RESUMEN
Green fluorescent protein (GFP)-expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha-1) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post-penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola. The contribution of post-penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed.
Asunto(s)
Acrilatos/farmacología , Ascomicetos/efectos de los fármacos , Fungicidas Industriales/farmacología , Pirimidinas/farmacología , Ascomicetos/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes , Hifa/efectos de los fármacos , Hifa/crecimiento & desarrollo , Técnicas In Vitro , Proteínas Luminiscentes/genética , Metacrilatos , Microscopía Fluorescente , Enfermedades de las Plantas/microbiología , Estrobilurinas , Factores de Tiempo , Transformación Genética , Triticum/genética , Triticum/microbiologíaRESUMEN
In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.
Asunto(s)
Transporte de Electrón , Hongos/fisiología , Secuencia de Aminoácidos , Complejo I de Transporte de Electrón , Complejo II de Transporte de Electrones , Complejo III de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/química , Hongos/química , Hongos/genética , Regulación de la Expresión Génica , Proteínas Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/deficiencia , Oxidorreductasas/química , Oxidorreductasas/genética , Proteínas de Plantas , Plantas/enzimología , Fuerza Protón-Motriz , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Succinato Deshidrogenasa/químicaRESUMEN
A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.
Asunto(s)
Ascomicetos/genética , Metabolismo de los Hidratos de Carbono , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Luminiscentes/genética , Transformación Genética , Triticum/microbiología , Western Blotting , Fluorometría , Proteínas Fluorescentes Verdes , Hojas de la Planta/microbiología , Reacción en Cadena de la PolimerasaRESUMEN
ABSTRACT Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.
RESUMEN
Respiratory rates involving the alternative oxidase (AO) were studied in mitochondria from Tapesia acuformis. There was no evidence for regulation by pyruvate, in contrast with plant AO. The site of interaction of pyruvate with the plant AO is a conserved cysteine. The primary sequence was obtained for AO from Magnaporthe grisea and compared with four published sequences for fungal AO. In all cases this cysteine was absent. Sequence data were obtained for the C-terminal domain of a further five fungal AOs. In this region the fungal sequences were all consistent with a four-helix, di-iron binding structure as in the ferritin-fold family. A molecular model of this domain was deduced from the structure of Delta-9 desaturase. This is in general agreement with that developed for plant AOs, despite very low sequence identity between the two kingdoms. Further modelling indicated an appropriate active site for binding of ubiquinol, required in the AO redox reaction.
Asunto(s)
Hongos/enzimología , Mitocondrias/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Ácido Pirúvico/farmacología , Ubiquinona/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia Conservada/genética , Cisteína/genética , Cisteína/metabolismo , Dimerización , Hongos/genética , Holoenzimas/química , Holoenzimas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxígeno/metabolismo , Proteínas de Plantas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ácido Pirúvico/metabolismo , Alineación de Secuencia , Ubiquinona/metabolismoRESUMEN
Triadimenol and tebuconazole are potent inhibitors of the sterol 14 alpha-demethylation reaction in fungi which is catalysed by CYP51, a haem-thiolate containing enzyme belonging to the cytochrome P450 monooxygenase superfamily. Using CYP51 from the phytopathogen Ustilago maydis, a comparison of the sensitivity of the fungal enzyme to triadimenol and tebuconazole has been carried out. U. maydis CYP51 was purified to homogeneity as determined by SDS-PAGE and specific haem content. Catalytic activity was investigated following reconstitution with its respective NADPH cytochrome P450 reductase and proposed endogenous substrate, 24-methylenedihydrolanosterol. Addition of the triadimenol and tebuconazole induced type II spectral changes in the enzyme, with saturation occurring at equimolar azole concentrations. Inhibition of reconstituted activities showed a one-to-one sensitivity of the fungal CYP51 as judged by IC50 values. The implications for fungicide mode of action and treatment are discussed.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Oxidorreductasas , Ustilago/enzimología , Secuencia de Aminoácidos , Azoles/farmacología , Clonación Molecular , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/fisiología , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/fisiología , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Espectrofotometría , Esterol 14-Desmetilasa , Triazoles/farmacologíaRESUMEN
The alpha-tubulin genes from Septoria tritici and Rhynchosporium secalis have been cloned and sequenced. The predicted amino acid sequence and intron structure showed strong homology with other known filamentous fungal alpha-tubulins. Comparison of sixteen fungal alpha-tubulin sequences based on amino acid sequence homology and intron structure identified five groups of proteins. Group 1 consists of filamentous fungi, including S. tritici and R. secalis, the dimorphic fungus Histoplasma capsulatum, and Pneumocystis carinii. Group 2 includes two divergent isoforms from Neurospora crassa and Aspergillus nidulans. Group 3 includes the yeast Saccharomyces cerevisiae and the dimorphic fungus Candida albicans. Group 4 contains the single yeast Schizosaccharomyces pombe. Group 5 includes the only Basidiomycete, Schizophyllum commune. This analysis supports the classification of P carinii as a primitive Ascomycete. The presence of an additional glycine residue between the second and third amino acid found only in Group 2 proteins may indicate a functionally distinct fungal isotype. Implications in terms of structure-function relationships for alpha-tubulin molecules are discussed.
Asunto(s)
Ascomicetos/genética , Hongos Mitospóricos/genética , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in beta-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing beta-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to beta-tubulin. This binding is reduced by the mutation Glu198-->Gly198, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.
Asunto(s)
Bencimidazoles/metabolismo , Carbamatos/metabolismo , Fungicidas Industriales/metabolismo , Fenilcarbamatos , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteínas de Unión a Maltosa , Datos de Secuencia MolecularRESUMEN
The fungal wheat pathogen Mycosphaerella graminicola was transformed to carbendazim and hygromycin B resistance. A beta-tubulin gene from a M. graminicola strain resistant to the fungicide carbendazim was cloned and used to transform a sensitive strain to carbendazim resistance. Hygromycin B-resistant transformants (up to 8 per microgram of transforming DNA) arose at a higher rate than beta-tubulin transformants (up to 0.6 per microgram of DNA). Transformants were able to infect wheat and were morphologically similar to recipient strains.
Asunto(s)
Ascomicetos/efectos de los fármacos , Ascomicetos/genética , Bencimidazoles/farmacología , Carbamatos , Farmacorresistencia Microbiana/genética , Higromicina B/farmacología , Transformación Genética , Antifúngicos/farmacología , Ascomicetos/patogenicidad , Secuencia de Bases , Cartilla de ADN/genética , ADN de Hongos/genética , Genes Fúngicos , Técnicas Genéticas , Triticum/microbiología , Tubulina (Proteína)/genéticaRESUMEN
This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Deltapsi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.
Asunto(s)
Ascomicetos/metabolismo , Triticum/microbiología , Antifúngicos/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Ascomicetos/enzimología , Carboxina/farmacología , Citocromos/metabolismo , Transporte de Electrón , Pruebas de Sensibilidad Microbiana , Oligomicinas/farmacología , Consumo de Oxígeno , Cianuro de Potasio/farmacología , Salicilamidas/farmacologíaRESUMEN
This paper reviews the current status of our understanding of azole antifungal resistance mechanisms at the molecular level and explores their implications. Extensive biochemical studies have highlighted a significant diversity in mechanisms conferring resistance to azoles, which include alterations in sterol biosynthesis, target site, uptake and efflux. In stark contrast, few examples document the molecular basis of azole resistance. Those that do refer almost exclusively to mechanisms in laboratory mutants, with the exception of the role of multi-drug resistance proteins in clinical isolates of Candida albicans. It is clear that the technologies required to examine and define azole resistance mechanisms at the molecular level exist, but research appears distinctly lacking in this most important area.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/genética , Antifúngicos/química , Azoles/química , Candida albicans/efectos de los fármacos , Farmacorresistencia Microbiana , Proteínas Fúngicas/genética , Mutagénesis/fisiologíaRESUMEN
Amphotericin B resistant mutants of Cryptococcus neoformans were isolated accumulating mainly ergosterol. Cross-resistance to azole antifungals was not observed. Together with previous data this indicates that at least three categories of amphotericin B resistance can arise: sterol mutants, amphotericin B and azole cross-resistant mutants and amphotericin B resistant mutants with no azole cross-resistance.
Asunto(s)
Anfotericina B/farmacología , Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/metabolismo , Esteroles/biosíntesis , Cryptococcus neoformans/crecimiento & desarrollo , Farmacorresistencia MicrobianaRESUMEN
We report here a biochemical study of resistance to azole antifungal agents in a field isolate (S-27) of a fungal phytopathogen. Isolates of Septoria tritici were compared in vitro, and their responses reflected that observed in the field, with S-27 exhibiting resistance relative to RL2. In untreated cultures, both RL2 and S-27 contained isomers of ergosterol and ergosta-5,7-dienol, although in differing concentrations. Under azole treatment, this phytopathogen exhibited a response similar to that of other pathogenic fungi, with a reduction in desmethyl sterols and an accumulation of 14(alpha)-methyl sterols, indicative of inhibition of the P450-mediating sterol 14(alpha)-demethylase. Growth arrest was attributed to the reduction of ergosterol combined with an accumulation of nonutilizable sterols. Strain S-27 exhibited an azole-resistant phenotype which was correlated with decreased cellular content of azole.
RESUMEN
Azole antifungals inhibit CYP51A1-mediated sterol 14 alpha-demethylation and the mechanism(s) of resistance to such compounds in Ustilago maydis were examined. The inhibition of growth was correlated with the accumulation of the substrate, 24-methylene-24,25-dihydrolanosterol (eburicol), and depletion of ergosterol. Mutants overcoming the effect of azole antifungal treatment exhibited a unique phenotype with leaky CYP51A1 activity which was resistant to inhibition. The results demonstrate that alterations at the level of inhibitor binding to the target site can produce azole resistance. Similar changes may account for fungal azole resistance phenomena in agriculture, and also in medicine where resistance has become a problem in immunocompromised patients suffering from AIDS.
Asunto(s)
Azoles/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidorreductasas/metabolismo , Ustilago/efectos de los fármacos , Ustilago/enzimología , Radioisótopos de Carbono , Farmacorresistencia Microbiana , Estructura Molecular , Mutación , Esterol 14-Desmetilasa , Esteroles/metabolismo , Especificidad por Sustrato , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
Fluconazole was observed to inhibit sterol 14 alpha-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Cryptococcus neoformans/efectos de los fármacos , Polienos/farmacología , Anfotericina B/farmacología , Cryptococcus neoformans/metabolismo , Farmacorresistencia Microbiana , Pruebas de Sensibilidad Microbiana , Esteroles/biosíntesisRESUMEN
Resistance to azole antifungals in Ustilago maydis was associated with a leaky defect in sterol delta 5(6)desaturase. This defect resulted in reduced accumulation of 14 alpha-methylergosta-24(28)-diene-3 beta,6 alpha-diol and an increase in the proportion of 14 alpha-methylfecosterol in treated cells when compared to the parent strain. The results demonstrate the importance of this mechanism in pathogenic fungi.