RESUMO
The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.
Assuntos
Morte Celular/efeitos dos fármacos , Magnaporthe/química , Micotoxinas/farmacologia , Espécies Reativas de Oxigênio , Avena/microbiologia , Morte Celular/efeitos da radiação , Clorofila , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados , Luz , Micotoxinas/química , Micotoxinas/isolamento & purificação , Oryza/microbiologia , Folhas de Planta/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiaçãoRESUMO
We developed an RNA-silencing vector, pSilent-Dual1 (pSD1), with a convergent dual promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice-blast fungus Magnaporthe oryzae, 37 calcium signalling-related genes that include almost all known calcium-signalling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35 and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M. oryzae. These included several novel findings such as that Pmc1-, Spf1- and Neo1-like Ca(2+) pumps, calreticulin and calpactin heavy chain were essential for fungal pathogenicity.
Assuntos
Sinalização do Cálcio , Proteínas Fúngicas/genética , Genoma Fúngico , Magnaporthe/genética , Oryza/microbiologia , Interferência de RNA , Proteínas Fúngicas/metabolismo , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hordeum/microbiologia , Magnaporthe/patogenicidade , Magnaporthe/fisiologia , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Triticum/microbiologiaRESUMO
The LTR-retrotransposon MAGGY was introduced into naive genomes of Magnaporthe oryzae with different genetic backgrounds (wild-type, and MoDcl1 [mdl1] and MoDcl2 [mdl2] dicer mutants). The MoDcl2 mutants deficient in MAGGY siRNA biogenesis generally showed greater MAGGY mRNA accumulation and more rapid increase in MAGGY copy number than did the wild-type and MoDcl1 mutants exhibiting normal MAGGY siRNA accumulation, indicating that RNA silencing functioned as an effective defense against the invading element. Interestingly, however, regardless of genetic background, the rate of MAGGY transposition drastically decreased as its copy number in the genome increased. Notably, in the MoDcl2 mutant, copy-number-dependent MAGGY suppression occurred without a reduction in its mRNA accumulation, and therefore by a silencing mechanism distinct from both transcriptional gene silencing and siRNA-mediated RNA silencing. This might imply that some mechanism possibly similar to post-transcriptional cosuppression of Ty1 retrotransposition in Saccharomyces cerevisiae, which operates regardless of the abundance of target transcript and independent of RNA silencing, would also function in M. oryzae that possesses the RNA silencing machinery.
Assuntos
Magnaporthe/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retroelementos , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Magnaporthe/enzimologia , Mutação , Micélio/genética , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Sequências Repetidas TerminaisRESUMO
Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.
Assuntos
Apoptose , Doenças das Plantas/microbiologia , Pseudomonas/patogenicidade , Ânions/metabolismo , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , DNA de Plantas/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Sequestradores de Radicais Livres/metabolismo , Lactuca/efeitos dos fármacos , Lactuca/microbiologia , Lactuca/ultraestrutura , Peptídeo Hidrolases/metabolismo , Peroxidase/metabolismo , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Polissacarídeo-Liases/metabolismo , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Fatores de TempoRESUMO
Pseudomonas fluorescens FPT9601-T5 was originally identified as an endophytic plant growth-promoting rhizobacteria (PGPR) on tomato. To perform a molecular dissecttion of physiological and biochemical changes occurring in the host triggered by P. fluorescens FPT9601-T5 colonization, the model plant Arabidopsis was used in this study. Root colonization of Arabidopsis with P. fluorescens FPT9601-T5 promoted plant growth later than three weeks after inoculation and partially suppressed disease symptoms caused by Pseudomonas syringae pv. tomato DC3000, indicating that P. fluorescens FPT9601-T5 acted as a PGPR on Arabidopsis. To obtain a global view on transcript modification during the Arabidopsis-FPT9601-T5 interaction, we performed microarray analysis using Affymetrix Genechip probe arrays representing approximately 22,800 genes. The results showed that 95 and 105 genes were up- or down-regulated, respectively, more than twofold in FPT9601-T5-treated Arabidopsis plants as compared with control plants. Those up-regulated included genes involved in metabolism, signal transduction, and stress response. Noteworthy, upon FPT9601-T5 colonization, putative auxin-regulated genes and nodulin-like genes were up-regulated, and some ethylene-responsive genes were down-regulated. Our results suggest that P. fluorescens FPT9601-T5 triggered plant responses in a manner similar to known PGPR and, at least in some aspects, to rhizobia.
Assuntos
Arabidopsis/microbiologia , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Pseudomonas fluorescens/fisiologia , Arabidopsis/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/microbiologia , Regulação para CimaRESUMO
The significance of AVR1-CO39, an avirulence gene of the blast fungus corresponding to Pi-CO39(t) in rice cultivars, during the evolution and differentiation of the blast fungus was evaluated by studying its function and distribution in Pyricularia spp. When the presence or absence of AVR1-CO39 was plotted on a dendrogram constructed from ribosomal DNA sequences, a perfect parallelism was observed between its distribution and the phylogeny of Pyricularia isolates. AVR1-CO39 homologs were exclusively present in one species, Pyricularia oryzae, suggesting that AVR1-CO39 appeared during the early stage of evolution of P. oryzae. Transformation assays showed that all the cloned homologs tested are functional as an avirulence gene, indicating that selection has maintained their function. Nevertheless, Oryza isolates (isolates virulent on Oryza spp.) in P. oryzae were exceptionally noncarriers of AVR1-CO39. All Oryza isolates suffered from one of the two types of known rearrangements at the Avr1-CO39 locus (i.e., G type and J type). These types were congruous to the two major lineages of Oryza isolates from Japan determined by MGR586 and MAGGY. These results indicate that AVR1-CO39 was lost during the early stage of evolution of the Oryza-specific subgroup of P. oryzae. Interestingly, its corresponding resistance gene, Pi-CO39(t), is not widely distributed in Oryza spp.
Assuntos
Evolução Molecular , Genes Fúngicos , Magnaporthe/genética , Magnaporthe/patogenicidade , Frequência do Gene , Oryza/microbiologia , Filogenia , Poaceae/microbiologia , Virulência/genéticaRESUMO
MAGGY is a Ty3/Gypsy retrotransposon, which was identified in the rice blast fungus Magnaporthe oryzae. Some Ty3/Gypsy retrotransposons, including MAGGY, contain a chromodomain-like module (CLM) in the C-terminus of the integrase domain. We have made a series of MAGGY mutants to examine the role of the CLM in the transposition activity of the element. Introduction of a mutation at different positions in the MAGGY integrase revealed that a loss or alteration of the CLM resulted in a drastic decrease in the transposition activity of the element. Our results indicate that the CLM may confer high transposition activity to the element.
Assuntos
Integrases/química , Magnaporthe/enzimologia , Magnaporthe/genética , Retroelementos , Sequência de Aminoácidos , DNA Fúngico/genética , Integrases/genética , Íntrons/genética , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína/genética , Recombinação Genética/genética , Sequências Repetidas Terminais/genética , Dedos de Zinco/genéticaRESUMO
Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.
Assuntos
Magnaporthe/genética , Interferência de RNA , Sequência de Bases , Northern Blotting , Southern Blotting , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Magnaporthe/patogenicidade , Microscopia de Fluorescência , Plantas/microbiologia , RNA Interferente Pequeno/genéticaRESUMO
We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-terminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.
Assuntos
Apoptose/fisiologia , Avena/microbiologia , Avena/ultraestrutura , Avena/virologia , Bactérias/crescimento & desenvolvimento , Fungos/crescimento & desenvolvimento , Marcação In Situ das Extremidades Cortadas , Magnaporthe/crescimento & desenvolvimento , Microscopia Eletrônica , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Vírus de Plantas/crescimento & desenvolvimento , Pseudomonas/crescimento & desenvolvimentoRESUMO
The AVR1-CO39 gene that came from a Magnaporthe grisea isolate from weeping lovegrass controls avirulence on the rice cultivar CO39. AVR1-CO39 was not present in the genome of the rice-infecting M. grisea isolate Guyll from French Guyana, suggesting that the gene had been deleted. Molecular analysis of the deletion breakpoints in the AVR1-CO39 locus revealed the presence of a truncated copy of a previously unknown retrotransposon at the left-hand border. At the right-hand border was a truncated copy of another repetitive element that is present at multiple locations in the genome of Guyll. The structures of avr1-CO39 loci were further examined in 45 rice-infecting isolates collected in Brazil, China, Japan, India, Indonesia, Mali, and the Philippines. Most isolates showed no hybridization signal with the AVR1-CO39 probe and had the same locus structure as Guyll. Some isolates from Japan showed a signal with the AVR1-CO39 probe, but the region specifying avirulence activity was rearranged. These findings suggest that widespread virulence to 'CO39' among rice-infecting M. grisea isolates is due to ancestral rearrangements at the AVR1-CO39 locus that may have occurred early in the evolution of pathogenicity to rice.
Assuntos
Mapeamento Cromossômico , Magnaporthe/genética , Magnaporthe/patogenicidade , Oryza/microbiologia , Virulência/genética , Sequência de Bases , Brasil , China , Genes Fúngicos , Geografia , Japão , Magnaporthe/isolamento & purificação , Dados de Sequência MolecularRESUMO
Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2- in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2- but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.
Assuntos
Apoptose/fisiologia , Avena/metabolismo , Óxido Nítrico/metabolismo , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Avena/citologia , Avena/efeitos dos fármacos , Avena/microbiologia , Benzoatos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Fungos/crescimento & desenvolvimento , Peróxido de Hidrogênio/metabolismo , Imidazóis/farmacologia , Imunidade Inata/genética , Imunidade Inata/fisiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Superóxido Dismutase/farmacologiaRESUMO
Two oat genes encoding hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT) and S-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), both of which are possibly involved in the biosynthesis of oat avenanthramide phytoalexins, were cloned and their expression profiles in response to biological stress were studied. Four distinct cDNAs of oat HHT (AsHHT1-4) were isolated with the degenerative polymerase chain reaction method. The enzymatic activity of AsHHT1 expressed in E. coli was found using hydroxyanthranilate and hydroxycinnamoyl-CoAs as cosubstrates. Cloned oat CCoAOMT (AsCCoAOMT) encoded a polypeptide of 130 amino acid residues with 77.7 to 80.8% identities to the CCoAOMT sequences from other plant species. The accumulation of AsHHT1 and AsCCoAOMT transcripts increased concomitantly with phytoalexin accumulation by the treatment of victorin, a specific elicitor in oat lines carrying the Pc-2/Vb gene. Pharmacological approaches indicated the involvement of Ca2+, NO, and protein kinases in the signaling pathways of AsHHT1 and AsCCoAOMT mRNA induction. When oat leaves were inoculated with Puccinia coronata, the mRNA expression of AsHHT1 and AsCCOAOMT increased in both incompatible and compatible interactions but more rapidly in incompatible interaction. Interestingly, however, significant phytoalexin accumulation was observed only in incompatible interaction during the experimental period, suggesting that phytoalexin accumulation may be inhibited in one or more posttranscriptional processes in the compatible interaction.
Assuntos
Aciltransferases/genética , Avena/enzimologia , Fungos/crescimento & desenvolvimento , Metiltransferases/genética , Extratos Vegetais/biossíntese , Folhas de Planta/enzimologia , Aciltransferases/metabolismo , Sequência de Aminoácidos , Avena/genética , Avena/microbiologia , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metiltransferases/metabolismo , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sesquiterpenos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Terpenos , FitoalexinasRESUMO
We have developed an oat cell-free apoptosis system to investigate the execution mechanisms of plant apoptosis. Cell extracts derived from oat tissues undergoing toxin (victorin)-induced apoptosis caused nuclear collapse and internucleosomal DNA fragmentation in isolated nuclei. Pharmacological studies revealed that cysteine protease, which is E-64-sensitive but insensitive to caspase-specific inhibitors, is a crucial component in the morphological change of isolated nuclei, and that nuclease and the cysteine protease act cooperatively to induce the apoptotic DNA laddering. Interestingly, this finding is contrasted with those in well-studied animal cell-free systems in which an apoptotic endonuclease is solely responsible for the DNA fragmentation.
Assuntos
Apoptose/fisiologia , Avena/enzimologia , Cisteína Endopeptidases/metabolismo , Endonucleases/metabolismo , Leucina/análogos & derivados , Apoptose/efeitos dos fármacos , Ácido Aurintricarboxílico/farmacologia , Avena/citologia , Avena/metabolismo , Extratos Celulares , Núcleo Celular/efeitos dos fármacos , Sistema Livre de Células , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , DNA de Plantas/efeitos dos fármacos , Interações Medicamentosas , Endonucleases/farmacologia , Leucina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Toxinas Biológicas/farmacologiaRESUMO
The chitinase gene-transformed strain KPM-007E/chi of Enterobacter cloacae was vitally entrapped in sodium alginate gel beads with its specific virulent bacteriophage EcP-01 to provide a new method for microbially digesting chitinous peritrophic membranes of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, chitinase SH1 from a gram-positive bacterium Kurthia zopfii was overproduced by Escherichia coli cells and purified by affinity column chromatography. The purified enzyme effectively digested peritrophic membranes dissected from the ladybird beetles to expose epithelial tissues beneath the peritrophic membrane, and the beetles that had ingested chitinase after submergence in chitinase solution had considerably reduced their feeding on tomato leaves. KPM-007E/chi, entrapped in the alginate beads, released the chitinase. More chitinase was released when KPM-007E/chi was present with their specific virulent bacteriophage EcP-01 in the beads because of lysis of bacterial cells infected with the bacteriophages. This chitinase release from the microbial beads (containing KPM-007E/chi and EcP-01) was sufficient to digest the peritrophic membrane as well as to suppress feeding of bead-sprayed tomato leaves by the ladybird beetles. A daily supply of tomato leaves treated with the microbial beads considerably suppressed leaf feeding and oviposition by the ladybird beetles, suggesting a possible application of chitinase-secreting bacteria for suppressing herbivorous insect pests.
Assuntos
Quitinases/metabolismo , Besouros/fisiologia , Enterobacter cloacae/enzimologia , Enterobacter cloacae/virologia , Comportamento Alimentar , Controle de Insetos/métodos , Oviposição , Solanum lycopersicum/parasitologia , Alginatos , Animais , Bacteriófagos/fisiologia , Quitinases/genética , Enterobacter cloacae/genética , Géis , Microesferas , Folhas de Planta , Transformação BacterianaRESUMO
Victorin, the host-selective toxin produced by the fungus Cochliobolus victoriae, induces programmed cell death (PCD) in victorin-sensitive oat lines with characteristic features of animal apoptosis, such as mitochondrial permeability transition, chromatin condensation, nuclear DNA laddering and rRNA/mRNA degradation. In this study, we characterized a calcium-binding protein, namely AsALG-2, which might have a role in the victorin-induced PCD. AsALG-2 is homologous to the Apoptosis-Linked Gene ALG-2 identified in mammalian cells. Northern blot analysis revealed that the accumulation of AsALG-2 transcripts increased during victorin-induced PCD, but not during necrotic cell death. Salicylic acid, chitosan and chitin strongly activated the expression of general defence response genes, such as PR-10; however, neither induced cell death nor the accumulation of AsALG-2 mRNA. Pharmacological studies indicated that victorin-induced DNA laddering and AsALG-2 expression were regulated through similar pathways. The calcium channel blocker, nifedipine, moderately inhibited the accumulation of AsALG-2 mRNA during cell death. Trifluoperazine (calmodulin antagonist) and K252a (serine-threonine kinase inhibitor) reduced the victorin-induced phytoalexin accumulation, but did not prevent the victorin-induced DNA laddering or accumulation of AsALG-2 mRNA. Taken together, our investigations suggest that there is a calcium-mediated signalling pathway in animal and plant PCD in common.
Assuntos
Apoptose/fisiologia , Avena/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Plantas/metabolismo , Apoptose/genética , Avena/genética , Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , Proteínas de Plantas/genéticaRESUMO
Pyricularia isolates from various host plants were subjected to a multilocus phylogenetic analysis based on rDNA-ITS, actin, beta-tubulin, and calmodulin loci. A combined gene tree resolved seven groups with 100% BS support, suggesting that they are monophyletic groups supported concordantly by all four loci. By incorporating biological and morphological species criteria, each of the seven groups was considered to be a current species. However, phylogenetic relationships among these species were unresolved in the single-gene trees and in the combined tree. Furthermore, the transition from concordance to conflict occurred more than once in the combined gene tree. They were interpreted by assuming that Pyricularia has evolved through repeated species radiation. The transition point other than the current species limit was considered to be the limit of the former species.
Assuntos
Ascomicetos/classificação , DNA Espaçador Ribossômico/análise , Proteínas Fúngicas/genética , Especiação Genética , Filogenia , Actinas/genética , Ascomicetos/genética , Calmodulina/genética , DNA Fúngico/análise , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Técnicas de Tipagem Micológica , Análise de Sequência de DNA , Especificidade da Espécie , Tubulina (Proteína)/genéticaRESUMO
Comprehensive phylogenetic analyses of fungal Argonaute, Dicer, and RNA-dependent RNA polymerase-like proteins have been performed to gain insights into the diversification of RNA silencing pathways during the evolution of fungi. A wide range of fungi including ascomycetes, basidiomycetyes, and zygomycetes possesses multiple RNA silencing components in the genome, whereas a portion of ascomycete and basidiomycete fungi apparently lacks the whole or most of the components. The number of paralogous silencing proteins in the genome differs considerably among fungal species, suggesting that RNA silencing pathways have diversified significantly during evolution in parallel with developing the complexity of life cycle or in response to environmental conditions. Interestingly, orthologous silencing proteins from different fungal clades are often clustered more closely than paralogous proteins in a fungus, indicating that duplication events occurred before speciation events. Therefore, the origin of multiple RNA silencing pathways seems to be very ancient, likely having occurred prior to the divergence of the major fungal lineages.
Assuntos
Endorribonucleases/genética , Evolução Molecular , Genes Fúngicos , Variação Genética , Interferência de RNA , DNA Polimerase Dirigida por RNA/genética , Ascomicetos/metabolismo , Basidiomycota/metabolismo , Linhagem da Célula , Endorribonucleases/metabolismo , Filogenia , DNA Polimerase Dirigida por RNA/metabolismo , Transdução de SinaisRESUMO
Here we report that rRNA and mRNA are specifically degraded in oat (Avena sativa L.) cells during apoptotic cell death induced by victorin, a host-selective toxin produced by Cochliobolus victoriae. Northern analysis indicated that rRNA species from the cytosol, mitochondria and chloroplasts were all degraded via specific degradation intermediates during victorin-induced apoptotic cell death but, in contrast, they were randomly digested in necrotic cell death induced by 30 mM CuSO(4) and heat shock. This indicates that specific rRNA cleavage could be controlled by an intrinsic program. We also observed specific cleavage of mRNA of housekeeping genes such as actin and ubiquitin during victorin-induced cell death. Interestingly, no victorin-induced mRNA degradation was detected with stress-responding genes such as PR-1, PR-10 and GPx throughout the experimental period. The RNA degradation mostly, but not always, occurred in parallel with DNA laddering, but pharmacological studies indicated that these processes are regulated by different signaling pathways with some overlapping upstream signals.
Assuntos
Apoptose/efeitos dos fármacos , Avena/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Micotoxinas/farmacologia , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , RNA Ribossômico/metabolismo , Apoptose/fisiologia , Avena/citologia , Avena/genética , DNA de Plantas/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Folhas de Planta/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , RNA Ribossômico/genética , Transdução de SinaisRESUMO
SUMMARY Culture filtrates from 72 isolates of Pyricularia, grouped into 13 rDNA types, were analysed via HPLC. Of these isolates, 31 (r9 DNA type) from crabgrass (Digitaria sanguinalis), one (r9 DNA type) from pangolagrass (Digitaria smutsii) and six (r8 DNA type) from Digitaria horizontalis produced 20-280 microg pyrichalasin H per millilitre of culture. These same isolates were pathogenic on five Digitaria species. Interestingly, two isolates, KM-1 and Br 29, which were originally isolated from Digitaria plants, did not produce pyrichalasin H, nor caused blast lesion on Digitaria plants. Because these two isolates were identified as Digitaria pathogens by PCR analysis using Digitaria-specific primers, they are likely to be mutants lacking pyrichalasin H production. Isolates that belonged to the remaining 11 rDNA types did not produce pyrichalasin H and were avirulent to Digitaria plants. Therefore, the virulence of Pyricularia on Digitaria plants correlates with pyrichalasin H production. Pyrichalasin H was also present in spore germination fluid of a crabgrass isolate (IBDS 5-1-1), but not in that of isolates from rice, foxtail millet, finger millet, common millet and wheat. In addition, pyrichalasin H was detected in host leaves infected with IBDS 5-1-1, but not in leaves from other plants infected with compatible Pyricularia isolates. Pretreatment of leaf sheaths of crabgrass with 3 microg/mL pyrichalasin H led to the penetration and colonization by non-host isolates. Overall, these results indicate that production of pyrichalasin H is responsible for the genus-specific pathogenicity of Digitaria isolates.
RESUMO
In potato (Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H2O2 accumulation were induced by treatment with beta-glucan elicitor. The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H2O2, whereas the ascorbate peroxidase inhibitor did not have any effect on these responses. Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H2O2 accumulation, leading to the disruption of mitochondrial membrane potential (deltapsi(m)) and programmed cell death (PCD). The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial deltapsi(m) breakdown and PCD induced by beta-glucan elicitor.