin in the caterpillar secretion, whereas an older version of the protein, which had a slightly different set of building blocks, could not. This suggests that INR evolved the ability to respond to inceptin as a result of small mutations in the gene encoding a more ancient receptor. The work of Snoeck et al. reveals how the Phaseoloids evolved to respond to caterpillars, and how this ability has been lost in soybeans and other members of the subgroup. In the future, these findings may aid plant breeding or genetic engineering approaches for enhancing soybeans and other crops resistance to caterpillar pests.
Assuntos
Imunidade Inata , Receptores de Reconhecimento de Padrão , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Plantas/genética , Plantas/metabolismo , SinteniaRESUMO
Chewing herbivores activate plant defense responses through a combination of mechanical wounding and elicitation by herbivore-associated molecular patterns (HAMPs). HAMPs are wound response amplifiers; however, specific defense outputs may also exist that strictly require HAMP-mediated defense signaling. To investigate HAMP-mediated signaling and defense responses, we characterized cowpea (Vigna unguiculata) transcriptome changes following elicitation by inceptin, a peptide HAMP common in Lepidoptera larvae oral secretions. Following inceptin treatment, we observed large-scale reprogramming of the transcriptome consistent with three different response categories: (i) amplification of mechanical wound responses, (ii) temporal extension through accelerated or prolonged responses, and (iii) examples of inceptin-specific elicitation and suppression. At both early and late timepoints, namely 1 and 6 h, large sets of transcripts specifically accumulated following inceptin elicitation. Further early inceptin-regulated transcripts were classified as reversing changes induced by wounding alone. Within key signaling- and defense-related gene families, inceptin-elicited responses included target subsets of wound-induced transcripts. Transcripts displaying the largest inceptin-elicited fold changes included transcripts encoding terpene synthases (TPSs) and peroxidases (POXs) that correspond with induced volatile production and increased POX activity in cowpea. Characterization of inceptin-elicited cowpea defenses via heterologous expression in Nicotiana benthamiana demonstrated that specific cowpea TPSs and POXs were able to confer terpene emission and the reduced growth of beet armyworm (Spodoptera exigua) herbivores, respectively. Collectively, our present findings in cowpea support a model where HAMP elicitation both amplifies concurrent wound responses and specifically contributes to the activation of selective outputs associated with direct and indirect antiherbivore defenses.
Assuntos
Fabaceae , Vigna , Animais , Fabaceae/genética , Regulação da Expressão Gênica de Plantas , Herbivoria/fisiologia , Plantas , Spodoptera , Terpenos/metabolismo , Vigna/genéticaRESUMO
Plant defense responses against insect herbivores are induced through wound-induced signaling and the specific perception of herbivore-associated molecular patterns (HAMPs). In addition, herbivores can deliver effectors that suppress plant immunity. Here we review plant immune recognition of HAMPs and effectors, and argue that these initial molecular interactions upon a plant-herbivore encounter mediate and structure effective resistance. While the number of distinct HAMPs and effectors from both chewing and piercing-sucking herbivores has expanded rapidly with omics-enabled approaches, paired receptors and targets in the host are still not well characterized. Herbivore-derived effectors may also be recognized as HAMPs depending on the host plant species, potentially through the evolution of novel immune receptor functions. We compile examples of HAMPs and effectors where natural variation between species may inform evolutionary patterns and mechanisms of plant-herbivore interactions. Finally, we discuss the combined effects of wounding and HAMP recognition, and review potential signaling hubs, which may integrate both sensing functions. Understanding the precise mechanisms for plant sensing of herbivores will be critical for engineering resistance in agriculture.
Assuntos
Herbivoria , Plantas , Animais , Herbivoria/fisiologia , Insetos/fisiologia , Imunidade Vegetal/genética , Plantas/genética , Transdução de SinaisRESUMO
Many biological systems across scales of size and complexity exhibit a time-varying complex network structure that emerges and self-organizes as a result of interactions with the environment. Network interactions optimize some intrinsic cost functions that are unknown and involve for example energy efficiency, robustness, resilience, and frailty. A wide range of networks exist in biology, from gene regulatory networks important for organismal development, protein interaction networks that govern physiology and metabolism, and neural networks that store and convey information to networks of microbes that form microbiomes within hosts, animal contact networks that underlie social systems, and networks of populations on the landscape connected by migration. Increasing availability of extensive (big) data is amplifying our ability to quantify biological networks. Similarly, theoretical methods that describe network structure and dynamics are being developed. Beyond static networks representing snapshots of biological systems, collections of longitudinal data series can help either at defining and characterizing network dynamics over time or analyzing the dynamics constrained to networked architectures. Moreover, due to interactions with the environment and other biological systems, a biological network may not be fully observable. Also, subnetworks may emerge and disappear as a result of the need for the biological system to cope with for example invaders or new information flows. The confluence of these developments renders tractable the question of how the structure of biological networks predicts and controls network dynamics. In particular, there may be structural features that result in homeostatic networks with specific higher-order statistics (e.g., multifractal spectrum), which maintain stability over time through robustness and/or resilience to perturbation. Alternative, plastic networks may respond to perturbation by (adaptive to catastrophic) shifts in structure. Here, we explore the opportunity for discovering universal laws connecting the structure of biological networks with their function, positioning them on the spectrum of time-evolving network structure, that is, dynamics of networks, from highly stable to exquisitely sensitive to perturbation. If such general laws exist, they could transform our ability to predict the response of biological systems to perturbations-an increasingly urgent priority in the face of anthropogenic changes to the environment that affect life across the gamut of organizational scales.
Assuntos
Algoritmos , Animais , HomeostaseRESUMO
Crop damage by herbivorous insects remains a significant contributor to annual yield reductions. Following attack, maize (Zea mays) responds to herbivore-associated molecular patterns (HAMPs) and damage-associated molecular patterns (DAMPs), activating dynamic direct and indirect antiherbivore defense responses. To define underlying signaling processes, comparative analyses between plant elicitor peptide (Pep) DAMPs and fatty acid-amino acid conjugate (FAC) HAMPs were conducted. RNA sequencing analysis of early transcriptional changes following Pep and FAC treatments revealed quantitative differences in the strength of response yet a high degree of qualitative similarity, providing evidence for shared signaling pathways. In further comparisons of FAC and Pep responses across diverse maize inbred lines, we identified Mo17 as part of a small subset of lines displaying selective FAC insensitivity. Genetic mapping for FAC sensitivity using the intermated B73 × Mo17 population identified a single locus on chromosome 4 associated with FAC sensitivity. Pursuit of multiple fine-mapping approaches further narrowed the locus to 19 candidate genes. The top candidate gene identified, termed FAC SENSITIVITY ASSOCIATED (ZmFACS), encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that belongs to the same family as a rice (Oryza sativa) receptor gene previously associated with the activation of induced responses to diverse Lepidoptera. Consistent with reduced sensitivity, ZmFACS expression was significantly lower in Mo17 as compared to B73. Transient heterologous expression of ZmFACS in Nicotiana benthamiana resulted in a significantly increased FAC-elicited response. Together, our results provide useful resources for studying early elicitor-induced antiherbivore responses in maize and approaches to discover gene candidates underlying HAMP sensitivity in grain crops.
Assuntos
Regulação da Expressão Gênica de Plantas , Lepidópteros/fisiologia , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Transdução de Sinais , Zea mays/genética , Animais , Mapeamento Cromossômico , Loci Gênicos/genética , Herbivoria , Peptídeos/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Nicotiana/genética , Nicotiana/fisiologia , Zea mays/fisiologiaRESUMO
Plants are resistant to most pathogens because of an immune system that perceives invading microbes and activates defense. A large repertoire of innate immune receptors mediates specific direct or indirect recognition of pathogen-derived molecules. Disease is often a consequence of insufficient immune surveillance, and the transfer of immune receptor genes from resistant plants to susceptible crop varieties is an effective strategy for combating disease outbreaks. We discuss approaches for identifying intracellular and cell surface immune receptors, with particular focus on recently developed and emerging methodologies. We also review considerations for the transfer of immune receptor genes into crop species, including additional host factors that may be required for immune receptor function. Together, these concepts lay out a broadly applicable playbook for developing crop varieties with durable disease resistance.
Assuntos
Doenças das Plantas , Imunidade Vegetal , Produtos Agrícolas/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Imunidade Vegetal/genéticaRESUMO
Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.
Assuntos
Herbivoria/fisiologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Plantas Geneticamente Modificadas , Spodoptera/fisiologia , Nicotiana/imunologia , Vigna/imunologiaRESUMO
To recognize diverse threats, plants monitor extracellular molecular patterns and transduce intracellular immune signaling through receptor complexes at the plasma membrane. Pattern recognition occurs through a prototypical network of interacting proteins, comprising A) receptors that recognize inputs associated with a growing number of pest and pathogen classes (bacteria, fungi, oomycetes, caterpillars), B) co-receptor kinases that participate in binding and signaling, and C) cytoplasmic kinases that mediate first stages of immune output. While this framework has been elucidated in reference accessions of model organisms, network components are part of gene families with widespread variation, potentially tuning immunocompetence for specific contexts. Most dramatically, variation in receptor repertoires determines the range of ligands acting as immunogenic inputs for a given plant. Diversification of receptor kinase (RK) and related receptor-like protein (RLP) repertoires may tune responses even within a species. Comparative genomics at pangenome scale will reveal patterns and features of immune network variation.
Assuntos
Oomicetos , Plantas , Membrana Celular , Imunidade Vegetal/genética , Plantas/genética , Receptores de Reconhecimento de Padrão/genética , Transdução de SinaisRESUMO
Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely-related Nicotiana sylvestris and the distantly-related beet plant (Beta vulgaris). Roq1 was found to co-immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.
Assuntos
Resistência à Doença , Nicotiana/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Pseudomonas/metabolismo , Xanthomonas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Nucleotide-binding leucine-rich repeat (NB-LRR, or NLR) receptors mediate pathogen recognition. The Arabidopsis thaliana NLR RPP1 recognizes the tandem WY-domain effector ATR1 from the oomycete Hyaloperonospora arabidopsidis through direct association with C-terminal LRRs. We isolated and characterized homologous NLR genes RPP1-EstA and RPP1-ZdrA from two Arabidopsis ecotypes, Estland (Est-1) and Zdarec (Zdr-1), responsible for recognizing a novel spectrum of ATR1 alleles. RPP1-EstA and -ZdrA encode nearly identical NLRs that are phylogenetically distinct from known immunity-activating RPP1 homologs and possess greatly expanded LRR domains. Site-directed mutagenesis and truncation analysis of ATR1 suggests that these homologs recognize a novel surface of the 2(nd) WY domain of ATR1, partially specified by a C-terminal region of the LRR domain. Synteny comparison with RPP1 loci involved in hybrid incompatibility suggests that these functions evolved independently. Closely related RPP1 homologs have diversified their recognition spectra through LRR expansion and sequence variation, allowing them to detect multiple surfaces of the same pathogen effector. Engineering NLR receptor specificity may require a similar combination of repeat expansion and tailored amino acid variation.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Oomicetos/fisiologia , Proteínas/química , Proteínas/metabolismo , Alelos , Sequência de Aminoácidos , Segregação de Cromossomos , Cruzamentos Genéticos , Ecótipo , Loci Gênicos , Genoma de Planta , Proteínas de Repetições Ricas em Leucina , Anotação de Sequência Molecular , Fenótipo , Filogenia , Polimorfismo Genético , Domínios Proteicos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de AminoácidosRESUMO
In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function.
Assuntos
Alelos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Oomicetos/fisiologia , Doenças das Plantas/parasitologia , Receptores de Superfície Celular/metabolismo , Arabidopsis/genética , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Doenças das Plantas/genética , Receptores de Superfície Celular/genéticaRESUMO
UNLABELLED: Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-ß expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCE: The Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.
Assuntos
Fígado/virologia , Vírus da Hepatite Murina/fisiologia , Proteínas da Matriz Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Tropismo Viral , Animais , Linhagem Celular , Proteínas M de Coronavírus , Cricetinae , Hepatite Viral Animal/virologia , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina/genética , Genética Reversa , Proteínas da Matriz Viral/genética , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Induced changes in primary metabolism are important plant responses to herbivory, providing energy and metabolic precursors for defense compounds. Metabolic shifts also can lead to reallocation of leaf resources to storage tissues, thus increasing a plant's tolerance. We characterized whole-plant metabolic responses of tomato (Solanum lycopersicum) 24 h after leaf herbivory by two caterpillars (the generalist Helicoverpa zea and the specialist Manduca sexta) by using GC-MS. We measured 56 primary metabolites across the leaves, stems, roots, and apex, comparing herbivore-attacked plants to undamaged plants and mechanically damaged plants. Induced metabolic change, in terms of magnitude and number of individual concentration changes, was stronger in the apex and root tissues than in undamaged leaflets of damaged leaves, indicating rapid and significant whole-plant responses to damage. Helicoverpa zea altered many more metabolites than M. sexta across most tissues, suggesting an enhanced plant response to H. zea herbivory. Helicoverpa zea herbivory strongly affected concentrations of defense-related metabolites (simple phenolics and precursor amino acids), while M. sexta altered metabolites associated with carbon and nitrogen transport. We conclude that herbivory induces many systemic primary metabolic changes in tomato, and that changes often are specific to a single tissue or type of herbivore. The potential implications of primary metabolic changes are discussed in relation to resistance and tolerance.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Herbivoria , Mariposas/fisiologia , Solanum lycopersicum/metabolismo , Análise de Variância , Animais , Regulação da Expressão Gênica de Plantas , Larva/crescimento & desenvolvimento , Larva/fisiologia , Solanum lycopersicum/genética , Manduca/crescimento & desenvolvimento , Manduca/fisiologia , Metaboloma , Mariposas/crescimento & desenvolvimento , Especificidade de Órgãos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Especificidade da EspécieRESUMO
The in planta association of the Hyaloperonospora arabidopsidis effector ATR1 with the cognate Arabidopsis thaliana RPP1 immune receptor activates a disease-resistance signaling pathway that inhibits pathogen growth. To define the molecular events specifying effector recognition by RPP1, we determined the crystal structure of ATR1 and assayed in planta the effects of surface polymorphisms that are critical to activating plant immunity. ATR1 adopts an elongated, all-helical, two-domain, seahorse-like structure with an overall architecture unlike any previously described fold. Structural comparisons highlight a tandemly duplicated, five-helix motif in the C-terminal domain that creates a structural framework for rapid diversification. Identification and mapping of critical recognition sites suggest that ATR1 detection by the RPP1 resistance protein is mediated by several distinct protein surfaces that allow the effectors to escape recognition through diverse surface polymorphisms. ATR1 gain-of-recognition mutants demonstrate that multiple amino acid substitutions are necessary for recognition and that surface polymorphisms exert additive effects. These results suggest that ATR1 is a modular repeat protein belonging to an ancient family of oomycete effectors that rapidly evolves to escape host detection and adopt diverse virulence functions.