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1.
Plant Cell ; 35(4): 1186-1201, 2023 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-36625683

RESUMO

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.


Assuntos
Phytophthora , Solanum , Proteínas/metabolismo , Plantas/metabolismo , Phytophthora/genética , Phytophthora/metabolismo , Nicotiana/metabolismo , Solanum/metabolismo , Doenças das Plantas
2.
Semin Cell Dev Biol ; 148-149: 13-21, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36792439

RESUMO

Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.


Assuntos
Phytophthora , Plantas
3.
Artigo em Inglês | MEDLINE | ID: mdl-38648121

RESUMO

The selective pressure of pathogen-host symbiosis drives adaptations. How these interactions shape the metabolism of pathogens is largely unknown. Here, we use comparative genomics to systematically analyse the metabolic networks of oomycetes, a diverse group of eukaryotes that includes saprotrophs as well as pathogens of animal- and plant pathogens, the latter causing devastating diseases with significant economic and/or ecological impact. In our analyses of 44 oomycete species, we uncover considerable variation in metabolism that can be linked to lifestyle differences. Comparisons of metabolic gene content reveal that plant pathogenic oomycetes have a bipartite metabolism consisting of a conserved core and an accessory set. The accessory set can be associated with the degradation of defence compounds produced by plants when challenged by pathogens. Obligate biotrophic oomycetes have smaller metabolic networks, and taxonomically distantly related biotrophic lineages display convergent evolution by repeated gene losses in both the conserved as well as the accessory set of metabolism. When investigating to what extent the metabolic networks in obligate biotrophs differ from those in hemibiotrophic plant pathogens, we observe that the losses of metabolic enzymes in obligate biotrophs are not random and that gene losses predominantly influence the terminal branches of the metabolic networks. Our analyses represent the first metabolism-focused comparison of oomycetes at this scale and will contribute to a better understanding of the evolution of oomycete metabolism in relation to lifestyle adaptation.

4.
New Phytol ; 238(2): 781-797, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36653957

RESUMO

Ubiquitin-like domain-containing proteins (UDPs) are involved in the ubiquitin-proteasome system because of their ability to interact with the 26S proteasome. Here, we identified potato StUDP as a target of the Phytophthora infestans RXLR effector Pi06432 (PITG_06432), which supresses the salicylic acid (SA)-related immune pathway. By overexpressing and silencing of StUDP in potato, we show that StUDP negatively regulates plant immunity against P. infestans. StUDP interacts with, and destabilizes, the 26S proteasome subunit that is referred to as REGULATORY PARTICLE TRIPLE-A ATP-ASE (RPT) subunit StRPT3b. This destabilization represses the proteasome activity. Proteomic analysis and Western blotting show that StUDP decreases the stability of the master transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) in SA biosynthesis. StUDP negatively regulates the SA signalling pathway by repressing the proteasome activity and destabilizing StSARD1, leading to a decreased expression of the SARD1-targeted gene ISOCHORISMATE SYNTHASE 1 and thereby a decrease in SA content. Pi06432 stabilizes StUDP, and it depends on StUDP to destabilize StRPT3b and thereby supress the proteasome activity. Our study reveals that the P. infestans effector Pi06432 targets StUDP to hamper the homeostasis of the proteasome by the degradation of the proteasome subunit StRPT3b and thereby suppresses SA-related immunity.


Assuntos
Phytophthora infestans , Solanum tuberosum , Phytophthora infestans/metabolismo , Ubiquitinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica , Imunidade Vegetal , Doenças das Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
5.
PLoS Pathog ; 16(1): e1008138, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961913

RESUMO

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Phytophthora/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Esporos/crescimento & desenvolvimento , Núcleo Celular/genética , Núcleo Celular/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Phytophthora/genética , Phytophthora/crescimento & desenvolvimento , Phytophthora/patogenicidade , Doenças das Plantas/parasitologia , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Glycine max/parasitologia , Esporos/enzimologia , Esporos/genética , Esporos/metabolismo , Virulência
6.
Mol Plant Microbe Interact ; 33(5): 742-753, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32237964

RESUMO

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.


Assuntos
Genoma Mitocondrial , Peronospora/genética , Genômica , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas
7.
J Exp Bot ; 71(3): 837-849, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31665494

RESUMO

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.


Assuntos
Bryopsida/metabolismo , Exocitose , Interações Hospedeiro-Parasita , Microscopia Confocal/métodos , Phytophthora/fisiologia , Bryopsida/microbiologia
8.
Mol Cell Proteomics ; 16(11): 1958-1971, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28935716

RESUMO

The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1-3 Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.


Assuntos
Phytophthora infestans/patogenicidade , Proteômica/métodos , Solanum tuberosum/parasitologia , Fatores de Virulência/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Espectrometria de Massas , Phytophthora infestans/crescimento & desenvolvimento , Phytophthora infestans/metabolismo , Doenças das Plantas/parasitologia , Fatores de Virulência/genética
9.
Fungal Genet Biol ; 116: 73-82, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29704555

RESUMO

The oomycete Phytophthora infestans is a notorious plant pathogen with potato and tomato as its primary hosts. Previous research showed that the heterotrimeric G-protein subunits Gα and Gß have a role in zoospore motility and virulence, and sporangial development, respectively. Here, we present analyses of the gene encoding a Gγ subunit in P. infestans, Pigpg1. The overall similarity of PiGPG1 with non-oomycete Gγ subunits is low, with only the most conserved amino acids maintained, but similarity with its homologs in other oomycetes is high. Pigpg1 is expressed in all life stages and shows a similar expression profile as the gene encoding the Gß subunit, Pigpb1. To elucidate its function, transformants were generated in which Pigpg1 is silenced or overexpressed and their phenotypes were analyzed. Pigpg1-silenced lines produce less sporangia, which are malformed. Altogether, the results show that PiGPG1 is crucial for proper sporangia development and zoosporogenesis. PiGPG1 is a functional Gγ, and likely forms a dimer with PiGPB1 that mediates signaling.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Phytophthora infestans/crescimento & desenvolvimento , Esporângios/crescimento & desenvolvimento , Sequência Conservada , Subunidades gama da Proteína de Ligação ao GTP/genética , Micélio/metabolismo , Phytophthora infestans/genética , Interferência de RNA , Esporângios/genética , Esporos/metabolismo
11.
J Exp Bot ; 69(3): 655-666, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29329405

RESUMO

The exocyst, a multiprotein complex consisting of eight subunits, plays an essential role in many biological processes by mediating secretion of post-Golgi-derived vesicles towards the plasma membrane. In recent years, roles for plant exocyst subunits in pathogen defence have been uncovered, largely based on studies in the model plant Arabidopsis. Only a few studies have been undertaken to assign the role of exocyst subunits in plant defence in other plants species, including crops. In this study, predicted protein sequences from exocyst subunits were retrieved by mining databases from the Solanaceous plants Nicotiana benthamiana, tomato, and potato. Subsequently, their evolutionary relationship with Arabidopsis exocyst subunits was analysed. Gene silencing in N. benthamiana showed that several exocyst subunits are required for proper plant defence against the (hemi-)biotrophic plant pathogens Phytophthora infestans and Pseudomonas syringae. In contrast, some exocyst subunits seem to act as susceptibility factors for the necrotrophic pathogen Botrytis cinerea. Furthermore, the majority of the exocyst subunits were found to be involved in callose deposition, suggesting that they play a role in basal plant defence. This study provides insight into the evolution of exocyst subunits in Solanaceous plants and is the first to show their role in immunity against multiple unrelated pathogens.


Assuntos
Botrytis/fisiologia , Nicotiana/genética , Phytophthora infestans/fisiologia , Imunidade Vegetal/genética , Pseudomonas syringae/fisiologia , Solanum lycopersicum/genética , Solanum tuberosum/genética , Inativação Gênica , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Solanum tuberosum/imunologia , Solanum tuberosum/microbiologia , Nicotiana/imunologia , Nicotiana/microbiologia
12.
Cell Mol Life Sci ; 74(5): 909-920, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27714409

RESUMO

The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration.


Assuntos
Actinas/metabolismo , Parede Celular/metabolismo , Phytophthora infestans/citologia , Phytophthora infestans/metabolismo , Células Vegetais/metabolismo , Celulose/metabolismo , Meios de Cultura , Hifas/citologia , Hifas/metabolismo , Solanum lycopersicum/citologia , Solanum lycopersicum/microbiologia , Transporte Proteico
13.
Plant Physiol ; 169(3): 1975-90, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26336092

RESUMO

Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense.


Assuntos
Interações Hospedeiro-Patógeno , Nicotiana/parasitologia , Phytophthora infestans/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Fatores de Virulência/metabolismo , Morte Celular , Glucanos/metabolismo , Phytophthora infestans/metabolismo , Phytophthora infestans/patogenicidade , Doenças das Plantas/parasitologia , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Proteínas de Plantas/genética , Transporte Proteico , Nicotiana/imunologia , Técnicas do Sistema de Duplo-Híbrido , Fatores de Virulência/genética
14.
J Microsc ; 263(2): 171-80, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27027911

RESUMO

Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.


Assuntos
Bryopsida/citologia , Bryopsida/parasitologia , Espaço Intracelular , Microscopia/métodos , Phytophthora/patogenicidade , Doenças das Plantas/parasitologia , Actinas/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Parede Celular/metabolismo , Citoplasma/metabolismo , Phytophthora/fisiologia
15.
Plant Cell Rep ; 35(4): 845-55, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26795144

RESUMO

KEY MESSAGE: Transgenic Nicotiana benthamiana lines with constitutive expression of an Arabidopsis lectin receptor kinase gene (LecRK - I.9 or LecRK - IX.1) show enhanced resistance to Phytophthora pathogens, demonstrating conserved gene functionality after interfamily transfer. In plants, cell surface receptors mediate the first layer of innate immunity against pathogenic microbes. In Arabidopsis several L-type lectin receptor kinases (LecRKs) were previously found to function as Phytophthora resistance components. In this study, we determined the functionality of Arabidopsis LecRK-I.9 or LecRK-IX.1 in Phytophthora resistance when transferred into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines were generated for each LecRK gene and molecular analyses revealed variation in transgene copy number, transgene expression levels and LecRK protein accumulation. Infection assays showed that transgenic N. benthamiana plants expressing either Arabidopsis LecRK-I.9 or LecRK-IX.1 are more resistant to Phytophthora capsici and to Phytophthora infestans. These results demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora resistance function when transferred into N. benthamiana. Therefore, these LecRKs have the potential to function as a complementary Phytophthora resistance resource in distantly related plant species next to the canonical Phytophthora resistance genes encoding nucleotide-binding leucine-rich repeat proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Resistência à Doença , Genes de Plantas , Nicotiana/genética , Nicotiana/microbiologia , Phytophthora infestans/fisiologia , Doenças das Plantas/microbiologia , Proteínas Quinases/metabolismo , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Nicotiana/anatomia & histologia , Nicotiana/crescimento & desenvolvimento , Transgenes
16.
Mol Cell Proteomics ; 13(8): 2101-13, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24872595

RESUMO

Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.


Assuntos
Phytophthora infestans/metabolismo , Proteômica/métodos , Proteínas de Protozoários/isolamento & purificação , Parede Celular/metabolismo , Meios de Cultura/química , Bases de Dados de Proteínas , Espectrometria de Massas , Fosforilação , Phytophthora infestans/química , Proteínas de Protozoários/metabolismo
17.
PLoS Genet ; 9(6): e1003272, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23785293

RESUMO

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.


Assuntos
Transferência Genética Horizontal , Interações Hospedeiro-Parasita/genética , Oomicetos/genética , Saprolegnia/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Peixes/genética , Peixes/parasitologia , Genoma , Oomicetos/classificação , Oomicetos/patogenicidade , Filogenia , Plantas/parasitologia , Saprolegnia/classificação , Saprolegnia/patogenicidade
18.
Mol Plant Microbe Interact ; 28(12): 1271-80, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26313411

RESUMO

In biotrophic plant-microbe interactions, microbes infect living plant cells, in which they are hosted in a novel membrane compartment, the host-microbe interface. To create a host-microbe interface, arbuscular mycorrhizal (AM) fungi and rhizobia make use of the same endosymbiotic program. It is a long-standing hypothesis that pathogens make use of plant proteins that are dedicated to mutualistic symbiosis to infect plants and form haustoria. In this report, we developed a Phytophthora palmivora pathosystem to study haustorium formation in Medicago truncatula roots. We show that P. palmivora does not require host genes that are essential for symbiotic infection and host-microbe interface formation to infect Medicago roots and form haustoria. Based on these findings, we conclude that P. palmivora does not hijack the ancient intracellular accommodation program used by symbiotic microbes to form a biotrophic host-microbe interface.


Assuntos
Medicago truncatula/microbiologia , Micorrizas/fisiologia , Phytophthora/patogenicidade , Raízes de Plantas/microbiologia , Rhizobium/fisiologia , Simbiose , Genes de Plantas , Interações Hospedeiro-Patógeno , Medicago truncatula/genética
19.
Mol Plant Microbe Interact ; 28(9): 1032-48, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26011556

RESUMO

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Morte Celular/fisiologia , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética
20.
BMC Genomics ; 16: 741, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26438312

RESUMO

BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.


Assuntos
Genoma Fúngico , Helianthus/microbiologia , Oomicetos/genética , Evolução Biológica , Proteínas Fúngicas , Perfilação da Expressão Gênica , Genômica/métodos , Heterozigoto , Repetições de Microssatélites , Oomicetos/classificação , Oomicetos/metabolismo , Fosfolipídeos/metabolismo , Filogenia , Phytophthora/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Metabolismo Secundário , Transdução de Sinais , Fatores de Virulência/genética
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