RESUMEN
Innate immune responses to microbial pathogens are regulated by intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) in both the plant and animal kingdoms. Across plant innate immune systems, "helper" NLRs (hNLRs) work in coordination with "sensor" NLRs (sNLRs) to modulate disease resistance signaling pathways. Activation mechanisms of hNLRs based on structures are unknown. Our research reveals that the hNLR, known as NLR required for cell death 4 (NRC4), assembles into a hexameric resistosome upon activation by the sNLR Bs2 and the pathogenic effector AvrBs2. This conformational change triggers immune responses by facilitating the influx of calcium ions (Ca2+) into the cytosol. The activation mimic alleles of NRC2, NRC3, or NRC4 alone did not induce Ca2+ influx and cell death in animal cells, suggesting that unknown plant-specific factors regulate NRCs' activation in plants. These findings significantly advance our understanding of the regulatory mechanisms governing plant immune responses.
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Proteínas de Arabidopsis , Arabidopsis , Calcio , Calcio/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Inmunidad de la Planta , Proteínas NLR/metabolismo , Animales , Receptores Inmunológicos/metabolismo , Inmunidad Innata , Resistencia a la EnfermedadRESUMEN
Plant diseases cause famines, drive human migration, and present challenges to agricultural sustainability as pathogen ranges shift under climate change. Plant breeders discovered Mendelian genetic loci conferring disease resistance to specific pathogen isolates over 100 years ago. Subsequent breeding for disease resistance underpins modern agriculture and, along with the emergence and focus on model plants for genetics and genomics research, has provided rich resources for molecular biological exploration over the last 50 years. These studies led to the identification of extracellular and intracellular receptors that convert recognition of extracellular microbe-encoded molecular patterns or intracellular pathogen-delivered virulence effectors into defense activation. These receptor systems, and downstream responses, define plant immune systems that have evolved since the migration of plants to land â¼500 million years ago. Our current understanding of plant immune systems provides the platform for development of rational resistance enhancement to control the many diseases that continue to plague crop production.
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Resistencia a la Enfermedad , Enfermedades de las Plantas , Inmunidad de la Planta , Plantas , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Plantas/inmunología , Plantas/genética , Resistencia a la Enfermedad/genética , HumanosRESUMEN
Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregating populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6. We identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. This research enhances marker-assisted selection of bs5 and bs6 in breeding programs and is a crucial step towards elucidating the molecular mechanisms underlying the resistances.
RESUMEN
Many resistance genes deployed against pathogens in crops are intracellular nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs). The ability to rationally engineer the specificity of NLRs will be crucial in the response to newly emerging crop diseases. Successful attempts to modify NLR recognition have been limited to untargeted approaches or depended on previously available structural information or knowledge of pathogen-effector targets. However, this information is not available for most NLR-effector pairs. Here, we demonstrate the precise prediction and subsequent transfer of residues involved in effector recognition between two closely related NLRs without their experimentally determined structure or detailed knowledge about their pathogen effector targets. By combining phylogenetics, allele diversity analysis, and structural modeling, we successfully predicted residues mediating interaction of Sr50 with its cognate effector AvrSr50 and transferred recognition specificity of Sr50 to the closely related NLR Sr33. We created synthetic versions of Sr33 that contain amino acids from Sr50, including Sr33syn, which gained the ability to recognize AvrSr50 with 12 amino-acid substitutions. Furthermore, we discovered that sites in the LRR domain needed to transfer recognition specificity to Sr33 also influence autoactivity in Sr50. Structural modeling suggests these residues interact with a part of the NB-ARC domain, which we named the NB-ARC latch, to possibly maintain the inactive state of the receptor. Our approach demonstrates rational modifications of NLRs, which could be useful to enhance existing elite crop germplasm. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Proteínas de Plantas , Plantas , Proteínas de Plantas/metabolismo , Plantas/genética , Dominios Proteicos , Filogenia , Receptores Inmunológicos/genética , Enfermedades de las Plantas , Inmunidad de la PlantaRESUMEN
Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with â¼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.
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Oryza , Estomas de Plantas , Estomas de Plantas/fisiología , Resistencia a la Sequía , Fotosíntesis/genética , SequíasRESUMEN
Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.
RESUMEN
Plants and animals detect pathogen infection using intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8-angstrom-resolution cryo-electron microscopy structure of the activated ROQ1 (recognition of XopQ 1), an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain bound to the Xanthomonas euvesicatoria effector XopQ (Xanthomonas outer protein Q). ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.
Asunto(s)
Proteínas Bacterianas/química , Interacciones Huésped-Patógeno , Proteínas NLR/química , Nicotiana/inmunología , Nicotiana/microbiología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/química , Xanthomonas/patogenicidad , Microscopía por Crioelectrón , Resistencia a la Enfermedad , Unión Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.
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Proteínas del Dominio Armadillo/química , Proteínas del Citoesqueleto/química , NAD+ Nucleosidasa/química , NAD/metabolismo , Proteínas de Plantas/química , Dominios Proteicos , Receptores Inmunológicos/química , Animales , Proteínas del Dominio Armadillo/metabolismo , Axones/enzimología , Axones/patología , Sitios de Unión , Muerte Celular , Secuencia Conservada , Cristalografía por Rayos X , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ratones , NAD+ Nucleosidasa/metabolismo , NADP/metabolismo , Neuronas/enzimología , Proteínas de Plantas/metabolismo , Multimerización de Proteína , Receptores Inmunológicos/metabolismo , Degeneración Walleriana/enzimología , Degeneración Walleriana/patologíaRESUMEN
Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3-7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8-10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.
Asunto(s)
Arabidopsis/inmunología , Arabidopsis/metabolismo , Calmodulina/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Inmunidad de la Planta/inmunología , Animales , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio , Calmodulina/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/agonistas , Canales Catiónicos Regulados por Nucleótidos Cíclicos/antagonistas & inhibidores , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Femenino , Inmunidad Innata , Oocitos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , XenopusRESUMEN
The original version of this article contained an error in the author affiliations. Oliver J. Furzer was incorrectly associated with Department of Plant Sciences, College of Life Sciences, Wuhan University, 430072, Wuhan, China.This has now been corrected in the HTML version of the article. The PDF version of the article was correct at the time of publication.Furthermore, the original version of this article stated that correspondence and requests for materials should be addressed to Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany S.A. (email: shuta.asai@riken.jp) or to J.D.G.J. (email: jonathan.jones@tsl.ac.uk). The words "Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany" were introduced inadvertently.This has now been corrected in the PDF version of the article. The HTML version of the article was correct at the time of publication.
RESUMEN
Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Manihot/inmunología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Enfermedades de las Plantas/inmunología , Potyviridae/inmunología , Sistemas CRISPR-Cas , Factor 4E Eucariótico de Iniciación/metabolismo , Edición Génica , Interacciones Huésped-Patógeno , Manihot/genética , Manihot/virología , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/virología , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Pathogen co-evolution with plants involves selection for evasion of host surveillance systems. The oomycete Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis, and race-specific interactions between Arabidopsis accessions and Hpa isolates fit the gene-for-gene model in which host resistance or susceptibility are determined by matching pairs of plant Resistance (R) genes and pathogen Avirulence (AVR) genes. Arabidopsis Col-0 carries R gene RPP4 that confers resistance to Hpa isolates Emoy2 and Emwa1, but its cognate recognized effector(s) were unknown. We report here the identification of the Emoy2 AVR effector gene recognized by RPP4 and show resistance-breaking isolates of Hpa on RPP4-containing Arabidopsis carry the alleles that either are not expressed, or show cytoplasmic instead of nuclear subcellular localization.
Asunto(s)
Arabidopsis/microbiología , Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Oomicetos/genética , Oomicetos/metabolismo , Enfermedades de las Plantas/microbiología , Factores de Virulencia/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Núcleo Celular/genética , Citoplasma/genética , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/genética , Polimorfismo Genético , Transporte de Proteínas , Factores de Virulencia/genéticaRESUMEN
Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1-triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.
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Nicotiana/genética , Nicotiana/metabolismo , Subgrupos de Linfocitos B/metabolismo , Proteínas de Unión al ADN , Proteínas Repetidas Ricas en Leucina , Proteínas NLR/metabolismo , Neurregulina-1/genética , Neurregulina-1/fisiología , Enfermedades de las Plantas , Inmunidad de la Planta , Proteínas de Plantas/genética , Dominios Proteicos , Proteínas/genética , Pseudomonas , Transducción de Señal , Transcriptoma , XanthomonasRESUMEN
The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Proteínas de Plantas/química , Secuencia de Aminoácidos , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Sitios de Unión , Muerte Celular/genética , Muerte Celular/inmunología , Lino/genética , Lino/inmunología , Lino/microbiología , Interacciones Huésped-Patógeno , Modelos Moleculares , Mutación , Peronospora/patogenicidad , Peronospora/fisiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/microbiologíaRESUMEN
AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix-loop-helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1 By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.
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Proteínas Bacterianas/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Capsicum/microbiología , Regulación Bacteriana de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Nicotiana/microbiología , Enfermedades de las Plantas/microbiología , Polisacárido Liasas/genética , Solanum lycopersicum/microbiología , Factores de Transcripción/fisiología , Xanthomonas/patogenicidad , Proteínas Bacterianas/genética , Capsicum/metabolismo , Activación Enzimática , Solanum lycopersicum/metabolismo , Fenotipo , Enfermedades de las Plantas/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Nicotiana/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Regulación hacia Arriba , Virulencia , Agua/metabolismo , Xanthomonas/genética , Xanthomonas/fisiologíaRESUMEN
Bacterial disease management is a challenge for modern agriculture due to rapid changes in pathogen populations. Genome sequences for hosts and pathogens provide detailed information that facilitates effector-based breeding strategies. Tomato genotypes have gene-for-gene resistance to the bacterial spot pathogen Xanthomonas perforans. The bacterial spot populations in Florida shifted from tomato race 3 to 4, such that the corresponding tomato resistance gene no longer recognizes the effector protein AvrXv3. Genome sequencing showed variation in effector profiles among race 4 strains collected in 2006 and 2012 and compared with a race 3 strain collected in 1991. We examined variation in putative targets of resistance among Florida strains of X. perforans collected from 1991 to 2006. Consistent with race change, avrXv3 was present in race 3 strains but nonfunctional in race 4 strains due to multiple independent mutations. Effectors xopJ4 and avrBs2 were unchanged in all strains. The effector avrBsT was absent in race 3 strains collected in the 1990s but present in race 3 strains collected in 2006 and nearly all race 4 strains. These changes in effector profiles suggest that xopJ4 and avrBsT are currently the best targets for resistance breeding against bacterial spot in tomato.
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Genoma Bacteriano/genética , Genoma de Planta/genética , Enfermedades de las Plantas/inmunología , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Xanthomonas/genética , Secuencia de Bases , Cruzamiento , Genotipo , Solanum lycopersicum/microbiología , Enfermedades de las Plantas/microbiología , Análisis de Secuencia de ADNRESUMEN
Upon recognition of pathogen virulence effectors, plant nucleotide-binding leucine-rich repeat (NLR) proteins induce defense responses including localized host cell death. In an effort to understand the molecular mechanisms leading to this response, we examined the Arabidopsis thaliana NLR protein RECOGNITION OF PERONOSPORA PARASITICA1 (RPP1), which recognizes the Hyaloperonospora arabidopsidis effector ARABIDOPSIS THALIANA RECOGNIZED1 (ATR1). Expression of the N-terminus of RPP1, including the Toll/interleukin-1 receptor (TIR) domain ("N-TIR"), elicited an effector-independent cell death response, and we used allelic variation in TIR domain sequences to define the key residues that contribute to this phenotype. Further biochemical characterization indicated that cell death induction was correlated with N-TIR domain self-association. In addition, we demonstrated that the nucleotide-binding (NB)-ARC1 region of RPP1 self-associates and plays a critical role in cell death activation, likely by facilitating TIR:TIR interactions. Structural homology modeling of the NB subdomain allowed us to identify a putative oligomerization interface that was shown to influence NB-ARC1 self-association. Significantly, full-length RPP1 exhibited effector-dependent oligomerization and, although mutations at the NB-ARC1 oligomerization interface eliminated cell death induction, RPP1 self-association was unaffected, suggesting that additional regions contribute to oligomerization. Indeed, the leucine-rich repeat domain of RPP1 also self-associates, indicating that multiple interaction interfaces exist within activated RPP1 oligomers. Finally, we observed numerous intramolecular interactions that likely function to negatively regulate RPP1, and present a model describing the transition to an active NLR protein.
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Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Proteínas NLR/inmunología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Muerte Celular , Cromatografía en Gel , Inmunoprecipitación , Proteínas NLR/metabolismo , Peronospora/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Oomicetos/fisiología , Proteínas/química , Proteínas/metabolismo , Alelos , Secuencia de Aminoácidos , Segregación Cromosómica , Cruzamientos Genéticos , Ecotipo , Sitios Genéticos , Genoma de Planta , Proteínas Repetidas Ricas en Leucina , Anotación de Secuencia Molecular , Fenotipo , Filogenia , Polimorfismo Genético , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Estudios de Asociación Genética , Interacciones Huésped-Patógeno/genética , Manihot/microbiología , Xanthomonas axonopodis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Prueba de Complementación Genética , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Xanthomonas axonopodis/crecimiento & desarrolloRESUMEN
Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive 'hypersensitive response' (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.