RESUMO
Fusarium wilt of bananas (FWB) is a severe plant disease that leads to substantial losses in banana production worldwide. It remains a major concern for Cuban banana cultivation. The disease is caused by members of the soil-borne Fusarium oxysporum species complex. However, the genetic diversity among Fusarium species infecting bananas in Cuba has remained largely unexplored. In our comprehensive survey, we examined symptomatic banana plants across all production zones in the country, collecting 170 Fusarium isolates. Leveraging genotyping-by-sequencing and whole-genome comparisons, we investigated the genetic diversity within these isolates and compared it with a global Fusarium panel. Notably, typical FWB symptoms were observed in Bluggoe cooking bananas and Pisang Awak subgroups across 14 provinces. Our phylogenetic analysis revealed that F. purpurascens, F. phialophorum, and F. tardichlamydosporum are responsible for FWB in Cuba, with F. tardichlamydosporum dominating the population. Furthermore, we identified between five and seven distinct genetic clusters, with F. tardichlamydosporum isolates forming at least two subgroups. This finding underscores the high genetic diversity of Fusarium spp. contributing to FWB in the Americas. Our study sheds light on the population genetic structure and diversity of the FWB pathogen in Cuba and the broader Latin American and Caribbean regions.
Assuntos
Fusarium , Variação Genética , Musa , Filogenia , Doenças das Plantas , Fusarium/genética , Fusarium/classificação , Fusarium/patogenicidade , Fusarium/isolamento & purificação , Musa/microbiologia , Cuba , Doenças das Plantas/microbiologia , Região do Caribe , América LatinaRESUMO
Many pathogens evolved compartmentalized genomes with conserved core and variable accessory regions (ARs) that carry effector genes mediating virulence. The fungal plant pathogen Fusarium oxysporum has such ARs, often spanning entire chromosomes. The presence of specific ARs influences the host range, and horizontal transfer of ARs can modify the pathogenicity of the receiving strain. However, how these ARs evolve in strains that infect the same host remains largely unknown. We defined the pan-genome of 69 diverse F. oxysporum strains that cause Fusarium wilt of banana, a significant constraint to global banana production, and analyzed the diversity and evolution of the ARs. Accessory regions in F. oxysporum strains infecting the same banana cultivar are highly diverse, and we could not identify any shared genomic regions and in planta-induced effectors. We demonstrate that segmental duplications drive the evolution of ARs. Furthermore, we show that recent segmental duplications specifically in accessory chromosomes cause the expansion of ARs in F. oxysporum. Taken together, we conclude that extensive recent duplications drive the evolution of ARs in F. oxysporum, which contribute to the evolution of virulence.
Assuntos
Fusarium , Genoma Fúngico , Duplicações Segmentares Genômicas , Fusarium/genética , Especificidade de Hospedeiro , Genômica , Doenças das Plantas/genética , Doenças das Plantas/microbiologiaRESUMO
Bananas are major agricultural commodities in Cuba. One of the main constraints of banana production worldwide is Fusarium wilt of banana. Recent outbreaks in Colombia, Perú, and Venezuela have raised widespread concern in Latin America due to the potential devastating impact on the sustainability of banana production, food security, and livelihoods of millions of people in the region. Here, we phenotyped 18 important Cuban banana and plantain varieties with two Fusarium strains-Tropical Race 4 (TR4) and Race 1-under greenhouse conditions. These varieties represent 72.8% of the national banana acreage in Cuba and are also widely distributed in Latin America and the Caribbean region. A broad range of disease responses from resistant to very susceptible was observed against Race 1. On the contrary, not a single banana variety was resistant to TR4. These results underscore that TR4 potentially threatens nearly 56% of the contemporary Cuban banana production area, which is planted with susceptible and very susceptible varieties, and call for a preemptive evaluation of new varieties obtained in the national breeding program and the strengthening of quarantine measures to prevent the introduction of TR4 into the country.
Assuntos
Fusarium , Musa , Humanos , Fusarium/fisiologia , Doenças das Plantas/prevenção & controle , Melhoramento Vegetal , FenótipoRESUMO
Fusarium wilt of banana (FWB) is a serious soil-borne fungal disease. In the previous century, FWB already destroyed Gros Michel-based banana cultures in Central America, and currently, the disease threatens all major banana-producing regions of the world. The causal agents of these epidemics, however, are diverse. Gros Michel was infected by a wide range of Fusarium species, the so-called Race 1 strains, whereas the contemporary Cavendish-based cultures are affected by Fusarium odoratissimum, colloquially called Tropical Race 4 (TR4). TR4 was reported in Mozambique on two commercial banana farms in 2013, but no incursions were found outside the farm boundaries in 2015, suggesting that the disease was under control. Here we report the presence of TR4 outside of these farm boundaries. We obtained fungal samples from 13 banana plants in smallholder and roadside plantings at various locations throughout northern Mozambique. These samples tested positive for TR4 by molecular diagnostics and in greenhouse pathogenicity assays. The results were confirmed with reisolations, thereby completing Koch's postulates. To study the diversity of TR4 isolates in Mozambique, we selected five samples for whole-genome sequencing. Comparison with a global collection of TR4 samples revealed very little genetic variation, indicating that the fungus is clonally spreading in Mozambique. Furthermore, isolates from Mozambique are clearly genetically separated from other geographic incursions, and thus we cannot trace the origin of TR4 in Mozambique. Nevertheless, our data demonstrates the dissemination of TR4 in Mozambique, underscoring the failure of disease management strategies. This threatens African banana production.
Assuntos
Fusarium , Musa , Musa/microbiologia , Moçambique , Doenças das Plantas/microbiologiaRESUMO
Fusarium wilt of banana (FWB), caused by a suite of Fusarium fungi, is among the most devastating plant diseases. The iconic FWB epidemic in the previous century lasted decades and was caused by so-called Race 1 strains that wiped out the dominant 'Gros Michel' banana plantations across Central America. Eventually, it was stopped because the Race 1-resistant 'Cavendish' banana variety replaced 'Gros Michel', which dominates global production (>50%) and trade (>95%). However, presently, the so-called Tropical Race 4 (TR4) threatens plantations of 'Cavendish' and many other banana varieties around the globe. Prevention is the first line of defense against the spread of TR4. Therefore, many disinfection units are installed to prevent the entry of TR4 in banana plantations. These foot and tire baths are filled with disinfectants, but limited knowledge is available on their efficacy. In this project, we evaluated 13 disinfectants commonly used in the Philippines. Our results show that the efficacy of these products depends on the type of fungal spores, the exposure time, and the replenishment frequency of the disinfection units. The resting spores of TR4 were resistant to all but one - unfortunately corrosive - disinfectant. Furthermore, we show that the actual contact time with disinfectants was far below the thresholds determined in laboratory experiments. Finally, muddy disinfection units reduced the efficacy of disinfectants. Taken together, we conclude that practices are inadequate to prevent the dissemination of TR4.
Assuntos
Desinfetantes , Fusarium , Musa , Desinfetantes/farmacologia , Musa/microbiologia , Filipinas , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controleRESUMO
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/genéticaRESUMO
BACKGROUND: The course of illness in obsessive-compulsive disorder (OCD) varies significantly between patients. Little is known about factors predicting a chronic course of illness. The aim of this study is to identify factors involved in inducing and in maintaining chronicity in OCD. METHODS: The present study is embedded within the Netherlands Obsessive Compulsive Disorder Association (NOCDA) study, an ongoing multicenter naturalistic cohort study designed to identify predictors of long-term course and outcome in OCD. For this study, 270 subjects with a current diagnosis of OCD were included. Chronicity status at 2-year follow-up was regressed on a selection of baseline predictors related to OCD, to comorbidity and to stress and support. RESULTS: Psychotrauma [odds ratio (OR) 1.98, confidence interval (CI) 1.22-3.22, p = 0.006], recent negative life events (OR 1.42, CI 1.01-2.01, p = 0.043), and presence of a partner (OR 0.28, CI 0.09-0.85, p = 0.025) influenced the risk of becoming chronic. Longer illness duration (OR 1.46, CI 1.08-1.96, p = 0.013) and higher illness severity (OR 1.09, CI 1.03-1.16, p = 0.003) increased the risk of remaining chronic. CONCLUSIONS: External influences increase the risk of becoming chronic, whereas the factors involved in maintaining chronicity are illness-related. As the latter are potentially difficult to modify, treatment should be devoted to prevent chronicity from occurring in the first place. Therapeutic strategies aimed at alleviating stress and at boosting social support might aid in achieving this goal.
Assuntos
Progressão da Doença , Transtorno Obsessivo-Compulsivo/diagnóstico , Adulto , Doença Crônica , Comorbidade , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Transtorno Obsessivo-Compulsivo/epidemiologia , Fatores de Risco , Índice de Gravidade de Doença , Fatores de TempoRESUMO
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 ProteicoRESUMO
OBJECTIVE: Obsessive-compulsive symptoms (OCS) co-occur frequently with anxiety and depressive disorders, but the nature of their relationship and their impact on severity of anxiety and depressive disorders is poorly understood. In a large sample of patients with anxiety and depressive disorders, we assessed the frequency of OCS, defined as a Young Adult Self-Report Scale-obsessive-compulsive symptoms score >7. The associations between OCS and severity of anxiety and/or depressive disorders were examined, and it was investigated whether OCS predict onset, relapse, and persistence of anxiety and depressive disorders. METHODS: Data were obtained from the third (at 2-year follow-up) and fourth wave (at 4-year follow-up) of data collection in the Netherlands Study of Anxiety and Depression cohort, including 469 healthy controls, 909 participants with a remitted disorder, and 747 participants with a current anxiety and/or depressive disorder. RESULTS: OCS were present in 23.6% of the total sample, most notably in those with current combined anxiety and depressive disorders. In patients with a current disorder, OCS were associated with severity of this disorder. Moreover, OCS predicted (1) first onset of anxiety and/or depressive disorders in healthy controls (odds ratio [OR], 5.79; 95% confidence interval [CI], 1.15 to 29.14), (2) relapse in those with remitted anxiety and/or depressive disorders (OR, 2.31; 95% CI, 1.55 to 3.46), and (3) persistence in patients with the combination of current anxiety and depressive disorders (OR, 4.42; 95% CI, 2.54 to 7.70) within the 2-year follow-up period Conclusions: OCS are closely related to both the presence and severity of anxiety and depressive disorders and affect their course trajectories. Hence, OCS might be regarded as a course specifier signaling unfavorable outcomes. This specifier may be useful in clinical care to adapt and intensify treatment in individual patients.
Assuntos
Transtornos de Ansiedade/epidemiologia , Transtorno Depressivo/epidemiologia , Transtorno Obsessivo-Compulsivo/epidemiologia , Adulto , Transtornos de Ansiedade/diagnóstico , Transtornos de Ansiedade/fisiopatologia , Comorbidade , Transtorno Depressivo/diagnóstico , Transtorno Depressivo/fisiopatologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Transtorno Obsessivo-Compulsivo/diagnóstico , Transtorno Obsessivo-Compulsivo/fisiopatologia , Prevalência , Prognóstico , Índice de Gravidade de DoençaRESUMO
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/metabolismoRESUMO
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/patogenicidadeRESUMO
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/genéticaRESUMO
In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P. sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glycine max/metabolismo , Estresse Oxidativo , Phytophthora/metabolismo , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Phytophthora/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Glycine max/microbiologia , Fatores de Transcrição/genéticaRESUMO
The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required â¼30 s while disassembly was accomplished in â¼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.
Assuntos
Actinas/metabolismo , Phytophthora infestans/química , Phytophthora infestans/fisiologia , Multimerização Proteica , Hifas/química , Hifas/fisiologia , Microscopia de Fluorescência , Coloração e RotulagemRESUMO
Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.
Assuntos
Actinas/metabolismo , Morfolinas/farmacologia , Phytophthora infestans/efeitos dos fármacos , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Actinas/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Fluorescência Verde , Hifas , Phytophthora infestans/citologia , Phytophthora infestans/genética , Phytophthora infestans/crescimento & desenvolvimento , Tiazolidinas/farmacologiaRESUMO
The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).
Assuntos
Estágios do Ciclo de Vida/fisiologia , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Phytophthora infestans/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfoproteínas/análise , Fosfoproteínas/química , Fosforilação , Phytophthora infestans/química , Phytophthora infestans/fisiologia , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/química , Proteômica , Técnicas de Cultura de TecidosRESUMO
For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases/metabolismo , Phytophthora infestans/enzimologia , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Receptores Acoplados a Proteínas G/química , Esporângios/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Hifas/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosfotransferases/genética , Phytophthora infestans/crescimento & desenvolvimento , Phytophthora infestans/metabolismo , Folhas de Planta/microbiologia , Receptores Acoplados a Proteínas G/metabolismo , Solanum tuberosum/microbiologia , Esporos/crescimento & desenvolvimento , Nicotiana/microbiologia , Proteína Vermelha FluorescenteRESUMO
PURPOSE: Genetic variation in the cytochrome P450 2D6 (CYP2D6) enzyme is responsible for interindividual differences in the metabolism of many antipsychotic drugs, but the clinical relevance of polymorphisms in CYP2D6 for response to antipsychotic treatment is relatively unknown. In the Netherlands, clozapine is prescribed only when patients are non-responsive to or intolerant of at least two different antipsychotics. The aim of our study was to determine the association of the CYP2D6 genotype with switching to clozapine, which served as a surrogate outcome marker for treatment response to antipsychotics. METHODS: CYP2D6 genotype was assessed in patients who had been switched to clozapine and compared with antipsychotic users whose treatment regimen included no more than two different antipsychotic drugs and no clozapine. We also performed the analysis in patients who only used CYP2D6-dependent antipsychotics. RESULTS: A total of 528 patients were included in the study (222 cases, 306 controls). No statistically significant differences were found in the distribution of the polymorphisms among the case and control groups, both in all patients and in only those patients using CYP2D6-dependent antipsychotics. However, a trend was observed, suggesting an inverse association between CYP2D6 genotype and the switch to clozapine. (9.5 vs. 5.1 % poor metabolisers and 1.3 vs. 2.6 % ultrarapid metabolisers in cases vs. controls, respectively). CONCLUSIONS: Although the results of our study suggest that the CYP2D6 phenotype is not a major determining factor for patients to be switched to clozapine treatment, larger studies are warranted with a focus on the clinical consequences of the CYP2D6 ultrarapid metaboliser and poor metaboliser phenotypes.