RESUMEN
Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Inmunidad de la Planta , Factores de Transcripción/metabolismoRESUMEN
Heartworm disease, caused by Dirofilaria immitis, remains a significant threat to canines and felines. The development of parasites resistant to macrocyclic lactones (ML) has created a significant challenge to the control of the infection. The goal of this study was to determine if mice lacking a functional immune response would be susceptible to D. immitis. Immunodeficient NSG mice were susceptible to the infection, sustaining parasites for at least 15 weeks, with infective third-stage larvae molting and developing into the late fourth-stage larvae. Proteomic analysis of host responses to the infection revealed a complex pattern of changes after infection, with at least some of the responses directed at reducing immune control mechanisms that remain in NSG mice. NSG mice were infected with isolates of D. immitis that were either susceptible or resistant to MLs, as a population. The susceptible isolate was killed by ivermectin whereas the resistant isolate had improved survivability, while both isolates were affected by moxidectin. It was concluded that D. immitis survives in NSG mice for at least 15 weeks. NSG mice provide an ideal model for monitoring host responses to the infection and for testing parasites in vivo for susceptibility to direct chemotherapeutic activity of new agents.
Asunto(s)
Enfermedades de los Gatos , Dirofilaria immitis , Enfermedades de los Perros , Parásitos , Animales , Perros , Gatos , Ratones , Dirofilaria immitis/fisiología , Proteómica , Enfermedades de los Perros/parasitologíaRESUMEN
Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVRA1, AVRA7, AVRA9, AVRA13, and allelic AVRA10/AVRA22, which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVRA6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVRA1, AVRA7, AVRA10/AVRA22, and AVRA13, suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVRA1 and AVRA6 as well as allelic AVRA10 and AVRA22 is largely determined by the receptors' C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVRA10 and five specific AVRA22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVRA effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.
Asunto(s)
Ascomicetos/fisiología , Hordeum/genética , Leucina/química , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Receptores Inmunológicos/genética , Ribonucleasas/química , Alelos , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Variación Genética , Hordeum/inmunología , Hordeum/microbiología , Inmunidad Innata , Leucina/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Receptores Inmunológicos/metabolismo , Ribonucleasas/metabolismo , Homología de SecuenciaRESUMEN
Plant intracellular NLR receptors recognise pathogen interference to trigger immunity but how NLRs signal is not known. Enhanced disease susceptibility1 (EDS1) heterodimers are recruited by Toll-interleukin1-receptor domain NLRs (TNLs) to transcriptionally mobilise resistance pathways. By interrogating the Arabidopsis EDS1 É-helical EP-domain we identify positively charged residues lining a cavity that are essential for TNL immunity signalling, beyond heterodimer formation. Mutating a single, conserved surface arginine (R493) disables TNL immunity to an oomycete pathogen and to bacteria producing the virulence factor, coronatine. Plants expressing a weakly active EDS1R493A variant have delayed transcriptional reprogramming, with severe consequences for resistance and countering bacterial coronatine repression of early immunity genes. The same EP-domain surface is utilised by a non-TNL receptor RPS2 for bacterial immunity, indicating that the EDS1 EP-domain signals in resistance conferred by different NLR receptor types. These data provide a unique structural insight to early downstream signalling in NLR receptor immunity.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inmunidad de la Planta/genética , Inmunidad de la Planta/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors.
Asunto(s)
Alelos , Ascomicetos/metabolismo , Receptores Inmunológicos/metabolismo , Ascomicetos/genética , Genes de Plantas , Variación Genética , Enfermedades de las Plantas/microbiología , Polimorfismo de Nucleótido Simple , Unión Proteica , Receptores Inmunológicos/genéticaRESUMEN
Dynamic models analyzing gene regulation and metabolism face challenges when adapted to modeling signal transduction networks. During signal transduction, molecular reactions and mechanisms occur in different spatial and temporal frames and involve feedbacks. This impedes the straight-forward use of methods based on Boolean networks, Bayesian approaches, and differential equations. We propose a new approach, ProbRules, that combines probabilities and logical rules to represent the dynamics of a system across multiple scales. We demonstrate that ProbRules models can represent various network motifs of biological systems. As an example of a comprehensive model of signal transduction, we provide a Wnt network that shows remarkable robustness under a range of phenotypical and pathological conditions. Its simulation allows the clarification of controversially discussed molecular mechanisms of Wnt signaling by predicting wet-lab measurements. ProbRules provides an avenue in current computational modeling by enabling systems biologists to integrate vast amounts of available data on different scales.
Asunto(s)
Redes Reguladoras de Genes , Modelos Biológicos , Modelos Estadísticos , Transducción de Señal/genética , Biología de Sistemas/métodos , Teorema de Bayes , Retroalimentación , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Fosforilación , Transfección , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
The barley disease resistance (R) gene locus mildew locus A (Mla) provides isolate-specific resistance against the powdery mildew fungus Blumeria graminis hordei and has been introgressed into modern cultivars from diverse germplasms, including the wild relative Hordeum spontaneum. Known Mla disease resistance specificities to B. graminis hordei appear to encode allelic variants of the R gene homolog 1 (RGH1) family of nucleotide-binding domain and leucine-rich repeat (NLR) proteins. Here, we sequenced and assembled the transcriptomes of 50 H. spontaneum accessions representing nine populations distributed throughout the Fertile Crescent. The assembled Mla transcripts exhibited rich sequence diversity, linked neither to geographic origin nor population structure, and could be grouped into two similar-sized subfamilies based on two major N-terminal coiled-coil (CC) signaling domains that are both capable of eliciting cell death. The presence of positively selected sites located mainly in the C-terminal leucine-rich repeats of both MLA subfamilies, together with the fact that both CC signaling domains mediate cell death, implies that the two subfamilies are actively maintained in the population. Unexpectedly, known MLA receptor variants that confer B. graminis hordei resistance belong exclusively to one subfamily. Thus, signaling domain divergence, potentially as adaptation to distinct pathogen populations, is an evolutionary signature of functional diversification of an immune receptor. Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Asunto(s)
Ascomicetos , Resistencia a la Enfermedad , Hordeum , Ascomicetos/fisiología , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Hordeum/genética , Hordeum/inmunología , Hordeum/microbiología , Proteínas de Plantas/inmunologíaRESUMEN
During microbe-associated molecular pattern-triggered immunity more than 5000 Arabidopsis genes are significantly altered in their expression, and the question arises, how such an enormous reprogramming of the transcriptome can be regulated in a safe and robust manner? For the WRKY transcription factors (TFs), which are important regulators of numerous defense responses, it appears that they act in a complex regulatory sub-network rather than in a linear fashion, which would be much more vulnerable to gene function loss either by pathogen-derived effectors or by mutations. In this study we employed RNA-seq, mass spectrometry and chromatin immunoprecipitation-seq to find evidence for and uncover principles and characteristics of this network. Upon flg22-treatment, one can distinguish between two sets of WRKY genes: constitutively expressed and induced WRKY genes. Prior to elicitation the induced WRKY genes appear to be maintained in a repressed state mainly by the constitutively expressed WRKY factors, which themselves appear to be regulated by non-WRKY TFs. Upon elicitation, induced WRKYs rapidly bind to induced WRKY gene promoters and by auto- and cross-regulation build up the regulatory network. Maintenance of this flg22-induced network appears highly robust as removal of three key WRKY factors can be physically and functionally compensated for by other WRKY family members.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genoma de Planta/genética , Enfermedades de las Plantas/inmunología , Pseudomonas syringae/patogenicidad , Factores de Transcripción/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flagelina/farmacología , Mutación , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/efectos de los fármacos , Inmunidad de la Planta/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Bioactive natural products are important starting points for developing chemical tools for biological research. For elucidating their bioactivity profile, biological systems with concise complexity such as cell culture systems are frequently used, whereas unbiased investigations in more complex multicellular systems are only rarely explored. Here, we demonstrate with the natural product Rotihibinâ A and the plant research model system Arabidopsis thaliana that unbiased transcriptional profiling enables a rapid, label-free, and compound economic evaluation of a natural product's bioactivity profile in a complex multicellular organism. To this end, we established a chemical synthesis of Rotihibin A as well as that of structural analogues, followed by transcriptional profiling-guided identification and validation of Rotihibinâ A as a TOR signaling inhibitor (TOR=target of rapamycin). These findings illustrate that a combined approach of transcriptional profiling and natural product research may represent a technically simple approach to streamline the development of chemical tools from natural products even for biologically complex multicellular biological systems.
Asunto(s)
Oligopéptidos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Productos Biológicos , Expresión Génica , Perfilación de la Expresión Génica , Modelos Moleculares , Mutación , Oligopéptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sirolimus/farmacología , Bibliotecas de Moléculas PequeñasRESUMEN
The phytohormone network consisting of jasmonate, ethylene, PHYTOALEXIN-DEFICIENT4, and salicylic acid signaling is required for the two modes of plant immunity, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI). A previous study showed that during PTI, the transcriptional responses of over 5000 genes qualitatively depend on complex interactions between the network components. However, the role of the network in transcriptional reprogramming during ETI and whether it differs between PTI and ETI remain elusive. Here, we generated time-series RNA-sequencing data of Arabidopsis thaliana wild-type and combinatorial mutant plants deficient in components of the network upon challenge with virulent or ETI-triggering avirulent strains of the foliar bacterial pathogen Pseudomonas syringae Resistant plants such as the wild type achieved high-amplitude transcriptional reprogramming 4 h after challenge with avirulent strains and sustained this transcriptome response. Strikingly, susceptible plants including the quadruple network mutant showed almost identical transcriptome responses to resistant plants but with several hours delay. Furthermore, gene coexpression network structure was highly conserved between the wild type and quadruple mutant. Thus, in contrast to PTI, the phytohormone network is required only for achieving high-amplitude transcriptional reprogramming within the early time window of ETI against this bacterial pathogen.
Asunto(s)
Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Inmunidad de la Planta/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare). RESULTS: The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome. CONCLUSIONS: The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a "one-speed" genome that differs in its architecture and (co-)evolutionary pattern from the "two-speed" genomes reported for several other filamentous phytopathogens.
Asunto(s)
Ascomicetos/genética , Ascomicetos/fisiología , Elementos Transponibles de ADN/genética , Genoma Fúngico/genética , Hordeum/microbiología , Especificidad del Huésped/genética , Enfermedades de las Plantas/microbiología , Variaciones en el Número de Copia de ADN , Duplicación de Gen , Perfilación de la Expresión Génica , FilogeniaRESUMEN
Pattern recognition receptors (PRRs) and nucleotide-binding domain and leucine-rich repeat (LRR)-containing proteins (NLRs) initiate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively, each associated with the activation of an overlapping set of defence genes. The regulatory mechanism behind this convergence of PTI- and ETI-mediated defence gene induction remains elusive. We generated transgenic Arabidopsis plants that enable conditional NLR activation without pathogen infection to dissect NLR- and PRR-mediated transcriptional signals. A comparative analysis of over 40 transcriptome datasets linked calmodulin-binding transcription activators (CAMTAs) to the activation of overlapping defence genes in PTI and ETI. We used a dominant camta3 mutant (camta3-D) to assess CAMTA functions in the corresponding transcriptional regulation. Transcriptional regulation by NLRs, although highly similar to PTI responses, can be established independently of pathogen-associated molecular pattern (PAMP) perception, defence phytohormones and host cell death. Conditional expression of the N-terminal coiled-coil domain of the barley MLA (Mildew resistance locus A) NLR is sufficient to trigger similar transcriptional reprogramming as full-length NLRs. CAMTA-binding motifs are overrepresented in the 5' regulatory regions of the identified primary immune response genes, consistent with their altered expression and disease resistance responses in camta3-D plants. We propose that CAMTA-mediated transcriptional regulation defines an early convergence point in NLR- and PRR-mediated signalling.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes Dominantes , Espacio Intracelular/metabolismo , Mutación/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas NLR/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
Plant growth regulating properties of brevicompanines (Brvs), natural products of the fungus Penicillium brevicompactum, have been known for several years, but further investigations into the molecular mechanism of their bioactivity have not been performed. Following chemical synthesis of brevicompanine derivatives, we studied their activity in the model plant Arabidopsis by a combination of plant growth assays, transcriptional profiling, and numerous additional bioassays. These studies demonstrated that brevicompanines cause transcriptional misregulation of core components of the circadian clock, whereas other biological read-outs were not affected. Brevicompanines thus represent promising chemical tools for investigating the regulation of the plant circadian clock. In addition, our study also illustrates the potential of an unbiased -omics-based characterization of bioactive compounds for identifying the often cryptic modes of action of small molecules.
Asunto(s)
Productos Biológicos/farmacología , Ritmo Circadiano/efectos de los fármacos , Indoles/farmacología , Péptidos Cíclicos/farmacología , Raíces de Plantas/crecimiento & desarrollo , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Productos Biológicos/síntesis química , Indoles/síntesis química , Penicillium/química , Péptidos Cíclicos/síntesis química , Fenómenos Fisiológicos de las Plantas/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Plant defenses induced by salicylic acid (SA) are vital for resistance against biotrophic pathogens. In basal and receptor-triggered immunity, SA accumulation is promoted by Enhanced Disease Susceptibility1 with its co-regulator Phytoalexin Deficient4 (EDS1/PAD4). Current models position EDS1/PAD4 upstream of SA but their functional relationship remains unclear. In a genetic and transcriptomic analysis of Arabidopsis autoimmunity caused by constitutive or conditional EDS1/PAD4 overexpression, intrinsic EDS1/PAD4 signaling properties and their relation to SA were uncovered. A core EDS1/PAD4 pathway works in parallel with SA in basal and effector-triggered bacterial immunity. It protects against disabled SA-regulated gene expression and pathogen resistance, and is distinct from a known SA-compensatory route involving MAPK signaling. Results help to explain previously identified EDS1/PAD4 regulated SA-dependent and SA-independent gene expression sectors. Plants have evolved an alternative route for preserving SA-regulated defenses against pathogen or genetic perturbations. In a proposed signaling framework, EDS1 with PAD4, besides promoting SA biosynthesis, maintains important SA-related resistance programs, thereby increasing robustness of the innate immune system.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Hidrolasas de Éster Carboxílico/metabolismo , Proteínas de Unión al ADN/metabolismo , Inmunidad de la Planta , Ácido Salicílico/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/microbiología , Autoinmunidad/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Resistencia a la Enfermedad/efectos de los fármacos , Estradiol/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Transcripción Genética/efectos de los fármacosRESUMEN
During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genoma de Planta/genética , Factores de Transcripción/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Sitios de Unión/genética , Flagelina/farmacología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ontología de Genes , Immunoblotting , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/efectos de los fármacos , Inmunidad de la Planta/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.
Asunto(s)
Alelos , Ascomicetos/genética , Ascomicetos/inmunología , Hordeum/inmunología , Hordeum/microbiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Arabidopsis/genética , Ascomicetos/patogenicidad , Secuencia de Bases , Muerte Celular , Resistencia a la Enfermedad/inmunología , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Estudios de Asociación Genética , Genoma Fúngico , Genotipo , Interacciones Huésped-Patógeno/inmunología , Fenotipo , Células Vegetales , Hojas de la Planta/microbiología , Plantas Modificadas Genéticamente , Polimorfismo de Nucleótido Simple , Receptores Inmunológicos/genética , Transcriptoma , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.
Asunto(s)
Arabidopsis/metabolismo , Colletotrichum/metabolismo , Endófitos/metabolismo , Fosfatos/deficiencia , Raíces de Plantas/metabolismo , Arabidopsis/inmunología , Quitina/metabolismo , Colletotrichum/genética , Endófitos/genética , Genoma Fúngico/genética , Inanición , Simbiosis/inmunología , Simbiosis/fisiologíaRESUMEN
A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host's phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.
Asunto(s)
Arabidopsis/microbiología , Colletotrichum/aislamiento & purificación , Fosfatos/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Colletotrichum/fisiología , Endófitos , Proteínas de Transporte de Fosfato/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , España , SimbiosisRESUMEN
Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.