RESUMEN
Pathogens and pests secrete proteins (effectors) to interfere with plant immunity through modification of host target functions and disruption of immune signalling networks. The extent of convergence between pathogen and herbivorous insect virulence strategies is largely unexplored. We found that effectors from the oomycete pathogen, Phytophthora capsici, and the major aphid pest, Myzus persicae target the host immune regulator SIZ1, an E3 SUMO ligase. We used transient expression assays in Nicotiana benthamiana as well as Arabidopsis mutants to further characterize biological role of effector-SIZ1 interactions in planta. We show that the oomycete and aphid effector, which both contribute to virulence, feature different activities towards SIZ1. While M. persicae effector Mp64 increases SIZ1 protein levels in transient assays, P. capsici effector CRN83_152 enhances SIZ1-E3 SUMO ligase activity in vivo. SIZ1 contributes to host susceptibility to aphids and an oomycete pathogen. Knockout of SIZ1 in Arabidopsis decreased susceptibility to aphids, independent of SNC1, PAD4 and EDS1. Similarly SIZ1 knockdown in N. benthamiana led to reduced P. capsici infection. Our results suggest convergence of distinct pathogen and pest virulence strategies on an E3 SUMO ligase to enhance host susceptibility.
Asunto(s)
Áfidos , Proteínas de Arabidopsis , Arabidopsis , Phytophthora , Animales , Áfidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Herbivoria , Ligasas/metabolismo , Phytophthora/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , VirulenciaRESUMEN
Phytophthora spp. secrete vast arrays of effector molecules during infection to aid in host colonization. The crinkling and necrosis (CRN) protein family forms an extensive repertoire of candidate effectors that accumulate in the host nucleus to perturb processes required for immunity. Here, we show that CRN12_997 from Phytophthora capsici binds a TCP transcription factor, SlTCP14-2, to inhibit its immunity-associated activity against Phytophthora spp. Coimmunoprecipitation and bimolecular fluorescence complementation studies confirm a specific CRN12_997-SlTCP14-2 interaction in vivo. Coexpression of CRN12_997 specifically counteracts the TCP14-enhanced immunity phenotype, suggesting that CRN mediated perturbation of SlTCP14-2 function. We show that SlTCP14-2 associates with nuclear chromatin and that CRN12_997 diminishes SlTCP14-2 DNA binding. Collectively, our data support a model in which SlTCP14-2 associates with chromatin to enhance immunity. The interaction between CRN12_997 and SlTCP14-2 reduces DNA binding of the immune regulator. We propose that the modulation of SlTCP14-2 chromatin affinity, caused by CRN12-997, enhances susceptibility to P. capsici.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Phytophthora , Receptores de Superficie Celular , Solanum lycopersicum , Solanum lycopersicum/parasitología , Phytophthora/genética , Phytophthora/patogenicidad , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Virulencia/genéticaRESUMEN
The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world-wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor. Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome. PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes. PenSeq represents a cost-effective alternative to whole-genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens.
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Genómica , Oomicetos/genética , Análisis de Secuencia de ADN/métodos , Alelos , Secuencia de Bases , Genética de Población , Genoma , Heterocigoto , Nucleótidos/genética , Phytophthora infestans/genética , Polimorfismo Genético , Estándares de ReferenciaRESUMEN
Phytophthora spp. cause devastating disease epidemics on important crop plants and pose a grave threat to global crop production. Critically, Phytophthora pathogens represent a distinct evolutionary lineage in which pathogenicity has been acquired independently. Therefore, there is an urgent need to understand and disrupt the processes that drive infection if we aspire to defeat oomycete pathogens in the field. One area that has received little attention thus far in this respect is the regulation of Phytophthora gene expression during infection. Here, we characterize PcNMRAL1 (Phyca11_505845), a homolog of the Aspergillus nidulans nitrogen metabolite repression regulator NMRA and demonstrate a role for this protein in progression of the Phytophthora capsici infection cycle. PcNmrAL1 is coexpressed with the biotrophic marker gene PcHmp1 (haustorial membrane protein 1) and, when overexpressed, extends the biotrophic infection stage. Microarray analyses revealed that PcNmrAL1 overexpression in P. capsici leads to large-scale transcriptional changes during infection and in vitro. Importantly, detailed analysis reveals that PcNmrAL1 overexpression induces biotrophy-associated genes while repressing those associated with necrotrophy. In addition to factors controlling transcription, translation, and nitrogen metabolism, PcNMRAL1 helps regulate the expression of a considerable effector repertoire in P. capsici. Our data suggests that PcNMRAL1 is a transcriptional regulator that mediates the biotrophy to necrotrophy transition. PcNMRAL1 represents a novel factor that may drive the Phytophthora disease cycle on crops. This study provides the first insight into mechanisms that regulate infection-related processes in Phytophthora spp. and provides a platform for further studies aimed at disabling pathogenesis and preventing crop losses.
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Regulación Fúngica de la Expresión Génica/fisiología , Phytophthora/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas/metabolismo , Solanum lycopersicum/microbiología , Secuencia de Aminoácidos , Biomarcadores , Perfilación de la Expresión Génica , Phytophthora/genéticaRESUMEN
With the increasing availability of plant pathogen genomes, secreted proteins that aid infection (effectors) have emerged as key factors that help to govern plant-microbe interactions. The conserved CRN (CRinkling and Necrosis) effector family was first described in oomycetes by their capacity to induce host cell death. Despite recent advances towards the elucidation of CRN virulence functions, the relevance of CRN-induced cell death remains unclear. In planta over-expression of PcCRN83_152, a CRN effector from Phytophthora capsici, causes host cell death and boosts P. capsici virulence. We used these features to ask whether PcCRN83_152-induced cell death is linked to its virulence function. By randomly mutating this effector, we generated PcCRN83_152 variants with no cell death (NCD) phenotypes, which were subsequently tested for activity towards enhanced virulence. We showed that a subset of PcCRN83_152 NCD variants retained their ability to boost P. capsici virulence. Moreover, NCD variants were shown to have a suppressive effect on PcCRN83_152-mediated cell death. Our work shows that PcCRN83_152-induced cell death and virulence function can be separated. Moreover, if these findings hold true for other cell death-inducing CRN effectors, this work, in turn, will provide a framework for studies aimed at unveiling the virulence functions of these effectors.
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Pruebas Genéticas , Mutagénesis/genética , Phytophthora/genética , Phytophthora/patogenicidad , Secuencia de Aminoácidos , Secuencia de Bases , Muerte Celular , Cromatina/metabolismo , Fenotipo , Proteínas/química , Proteínas/genética , Nicotiana/citología , Nicotiana/microbiología , VirulenciaRESUMEN
Plant-pathogen interactions are complex associations driven by the interplay of host and microbe-encoded factors. With secreted pathogen proteins (effectors) and immune signalling components found in the plant nucleus, this compartment is a battleground where susceptibility is specified. We hypothesized that, by defining changes in the nuclear proteome during infection, we can pinpoint vital components required for immunity or susceptibility. We tested this hypothesis by documenting dynamic changes in the tomato (Solanum lycopersicum) nuclear proteome during infection by the oomycete pathogen Phytophthora capsici. We enriched nuclei from infected and noninfected tissues and quantitatively assessed changes in the nuclear proteome. We then tested the role of candidate regulators in immunity through functional assays. We demonstrated that the host nuclear proteome dynamically changes during P. capsici infection. We observed that known nuclear immunity factors were differentially expressed and, based on this observation, selected a set of candidate regulators that we successfully implicated in immunity to P. capsici. Our work exemplifies a powerful strategy to gain rapid insight into important nuclear processes that underpin complex crop traits such as resistance. We have identified a large set of candidate nuclear factors that may underpin immunity to pathogens in crops.
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Phytophthora/fisiología , Proteínas de Plantas/fisiología , Proteoma , Solanum lycopersicum/genética , Núcleo Celular/genética , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/parasitología , Phytophthora/inmunología , Phytophthora/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Plant associated microbes rely on secreted virulence factors (effectors) to modulate host immunity and ensure progressive infection. Amongst the secreted protein repertoires defined and studied in pathogens to date, the CRNs (for CRinkling and Necrosis) have emerged as one of only a few highly conserved protein families, spread across several kingdoms. CRN proteins were first identified in plant pathogenic oomycetes where they were found to be modular factors that are secreted and translocated inside host cells by means of a conserved N-terminal domain. Subsequent localization and functional studies have led to the view that CRN C-termini execute their presumed effector function in the host nucleus, targeting processes required for immunity. These findings have led to great interest in this large protein family and driven the identification of additional CRN-like proteins in other organisms. The identification of CRN proteins and subsequent functional studies have markedly increased the number of candidate CRN protein sequences, expanded the range of phenotypes tentatively associated with function and revealed some of their molecular functions toward virulence. The increased number of characterized CRNs also has presented a set of challenges that may impede significant progress in the future. Here, we summarize our current understanding of the CRNs and re-assess some basic assumptions regarding this protein family. We will discuss the latest findings on CRN biology and highlight exciting new hypotheses that have emanated from the field. Finally, we will discuss new approaches to study CRN functions that would lead to a better understanding of CRN effector biology as well as the processes that lead to host susceptibility and immunity.
RESUMEN
There are currently 151 plants with draft genomes available but levels of functional annotation for putative protein products are low. Therefore, accurate computational predictions are essential to annotate genomes in the first instance, and to provide focus for the more costly and time consuming functional assays that follow. DNA-binding proteins are an important class of proteins that require annotation, but current computational methods are not applicable for genome wide predictions in plant species. Here, we explore the use of species and lineage specific models for the prediction of DNA-binding proteins in plants. We show that a species specific support vector machine model based on Arabidopsis sequence data is more accurate (accuracy 81%) than a generic model (74%), and based on this we develop a plant specific model for predicting DNA-binding proteins. We apply this model to the tomato proteome and demonstrate its ability to perform accurate high-throughput prediction of DNA-binding proteins. In doing so, we have annotated 36 currently uncharacterised proteins by assigning a putative DNA-binding function. Our model is publically available and we propose it be used in combination with existing tools to help increase annotation levels of DNA-binding proteins encoded in plant genomes.
Asunto(s)
Proteínas de Unión al ADN/química , Genoma de Planta , Anotación de Secuencia Molecular , Proteínas de Plantas/química , Máquina de Vectores de Soporte , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Solanum lycopersicum/genética , Modelos Biológicos , Proteínas de Plantas/genética , Análisis de Secuencia de Proteína , Especificidad de la EspecieRESUMEN
Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant-microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants.
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Núcleo Celular/fisiología , Plantas/inmunología , Empalme Alternativo , Ensamble y Desensamble de Cromatina , Genoma de Planta , Plantas/genética , Plantas/microbiología , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Transcripción GenéticaRESUMEN
Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.
RESUMEN
BACKGROUND: Plant-microbe interactions feature complex signal interplay between pathogens and their hosts. Phytophthora species comprise a destructive group of fungus-like plant pathogens, collectively affecting a wide range of plants important to agriculture and natural ecosystems. Despite the availability of genome sequences of both hosts and microbes, little is known about the signal interplay between them during infection. In particular, accurate descriptions of coordinate relationships between host and microbe transcriptional programs are lacking. RESULTS: Here, we explore the molecular interaction between the hemi-biotrophic broad host range pathogen Phytophthora capsici and tomato. Infection assays and use of a composite microarray allowed us to unveil distinct changes in both P. capsici and tomato transcriptomes, associated with biotrophy and the subsequent switch to necrotrophy. These included two distinct transcriptional changes associated with early infection and the biotrophy to necrotrophy transition that may contribute to infection and completion of the P. capsici lifecycle CONCLUSIONS: Our results suggest dynamic but highly regulated transcriptional programming in both host and pathogen that underpin P. capsici disease and hemi-biotrophy. Dynamic expression changes of both effector-coding genes and host factors involved in immunity, suggests modulation of host immune signaling by both host and pathogen. With new unprecedented detail on transcriptional reprogramming, we can now explore the coordinate relationships that drive host-microbe interactions and the basic processes that underpin pathogen lifestyles. Deliberate alteration of lifestyle-associated transcriptional changes may allow prevention or perhaps disruption of hemi-biotrophic disease cycles and limit damage caused by epidemics.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Phytophthora/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Factores de Transcripción/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Phytophthora/patogenicidad , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Transducción de Señal , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions.
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Familia de Multigenes , Phytophthora/genética , Phytophthora/metabolismo , Secuencia de Aminoácidos , Muerte Celular , Análisis por Conglomerados , Biología Computacional , Perfilación de la Expresión Génica , Genoma , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Oomicetos/genética , Oomicetos/metabolismo , Fenotipo , Phytophthora/patogenicidad , Enfermedades de las Plantas/parasitología , Posición Específica de Matrices de Puntuación , Dominios y Motivos de Interacción de Proteínas , Nicotiana/parasitología , Virulencia/genéticaRESUMEN
Plant-pathogen interactions feature complex signaling exchanges between host and microbes that ultimately determine association outcomes. Plants deploy pattern recognition receptors to perceive pathogen-associated molecular patterns, mount pattern-triggered immunity (PTI), and fend off potential pathogens. In recent years an increasing number of defense-signaling components have been identified along with a mechanistic understanding of their regulation during immune responses. Post-translational modifications (PTMs) are now thought to play a crucial role in regulating defense signaling. In a bid to suppress PTI and infect their host, pathogens have evolved large repertoires of effectors that trigger susceptibility and allow colonization of host tissues. While great progress has been made in elucidating defense-signaling networks in plants and the activities of effectors in immune suppression, a critical gap exists in our understanding of effector mechanism-of-action. Given the importance of PTMs in the regulation of defense signaling, we will explore the question: how do effectors modify the post-translational status of host proteins and thus interfere with host processes required for immunity? We will consider how emerging proteomics-based experimental strategies may help us answer this important question and ultimately open the pathogens' effector black box.
RESUMEN
The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.
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Phytophthora/fisiología , Enfermedades de las Plantas/microbiología , Adaptación Fisiológica , Capsicum/microbiología , Mapeo Cromosómico , Cucurbita/microbiología , Regulación de la Expresión Génica , Ligamiento Genético , Genoma , Genotipo , Polimorfismo de Nucleótido SimpleRESUMEN
UNLABELLED: Phytophthora capsici is a highly dynamic and destructive pathogen of vegetables. It attacks all cucurbits, pepper, tomato and eggplant, and, more recently, snap and lima beans. The disease incidence and severity have increased significantly in recent decades and the molecular resources to study this pathogen are growing and now include a reference genome. At the population level, the epidemiology varies according to the geographical location, with populations in South America dominated by clonal reproduction, and populations in the USA and South Africa composed of many unique genotypes in which sexual reproduction is common. Just as the impact of crop loss as a result of P. capsici has increased in recent decades, there has been a similar increase in the development of new tools and resources to study this devastating pathogen. Phytophthora capsici presents an attractive model for understanding broad-host-range oomycetes, the impact of sexual recombination in field populations and the basic mechanisms of Phytophthora virulence. TAXONOMY: Kingdom Chromista; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Phytophthora; Species capsici. DISEASE SYMPTOMS: Symptoms vary considerably according to the host, plant part infected and environmental conditions. For example, in dry areas (e.g. southwestern USA and southern France), infection on tomato and bell or chilli pepper is generally on the roots and crown, and the infected plants have a distinctive black/brown lesion visible at the soil line (Fig. 1). In areas in which rainfall is more common (e.g. eastern USA), all parts of the plant are infected, including the roots, crown, foliage and fruit (Fig. 1). Root infections cause damping off in seedlings, whereas, in older plants, it is common to see stunted growth, wilting and, eventually, death. For tomatoes, it is common to see significant adventitious root growth just above an infected tap root, and the stunted plants, although severely compromised, may not die. For many cucurbit fruit, the expanding lesions produce fresh sporangia over days (or even weeks depending on the size of the fruit) and the fruit often look as if they have been dipped in white powdered confectioner's sugar (Fig. 1). Generally, hyphae do not emerge from infected plants or fruit (common with Pythium infections) and all that is visible on the surface of an infected plant is sporangia. IMPORTANCE: Phytophthora capsici presents an oomycete worst-case scenario to growers as it has a broad host range, often produces long-lived dormant sexual spores, has extensive genotypic diversity and has an explosive asexual disease cycle. It is becoming increasingly apparent that novel control strategies are needed to safeguard food production from P. capsici and other oomycetes. Considering that P. capsici is easy to grow, mate and manipulate in the laboratory and infects many plant species, this pathogen is a robust model for investigations, particularly those related to sexual reproduction, host range and virulence. USEFUL WEBSITES: Phytophthora capsici genome database: http://genome.jgi-psf.org/Phyca11/Phyca11.home.html. Molecular tools to identify Phytophthora isolates: http://phytophthora-id.org.
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Especificidad del Huésped/fisiología , Phytophthora/fisiología , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Genoma/genética , Especificidad del Huésped/genética , Phytophthora/clasificación , Phytophthora/genética , Phytophthora/patogenicidad , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/estadística & datos numéricos , Reproducción Asexuada/fisiologíaRESUMEN
In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.
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Péptido Hidrolasas/genética , Phytophthora infestans/metabolismo , Agrobacterium/metabolismo , Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Sistema Inmunológico , Microscopía Confocal , Modelos Biológicos , Células Vegetales/metabolismo , Enfermedades de las Plantas/inmunología , Fenómenos Fisiológicos de las Plantas , Plásmidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Nicotiana/genética , VirulenciaRESUMEN
Genome sequencing combined with high-throughput functional analyses has proved vital in our quest to understand oomycete-plant interactions. With the identification of effector molecules from Phytophthora spp. we can now embark on dissecting the mechanisms by which effectors modulate host processes and thus ensure parasite fitness. One of the key limitations, however, is to genetically modify Phytophthora and assess gene function during parasitism. Here, we describe a straightforward protocol that allows rapid transformation of Phytophthora capsici, an emerging model in oomycete biology. P. capsici is a broad host range pathogen that can infect a wide variety of plants under lab conditions making it a suitable model for detailed studies on oomycete-host interactions. This protocol relies on electroporation-assisted uptake of DNA in to motile zoospores and allows the rapid identification and characterization of genetically stable transformants.
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Electroporación/métodos , Phytophthora/citología , Phytophthora/genética , Esporas/genética , Transfección/métodos , Interacciones Huésped-Patógeno/genética , Phytophthora/patogenicidad , Plantas/microbiologíaRESUMEN
Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined.
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Proteínas Algáceas/metabolismo , Núcleo Celular/metabolismo , Oomicetos/metabolismo , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Oomicetos/genética , Oomicetos/patogenicidad , Organismos Modificados Genéticamente , Enfermedades de las Plantas/parasitología , Hojas de la Planta/parasitología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. RESULTS: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. CONCLUSIONS: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.