RESUMO
The necrotrophic fungus Pyrenophora tritici-repentis is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by P. tritici-repentis. ToxB induces chlorosis in toxin-sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 h in planta to induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin-sensitive and -insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be reisolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently labeled ToxB indicate that the toxin remains in the apoplast in toxin-sensitive and -insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.
Assuntos
Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Triticum/metabolismo , Espaço Extracelular/metabolismo , Micotoxinas/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Pressão , Transporte Proteico , Triticum/citologia , Triticum/microbiologiaRESUMO
Pyrenophora tritici-repentis, the causal agent of tan spot disease of wheat, mediates disease by the production of host-selective toxins (HST). The known toxins are recognized in an 'inverse' gene-for-gene manner, where each is perceived by the product of a unique locus in the host and recognition leads to disease susceptibility. Given the importance of HSTs in disease development, we would predict that the loss of any of these major pathogenicity factors would result in reduced virulence and disease development. However, after either deletion of the gene encoding the HST ToxA or, reciprocally, heterologous expression of ToxA in a race that does not normally produce the toxin followed by inoculation of ToxA-sensitive and insensitive wheat cultivars, we demonstrate that ToxA symptom development can be epistatic to other HST-induced symptoms. ToxA epistasis on certain ToxA-sensitive wheat cultivars leads to genotype-specific increases in total leaf area affected by disease. These data indicate a complex interplay between host responses to HSTs in some genotypes and underscore the challenge of identifying additional HSTs whose activity may be masked by other toxins. Also, through mycelial staining, we acquire preliminary evidence that ToxA may provide additional benefits to fungal growth in planta in the absence of its cognate recognition partner in the host.
Assuntos
Ascomicetos/fisiologia , Epistasia Genética , Proteínas Fúngicas/genética , Micotoxinas/genética , Doenças das Plantas/microbiologia , Triticum/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Proteínas Fúngicas/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno , Micotoxinas/metabolismo , Folhas de Planta/microbiologia , Triticum/genéticaRESUMO
Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a ß-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent ß-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.
Assuntos
Proteínas Fúngicas/química , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/microbiologia , Triticum/microbiologia , Cristalografia por Raios X , Evolução Molecular , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/toxicidade , Espectroscopia de Ressonância Magnética , Dobramento de Proteína , Estrutura Secundária de Proteína , Soluções/química , Triticum/genéticaRESUMO
Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was identified, cloned and functionally characterized as an alkane-oxidizing cytochrome P450 (GSPALK1). Analysis of CYP52L1 suggests that it is a member of the CYP52 cytochrome P450 family, which is comprised of medium- and long-chain alkane-oxidizing enzymes found in yeasts. However, phylogenetic analysis of GSPALK1 with other CYP52 members suggests they are not closely related. Post-transcriptional ds-RNA-mediated gene silencing of CYP52L1 severely reduced the ability of this fungus to oxidize alkanes and ethers, however, downstream metabolic steps in these pathways were unaffected. Collectively, the results of this study suggest that GSPALK1 is the enzyme that catalyzes the initial oxidation of alkanes and ethers but is not involved in the later steps of alkane or ether metabolism.
Assuntos
Alcanos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Poluentes Ambientais/metabolismo , Éteres/metabolismo , Saccharomycetales/enzimologia , Biodegradação Ambiental , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/metabolismo , Gases , Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Oxirredução , Filogenia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Saccharomycetales/genética , Análise de Sequência de DNARESUMO
Pyrenophora tritici-repentis is a necrotrophic fungus causal to the disease tan spot of wheat, whose contribution to crop loss has increased significantly during the last few decades. Pathogenicity by this fungus is attributed to the production of host-selective toxins (HST), which are recognized by their host in a genotype-specific manner. To better understand the mechanisms that have led to the increase in disease incidence related to this pathogen, we sequenced the genomes of three P. tritici-repentis isolates. A pathogenic isolate that produces two known HSTs was used to assemble a reference nuclear genome of approximately 40 Mb composed of 11 chromosomes that encode 12,141 predicted genes. Comparison of the reference genome with those of a pathogenic isolate that produces a third HST, and a nonpathogenic isolate, showed the nonpathogen genome to be more diverged than those of the two pathogens. Examination of gene-coding regions has provided candidate pathogen-specific proteins and revealed gene families that may play a role in a necrotrophic lifestyle. Analysis of transposable elements suggests that their presence in the genome of pathogenic isolates contributes to the creation of novel genes, effector diversification, possible horizontal gene transfer events, identified copy number variation, and the first example of transduplication by DNA transposable elements in fungi. Overall, comparative analysis of these genomes provides evidence that pathogenicity in this species arose through an influx of transposable elements, which created a genetically flexible landscape that can easily respond to environmental changes.
Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Evolução Molecular , Variação Genética , Genoma Fúngico/genética , Micotoxinas/genética , Triticum/microbiologia , Sequência de Bases , Mapeamento Cromossômico , Análise Citogenética , Primers do DNA/genética , Elementos de DNA Transponíveis/genética , Duplicação Gênica/genética , Genômica , Funções Verossimilhança , Modelos Genéticos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.
Assuntos
Ascomicetos/genética , Peptídeo Sintases/genética , Doenças das Plantas , Policetídeo Sintases/genética , Polimorfismo de Nucleotídeo Único/genética , Ascomicetos/patogenicidade , Sequência de Bases , Evolução Molecular , Variação Genética , Genoma Fúngico , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Virulência/genéticaRESUMO
Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot of wheat, produces one or a combination of host-selective toxins (HSTs) necessary for disease development. The two most studied toxins produced by Ptr, Ptr ToxA (ToxA) and Ptr ToxB (ToxB), are proteins that cause necrotic or chlorotic symptoms respectively. Investigation of host responses induced by HSTs provides better insight into the nature of the host susceptibility. Microarray analysis of ToxA has provided evidence that it can elicit responses similar to those associated with defense. In order to evaluate whether there are consistent host responses associated with susceptibility, a similar analysis of ToxB-induced changes in the same sensitive cultivar was conducted. Comparative analysis of ToxA- and ToxB-induced transcriptional changes showed that similar groups of genes encoding WRKY transcription factors, RLKs, PRs, components of the phenylpropanoid and jasmonic acid pathways are activated. ROS accumulation and photosystem dysfunction proved to be common mechanism-of-action for these toxins. Despite similarities in defense responses, transcriptional and biochemical responses as well as symptom development occur more rapidly for ToxA compared to ToxB, which could be explained by differences in perception as well as by differences in activation of a specific process, for example, ethylene biosynthesis in ToxA treatment. Results of this study suggest that perception of HSTs will result in activation of defense responses as part of a susceptible interaction and further supports the hypothesis that necrotrophic fungi exploit defense responses in order to induce cell death.
Assuntos
Ascomicetos/patogenicidade , Micotoxinas/farmacologia , Doenças das Plantas/genética , Triticum/genética , Triticum/microbiologia , Morte Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Família Multigênica/efeitos dos fármacos , Estresse Oxidativo/genética , Fotossíntese/efeitos dos fármacos , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.
Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Variação Genética , Genoma Fúngico/genética , Filogenia , Mutação Puntual/genética , Fatores de Transcrição/genética , Composição de Bases/genética , Sequência de Bases , Biologia Computacional , Elementos de DNA Transponíveis/genética , Anotação de Sequência Molecular , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Ptr ToxB, encoded by ToxB, is one of multiple host-selective toxins (HST) produced by the wheat pathogen Pyrenophora tritici-repentis. Homologs of ToxB are found in several ascomycetes, including sister species Pyrenophora bromi, causal agent of brownspot of bromegrass. Due to the close evolutionary relatedness of P. tritici-repentis and P. bromi and that of their grass hosts, we hypothesized that homologs of ToxB in P. bromi may act as HST in the disease interaction between P. bromi and bromegrass. A representative set of transcriptionally active P. bromi ToxB genes were heterologously expressed in Pichia pastoris and the resultant proteins tested for their ability to act as HST on bromegrass. The tested Pyrenophora bromi ToxB (Pb ToxB) proteins were not toxic to bromegrass; thus, Pb ToxB does not appear to function as an HST in the P. bromi-bromegrass interaction. Instead, we revealed that the Pb ToxB proteins can be toxic to Ptr ToxB-sensitive wheat, at levels similar to Ptr ToxB, and the corresponding P. bromi ToxB genes are expressed in P. bromi-inoculated wheat. Our data suggest that P. bromi possesses the potential to become a wheat pathogen and highlights the importance of investigating the interaction between P. bromi and wheat.
Assuntos
Ascomicetos/patogenicidade , Bromus/microbiologia , Proteínas Fúngicas/metabolismo , Micotoxinas/metabolismo , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/metabolismo , Bromus/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , Regulação Fúngica da Expressão Gênica , Micotoxinas/genética , Micotoxinas/farmacologia , Pichia/genética , Pichia/metabolismo , Sensibilidade e Especificidade , Triticum/efeitos dos fármacos , Triticum/microbiologiaRESUMO
Host-selective toxins (HSTs) are effectors produced by some necrotrophic pathogenic fungi that typically confer the ability to cause disease. Often, diseases caused by pathogens that produce HSTs follow an inverse gene-for-gene model where toxin production is required for the ability to cause disease and a single locus in the host is responsible for toxin sensitivity and disease susceptibility. Pyrenophora tritici-repentis represents an ideal pathogen for studying the biological significance of such inverse gene-for-gene interactions, because it displays a complex race structure based on its production of multiple HSTs. Ptr ToxA and Ptr ToxB are two proteinaceous HSTs produced by P. tritici-repentis that are structurally unrelated and appear to evoke different host responses, yet each toxin confers the ability to cause disease. This review will summarize the current knowledge of how these two dissimilar HSTs display distinct modes of action, yet each confers pathogenicity to P. tritici-repentis.
Assuntos
Ascomicetos/patogenicidade , Genes Fúngicos/fisiologia , Interações Hospedeiro-Patógeno/genética , Micotoxinas , Doenças das Plantas/microbiologia , Triticum/microbiologia , Ascomicetos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologiaRESUMO
*ToxA, a host-selective toxin of wheat, can be detected within ToxA-sensitive mesophyll cells, where it localizes to chloroplasts and induces necrosis. Interaction of ToxA with the chloroplast-localized protein ToxABP1 has been implicated in this process. Therefore, we hypothesized that silencing of ToxABP1 in wheat would lead to a necrotic phenotype. Also, because ToxABP1 is highly conserved in plants, internal expression of ToxA in plants that do not normally internalize ToxA should result in cell death. *Reduction of ToxABP1 expression was achieved using Barley stripe mosaic virus (BSMV)-mediated, viral-induced gene silencing. The BSMV system was modified for use as an internal expression vector for ToxA in monocots. Agrobacterium-mediated expression of ToxA in a dicot (tobacco-Nicotiana benthamiana) was also performed. *Viral-induced gene silencing of ToxABP1 partially recapitulates the phenotype of ToxA treatment and wheat plants with reduced ToxABP1 also have reduced sensitivity to ToxA. When ToxA is expressed in ToxA-insensitive wheat, barley (Hordeum vulgare) and tobacco, cell death ensues. *ToxA accumulation in any chloroplast-containing cell is likely to result in cell death. Our data indicate that the ToxA-ToxABP1 interaction alters ToxABP1 function. This interaction is a critical, although not exclusive, component of the ToxA-induced cell death cascade.
Assuntos
Ascomicetos/patogenicidade , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes de Plantas , Micotoxinas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Ascomicetos/genética , Morte Celular , Proteínas Fúngicas/genética , Expressão Gênica , Inativação Gênica , Hordeum/genética , Hordeum/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Vírus do Mosaico , Micotoxinas/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/metabolismo , Triticum/genética , Triticum/metabolismoRESUMO
To obtain greater insight into the molecular events underlying plant disease susceptibility, we studied transcriptome changes induced by a host-selective toxin of Pyrenophora tritici-repentis, Ptr ToxA (ToxA), on its host plant, wheat. Transcriptional profiling of ToxA-treated leaves of a ToxA-sensitive wheat cultivar was performed using the GeneChip Wheat Genome Array. An improved and up-to-date annotation of the wheat microarray was generated and a new tool for array data analysis (BRAT) was developed, and both are available for public use via a web-based interface. Our data indicate that massive transcriptional reprogramming occurs due to ToxA treatment, including cellular responses typically associated with defense. In addition, this study supports previous results indicating that ToxA-induced cell death is triggered by impairment of the photosynthetic machinery and accumulation of reactive oxygen species. Based on results of this study, we propose that ToxA acts as both an elicitor and a virulence factor.
Assuntos
Proteínas Fúngicas/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Micotoxinas/farmacologia , Triticum/efeitos dos fármacos , Triticum/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triticum/genéticaRESUMO
Ptr ToxA (ToxA) is a proteinaceous necrotizing host-selective toxin produced by Pyrenophora tritici-repentis, a fungal pathogen of wheat (Triticum aestivum). In this study, we have found that treatment of ToxA-sensitive wheat leaves with ToxA leads to a light-dependent accumulation of reactive oxygen species (ROS) that correlates with the onset of necrosis. Furthermore, the accumulation of ROS and necrosis could be inhibited by the antioxidant N-acetyl cysteine, providing further evidence that ROS production is required for necrosis. Microscopic evaluation of ToxA-treated whole-leaf tissue indicated that ROS accumulation occurs in the chloroplasts. Analysis of total protein extracts from ToxA-treated leaves showed a light-dependent reduction of the chloroplast protein RuBisCo. In addition, Blue native-gel electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that ToxA induces changes in photosystem I (PSI) and photosystem II (PSII) in the absence of light, and therefore, the absence of ROS. When ToxA-treated leaves were exposed to light, all proteins in both PSI and PSII were extremely reduced. We propose that ToxA induces alterations in PSI and PSII affecting photosynthetic electron transport, which subsequently leads to ROS accumulation and cell death when plants are exposed to light.
Assuntos
Ascomicetos/patogenicidade , Micotoxinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Triticum/microbiologia , Acetilcisteína/farmacologia , Ascomicetos/metabolismo , Morte Celular/efeitos dos fármacos , Cloroplastos/efeitos dos fármacos , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Sequestradores de Radicais Livres/farmacologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Proteínas de Plantas/metabolismo , Triticum/efeitos dos fármacosRESUMO
Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 x 10(6) per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Micotoxinas/química , Micotoxinas/metabolismo , Oligopeptídeos/metabolismo , Membrana Celular/metabolismo , Interações Hospedeiro-Parasita , Fosforilação , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Triticum/microbiologiaRESUMO
Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Micotoxinas/genética , Sequência de Aminoácidos , Ascomicetos/classificação , Southern Blotting , Bromus/microbiologia , DNA Fúngico/química , DNA Fúngico/genética , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Triticum/microbiologiaRESUMO
The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority ( approximately 95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.
Assuntos
Ascomicetos/metabolismo , Éteres Metílicos/antagonistas & inibidores , Éteres Metílicos/metabolismo , Biodegradação Ambiental , Micélio/metabolismo , Oxirredução , Propano/metabolismo , terc-Butil Álcool/metabolismoRESUMO
Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteinaceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.
Assuntos
Ascomicetos/metabolismo , Cloroplastos/metabolismo , Proteínas Fúngicas/metabolismo , Micotoxinas/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Ascomicetos/genética , Northern Blotting , Western Blotting , Proteínas Fúngicas/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Micotoxinas/genética , Proteínas de Plantas/genética , Ligação Proteica , Alinhamento de Sequência , Triticum/genética , Triticum/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
ABSTRACT Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces multiple host-selective toxins (HSTs), including Ptr ToxA, Ptr ToxB, and Ptr ToxC. The specific complement of HSTs produced by a particular isolate determines its host cultivar specificity. Each unique specificity profile, represented by the differential induction of necrosis or chlorosis on a standard set of wheat differentials, defines a unique race. Eight races of P. tritici-repentis have been formally published, although additional races are under investigation. Although visual assessment of disease phenotype is often used in race designation of P. tritici-repentis, our results suggest that it has the potential to be misleading. Inoculation of the P. tritici-repentis isolates SO3 and PT82 on the current wheat differential set indicated classification as race 2 and race 8, respectively; however, genetic characterization revealed that these isolates do not possess the associated HSTs expected for these race assignments. Despite sharing disease phenotypes similar to known races, SO3 and PT82 were genotypically distinct from these previously characterized races of P. tritici-repentis. To ensure detection of the breadth of physiological variation among the isolates of P. tritici-repentis, our results indicate that race classification, where possible, should include both phenotypic and genotypic analyses and eventual expansion of the differential set.
RESUMO
Tan spot of wheat (Triticum aestivum), caused by the fungus Pyrenophora tritici-repentis, has significant agricultural and economic impact. Ptr ToxA (ToxA), the first discovered proteinaceous host-selective toxin, is produced by certain P. tritici-repentis races and is necessary and sufficient to cause cell death in sensitive wheat cultivars. We present here the high-resolution crystal structure of ToxA in two different crystal forms, providing four independent views of the protein. ToxA adopts a single-domain, beta-sandwich fold of novel topology. Mapping of the existing mutation data onto the structure supports the hypothesized importance of an Arg-Gly-Asp (RGD) and surrounding sequence. Its occurrence in a single, solvent-exposed loop in the protein suggests that it is directly involved in recognition events required for ToxA action. Furthermore, the ToxA structure reveals a surprising similarity with the classic mammalian RGD-containing domain, the fibronectin type III (FnIII) domain: the two topologies are related by circular permutation. The similar topologies and the positional conservation of the RGD-containing loop raises the possibility that ToxA is distantly related to mammalian FnIII proteins and that to gain entry it binds to an integrin-like receptor in the plant host.
Assuntos
Proteínas Fúngicas/química , Fungos/química , Fungos/metabolismo , Micoses/microbiologia , Micotoxinas/química , Doenças das Plantas/microbiologia , Triticum/microbiologia , Sequência de Aminoácidos/fisiologia , Sequência Conservada/fisiologia , Cristalografia por Raios X , Evolução Molecular , Fibronectinas/química , Interações Hospedeiro-Parasita/fisiologia , Modelos Moleculares , Filogenia , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen.