RESUMEN
Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.
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Proteínas de Arabidopsis , Arabidopsis , Oomicetos , Enfermedades de las Plantas , Arabidopsis/genética , Arabidopsis/microbiología , Arabidopsis/inmunología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Oomicetos/patogenicidad , Proteínas NLR/metabolismo , Proteínas NLR/genética , Nicotiana/genética , Nicotiana/microbiología , Nicotiana/inmunología , Secuencia de Aminoácidos , AlelosRESUMEN
BACKGROUND: Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea, coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. METHODS AND RESULTS: In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida) was initiated to transfer resistance genes (WRR8Sf-2 and WRR9Hi-0) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. CONCLUSIONS: Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.
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Arabidopsis , Oomicetos , Planta de la Mostaza/genética , Genotipo , Agrobacterium , Arabidopsis/genética , CandidaRESUMEN
The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis (Arabidopsis thaliana) by forming structures called haustoria inside host cells. Previous transcriptome analyses have revealed that host genes are specifically induced during infection; however, RNA profiling from whole-infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells, where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a translating ribosome affinity purification system using 2 high-affinity binding proteins, colicin E9 and Im9 (immunity protein of colicin E9), applicable to pathogen-responsive promoters, thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing insights into the Arabidopsis-downy mildew interaction. We propose that our protocol for profiling cell-specific transcripts will apply to several stimulus-specific contexts and other plant-pathogen interactions.
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Arabidopsis , Colicinas , Oomicetos , Peronospora , Arabidopsis/genética , ARN/metabolismo , Colicinas/metabolismo , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genéticaRESUMEN
White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica crops. The Brassica relative Arabidopsis thaliana uses the paired immune receptor complex CSA1-CHS3/DAR4 to resist Albugo infection. The CHS3/DAR4 sensor NLR, which functions together with its partner, the helper NLR CSA1, carries an integrated domain (ID) with homology to DA1 peptidases. Using domain swaps with several DA1 homologs, we show that the LIM-peptidase domain of the family member CHS3/DAR4 functions as an integrated decoy for the family member DAR3, which interacts with and inhibits the peptidase activities of the three closely related peptidases DA1, DAR1, and DAR2. Albugo infection rapidly lowers DAR3 levels and activates DA1 peptidase activity, thereby promoting endoreduplication of host tissues to support pathogen growth. We propose that the paired immune receptor CSA1-CHS3/DAR4 detects the actions of a putative Albugo effector that reduces DAR3 levels, resulting in defense activation.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Péptido Hidrolasas , Dominios Proteicos , Productos Agrícolas , Enfermedades de las PlantasRESUMEN
The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their capacity to infect different host plant species. Each A. candida race encodes secreted proteins with a CX2 CX5 G ('CCG') motif that are polymorphic and show presence/absence variation, and are therefore candidate effectors. The White Rust Resistance 4 (WRR4) locus in Arabidopsis thaliana accession Col-0 contains three genes that encode intracellular nucleotide-binding domain leucine-rich repeat immune receptors. The Col-0 alleles of WRR4A and WRR4B confer resistance to multiple A. candida races, although both WRR4A and WRR4B can be overcome by the Col-0-virulent race 4 isolate AcEx1. Comparison of CCG candidate effectors in avirulent and virulent races, and transient co-expression of CCG effectors from four A. candida races in Nicotiana sp. or A. thaliana, revealed CCG effectors that trigger WRR4A- or WRR4B-dependent hypersensitive responses. We found eight WRR4A-recognised CCGs and four WRR4B-recognised CCGs, the first recognised proteins from A. candida for which the cognate immune receptors in A. thaliana are known. This multiple recognition capacity potentially explains the broad-spectrum resistance to several A. candida races conferred by WRR4 paralogues. We further show that of five tested CCGs, three confer enhanced disease susceptibility when expressed in planta, consistent with A. candida CCG proteins being effectors.
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Proteínas de Arabidopsis , Arabidopsis , Brassica , Oomicetos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas NLR/metabolismo , Brassica/metabolismo , Oomicetos/metabolismo , Enfermedades de las Plantas/genéticaRESUMEN
Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Inmunidad de la Planta , Inmunidad , HomeostasisRESUMEN
Some plant NLR immune receptors are encoded in head-to-head "sensor-executor" pairs that function together. Alleles of the NLR pair CHS3/CSA1 form three clades. The clade 1 sensor CHS3 contains an integrated domain (ID) with homology to regulatory domains, which is lacking in clades 2 and 3. In this study, we defined two cell-death regulatory modes for CHS3/CSA1 pairs. One is mediated by ID domain on clade 1 CHS3, and the other relies on CHS3/CSA1 pairs from all clades detecting perturbation of an associated pattern-recognition receptor (PRR) co-receptor. Our data support the hypothesis that an ancestral Arabidopsis CHS3/CSA1 pair gained a second recognition specificity and regulatory mechanism through ID acquisition while retaining its original specificity as a "guard" against PRR co-receptor perturbation. This likely comes with a cost, since both ID and non-ID alleles of the pair persist in diverse Arabidopsis populations through balancing selection.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Alelos , Receptores Inmunológicos/genética , Muerte Celular , Receptores de Reconocimiento de Patrones , Inmunidad de la Planta/genética , Proteínas NLR/genéticaRESUMEN
Nucleotide-binding and leucine-rich repeat receptors (NLRs) are intracellular plant immune receptors that recognize pathogen effectors secreted into the plant cell. Canonical NLRs typically contain three conserved domains including a central nucleotide binding (NB-ARC) domain, C-terminal leucine-rich repeats (LRRs) and an N-terminal domain. A subfamily of plant NLRs contain additional noncanonical domain(s) that have potentially evolved from the integration of the effector targets in the canonical NLR structure. These NLRs with extra domains are thus referred to as NLRs with integrated domains (NLR-IDs). Here, we first summarize our current understanding of NLR-ID activation upon effector binding, focusing on the NLR pairs Pik-1/Pik-2, RGA4/RGA5, and RRS1/RPS4. We speculate on their potential oligomerization into resistosomes as it was recently shown for certain canonical plant NLRs. Furthermore, we discuss how our growing understanding of the mode of action of NLR-ID continuously informs engineering approaches to design new resistance specificities in the context of rapidly evolving pathogens.
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Inmunidad de la Planta , Plantas , Leucina , Nucleótidos , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Dominios ProteicosRESUMEN
Albugo candida is an obligate oomycete pathogen that infects many plants in the Brassicaceae family. We resequenced the genome of isolate Ac2V using PacBio long reads and constructed an assembly augmented by Illumina reads. The Ac2VPB genome assembly is 10% larger and more contiguous compared with a previous version. Our annotation of the new assembly, aided by RNA-sequencing information, revealed a 175% expansion (40 to 110) in the CHxC effector class, which we redefined as "CCG" based on motif analysis. This class of effectors consist of arrays of phylogenetically related paralogs residing in gene sparse regions, and shows signatures of positive selection and presence/absence polymorphism. This work provides a resource that allows the dissection of the genomic components underlying A. candida adaptation and, particularly, the role of CCG effectors in virulence and avirulence on different hosts.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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Brassicaceae , Oomicetos , Candida/genética , Genoma , Oomicetos/genética , Enfermedades de las PlantasRESUMEN
The oomycete Albugo candida causes white rust of Brassicaceae, including vegetable and oilseed crops, and wild relatives such as Arabidopsis thaliana. Novel White Rust Resistance (WRR) genes from Arabidopsis enable new insights into plant/parasite co-evolution. WRR4A from Arabidopsis accession Columbia (Col-0) provides resistance to many but not all white rust races, and encodes a nucleotide-binding, leucine-rich repeat immune receptor. Col-0 WRR4A resistance is broken by AcEx1, an isolate of A. candida. We identified an allele of WRR4A in Arabidopsis accession Øystese-0 (Oy-0) and other accessions that confers full resistance to AcEx1. WRR4AOy-0 carries a C-terminal extension required for recognition of AcEx1, but reduces recognition of several effectors recognized by the WRR4ACol-0 allele. WRR4AOy-0 confers full resistance to AcEx1 when expressed in the oilseed crop Camelina sativa.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Resistencia a la Enfermedad/genética , Variación Genética , Oomicetos/fisiología , Enfermedades de las Plantas/inmunología , Alelos , Secuencia de Aminoácidos , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Camellia/genética , Camellia/inmunología , Hojas de la Planta , Plantas Modificadas Genéticamente , Alineación de Secuencia , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas NLR/genética , Alelos , Proteínas de Arabidopsis/metabolismo , Resistencia a la Enfermedad/genética , Variación Genética , Genoma de Planta , Proteínas NLR/metabolismo , Enfermedades de las Plantas/genética , Inmunidad de la Planta , Especificidad de la EspecieRESUMEN
Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens.
Asunto(s)
Arabidopsis/inmunología , Brassica/microbiología , Oomicetos/patogenicidad , Enfermedades de las Plantas/inmunología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Haplotipos , Inmunidad Innata , Enfermedades de las Plantas/microbiologíaRESUMEN
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
Physiological races of the oomycete Albugo candida are biotrophic pathogens of diverse plant species, primarily the Brassicaceae, and cause infections that suppress host immunity to other pathogens. However, A. candida race diversity and the consequences of host immunosuppression are poorly understood in the field. We report a method that enables sequencing of DNA of plant pathogens and plant-associated microbes directly from field samples (Pathogen Enrichment Sequencing: PenSeq). We apply this method to explore race diversity in A. candida and to detect A. candida-associated microbes in the field (91 A. candida-infected plants). We show with unprecedented resolution that each host plant species supports colonization by one of 17 distinct phylogenetic lineages, each with an unique repertoire of effector candidate alleles. These data reveal the crucial role of sexual and asexual reproduction, polyploidy and host domestication in A. candida specialization on distinct plant species. Our bait design also enabled phylogenetic assignment of DNA sequences from bacteria and fungi from plants in the field. This paper shows that targeted sequencing has a great potential for the study of pathogen populations while they are colonizing their hosts. This method could be applied to other microbes, especially to those that cannot be cultured.
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Brassicaceae/genética , Brassicaceae/microbiología , Variación Genética , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Ploidias , Secuencia de Bases , Brassicaceae/crecimiento & desarrollo , Frecuencia de los Genes/genética , Sitios Genéticos , Genética de Población , Genotipo , Heterocigoto , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Recombinación Genética/genéticaRESUMEN
Most land plant genomes carry genes that encode RPW8-NLR Resistance (R) proteins. Angiosperms carry two RPW8-NLR subclasses: ADR1 and NRG1. ADR1s act as 'helper' NLRs for multiple TIR- and CC-NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co-occurs with TIR-NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR-NLRs. Using CRISPR mutagenesis, we obtained an nrg1a-nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR-NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC-NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC-NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR-NLR function and contributes to the signalling of some CC-NLRs. Some NRG1-dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.
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Proteínas de Arabidopsis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas NLR/metabolismo , Inmunidad de la Planta , Receptores Inmunológicos/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteína 9 Asociada a CRISPR/metabolismo , Resistencia a la Enfermedad , Modelos Biológicos , Mutación/genética , Oomicetos/fisiología , Enfermedades de las Plantas/microbiología , Ácido Salicílico/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
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.
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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
Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R-RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector's authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY-domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY-domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a "reversibly closed" conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Proteínas Bacterianas/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Transferencia Resonante de Energía de Fluorescencia , Complejos Multiproteicos/inmunología , Mutación , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Plantas Modificadas Genéticamente , Conformación Proteica , Dominios Proteicos , Ralstonia solanacearum/patogenicidad , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
The pathosystem of Arabidopsis thaliana and diploid biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) has been a model for investigating the molecular basis of Flor's gene-for-gene hypothesis. The isolates Hpa-Noks1 and Hpa-Cala2 are virulent on Arabidopsis accession RMX-A02 whilst an F1 generated from a cross between these two isolates was avirulent. The F2 progeny segregated 3,1 (avirulent, virulent), indicating a single major effect AVR locus in this pathogen. SNP-based linkage mapping confirmed a single AVR locus within a 14 kb map interval containing two genes encoding putative effectors. The Hpa-Cala2 allele of one gene, designated H. arabidopsidiscryptic1 (HAC1), encodes a protein with a signal peptide and an RxLR/dEER motif, and triggers a defense response in RMX-A02. The second gene is heterozygous in Hpa-Cala2. One allele, designated Suppressor ofHAC1Cala2 (S-HAC1Cala2 ) encodes a protein with a signal peptide and a dKEE motif with no RxLR motif; the other allele (s-hac1Cala2 ) encodes a protein with a signal peptide, a dEEE motif and is divergent in sequence from the S-HAC1Cala2 allele. In selfed progeny from Hpa-Cala2, dominant S-HAC1Cala2 allele carrying progeny correlates with virulence in RMX-A02, whereas homozygous recessive s-hac1Cala2 carrying progeny were avirulent. Genetic investigations suggested other heterozygous suppressor loci might exist in the Hpa-Cala2 genome.
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Ground-based facilities, such as clinostats and random positioning machines aiming at simulating microgravity conditions, are tools to prepare space experiments and identify gravity-related signaling pathways. A prerequisite is that the facilities are operated in an appropriate manner and potentially induced non-gravitational effects, such as shearing forces, have to be taken into account. Dinoflagellates, here P. noctiluca, as fast and sensitive reporter system for shear stress and hydrodynamic gradients, were exposed on a clinostat (constant rotation around one axis, 60 rpm) or in a random positioning machine, that means rotating around two axes, whose velocity and direction were chosen at random. Deformation of the cell membrane of P. noctiluca due to shear stress results in a detectable bioluminescence emission. Our results show that the amount of mechanical stress is higher on an random positioning machine than during constant clinorotation, as revealed by the differences in photon counts. We conclude that one axis clinorotation induced negligible non-gravitational effects in the form of shear forces in contrast to random operation modes tested. For the first time, we clearly visualized the device-dependent occurrence of shear forces by means of a bioassay, which have to be considered during the definition of an appropriate simulation approach and to avoid misinterpretation of results.
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Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation.