RESUMO
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.
Assuntos
Resistência à Doença , Doenças das Plantas , Imunidade Vegetal , Plantas , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Plantas/imunologia , Plantas/genética , Resistência à Doença/genética , HumanosRESUMO
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.
RESUMO
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.
Assuntos
Arabidopsis/imunologia , Arabidopsis/metabolismo , Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Imunidade Vegetal/imunologia , Animais , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Calmodulina/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Feminino , Imunidade Inata , Oócitos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , XenopusRESUMO
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.
Assuntos
Oryza , Estômatos de Plantas , Estômatos de Plantas/fisiologia , Resistência à Seca , Fotossíntese/genética , SecasRESUMO
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.
Assuntos
Proteínas de Plantas , Plantas , Proteínas de Plantas/metabolismo , Plantas/genética , Domínios Proteicos , Filogenia , Receptores Imunológicos/genética , Doenças das Plantas , Imunidade VegetalRESUMO
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.
Assuntos
Nicotiana/genética , Nicotiana/metabolismo , Subpopulações de Linfócitos B/metabolismo , Proteínas de Ligação a DNA , Proteínas de Repetições Ricas em Leucina , Proteínas NLR/metabolismo , Neuregulina-1/genética , Neuregulina-1/fisiologia , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas/genética , Pseudomonas , Transdução de Sinais , Transcriptoma , XanthomonasRESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Capsicum/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Polissacarídeo-Liases/genética , Solanum lycopersicum/microbiologia , Fatores de Transcrição/fisiologia , Xanthomonas/patogenicidade , Proteínas de Bactérias/genética , Capsicum/metabolismo , Ativação Enzimática , Solanum lycopersicum/metabolismo , Fenótipo , Doenças das Plantas/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Nicotiana/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para Cima , Virulência , Água/metabolismo , Xanthomonas/genética , Xanthomonas/fisiologiaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Proteínas de Plantas/química , Sequência de Aminoácidos , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Sítios de Ligação , Morte Celular/genética , Morte Celular/imunologia , Linho/genética , Linho/imunologia , Linho/microbiologia , Interações Hospedeiro-Patógeno , Modelos Moleculares , Mutação , Peronospora/patogenicidade , Peronospora/fisiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
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.
Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Manihot/imunologia , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Doenças das Plantas/imunologia , Potyviridae/imunologia , Sistemas CRISPR-Cas , Fator de Iniciação 4E em Eucariotos/metabolismo , Edição de Genes , Interações Hospedeiro-Patógeno , Manihot/genética , Manihot/virologia , Complexo Proteico Nuclear de Ligação ao Cap/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/imunologia , Proteínas NLR/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Morte Celular , Cromatografia em Gel , Imunoprecipitação , Proteínas NLR/metabolismo , Peronospora/imunologiaRESUMO
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
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.
Assuntos
Resistência à Doença/genética , Éxons , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Transcrição Gênica , Processamento Alternativo , Arabidopsis/genética , Proteínas de Bactérias/genética , Dexametasona/farmacologia , Oomicetos/genética , Fenótipo , Plantas Geneticamente Modificadas/genética , Nicotiana/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
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
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.
Assuntos
Genoma Bacteriano/genética , Genoma de Planta/genética , Doenças das Plantas/imunologia , Solanum lycopersicum/genética , Solanum lycopersicum/imunologia , Xanthomonas/genética , Sequência de Bases , Cruzamento , Genótipo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Análise de Sequência de DNARESUMO
Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.
Assuntos
Arabidopsis/genética , Simulação por Computador , Regulação da Expressão Gênica de Plantas , Oomicetos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Imunidade Inata/genética , Modelos Genéticos , Modelos Moleculares , Oomicetos/patogenicidadeRESUMO
Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.
Assuntos
Manihot/metabolismo , Manihot/microbiologia , Doenças das Plantas/microbiologia , Análise de Sequência de DNA/métodos , Xanthomonas axonopodis/metabolismo , Área Sob a Curva , Progressão da Doença , Genoma Bacteriano , Genômica , Geografia , Imunidade Inata , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/genética , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Fatores de TempoRESUMO
Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.
Assuntos
Proteínas de Bactérias/metabolismo , Hordeum/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas fluorescens/metabolismo , Triticum/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Proteínas de Bactérias/genética , Basidiomycota/patogenicidade , Calmodulina/genética , Calmodulina/metabolismo , Engenharia Genética , Hordeum/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Caules de Planta/metabolismo , Caules de Planta/microbiologia , Plantas Geneticamente Modificadas , Transporte Proteico , Pseudomonas fluorescens/genética , Pseudomonas syringae/genética , Proteínas Recombinantes de Fusão , Triticum/microbiologia , Virulência , Xanthomonas/genéticaRESUMO
The gene-for-gene concept has historically been applied to describe a specific resistance interaction wherein single genes from the host and the pathogen dictate the outcome. These interactions have been observed across the plant kingdom and all known plant microbial pathogens. In recent years, this concept has been extended to susceptibility phenotypes in the context of transcription activator-like (TAL) effectors that target SWEET sugar transporters. However, because this interaction has only been observed in rice, it was not clear whether the gene-for-gene susceptibility was unique to that system. Here, we show, through a combined systematic analysis of the TAL effector complement of Xanthomonas axonopodis pv. manihotis and RNA sequencing to identify targets in cassava, that TAL20Xam668 specifically induces the sugar transporter MeSWEET10a to promote virulence. Designer TAL effectors (dTALE) complement TAL20Xam668 mutant phenotypes, demonstrating that MeSWEET10a is a susceptibility gene in cassava. Sucrose uptake-deficient X. axonopodis pv. manihotis bacteria do not lose virulence, indicating that sucrose may be cleaved extracellularly and taken up as hexoses into X. axonopodis pv. manihotis. Together, our data suggest that pathogen hijacking of plant nutrients is not unique to rice blight but also plays a role in bacterial blight of the dicot cassava.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica de Plantas , Manihot/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/patogenicidade , Proteínas de Bactérias/genética , Resistência à Doença , Expressão Gênica , Manihot/genética , Manihot/imunologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Doenças das Plantas/imunologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Sequência de RNA , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologia , Regulação para Cima , Virulência , Xanthomonas axonopodis/genéticaRESUMO
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.
Assuntos
Oomicetos/metabolismo , Proteínas/química , Proteínas/metabolismo , Sequências Repetitivas de Aminoácidos , Alelos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Arabidopsis/parasitologia , Proteínas de Arabidopsis , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Effectors of the bacterial type III secretion system provide invaluable molecular probes to elucidate the molecular mechanisms of plant immunity and pathogen virulence. In this report, we focus on the AvrBs2 effector protein from the bacterial pathogen Xanthomonas euvesicatoria (Xe), the causal agent of bacterial spot disease of tomato and pepper. Employing homology-based structural analysis, we generate a three-dimensional structural model for the AvrBs2 protein and identify catalytic sites in its putative glycerolphosphodiesterase domain (GDE). We demonstrate that the identified catalytic region of AvrBs2 was able to functionally replace the GDE catalytic site of the bacterial glycerophosphodiesterase BhGlpQ cloned from Borrelia hermsii and is required for AvrBs2 virulence. Mutations in the GDE catalytic domain did not disrupt the recognition of AvrBs2 by the cognate plant resistance gene Bs2. In addition, AvrBs2 activation of Bs2 suppressed subsequent delivery of other Xanthomonas type III effectors into the host plant cells. Investigation of the mechanism underlying this modulation of the type III secretion system may offer new strategies to generate broad-spectrum resistance to bacterial pathogens.