RESUMEN
Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.
Asunto(s)
Ascomicetos/patogenicidad , Farmacorresistencia Fúngica/genética , Polinización , Proteínas Quinasas/genética , Estrés Fisiológico , Estrobilurinas/farmacología , Triticum/genética , Agricultura , Ascomicetos/efectos de los fármacos , Mapeo Cromosómico , Cromosomas de las Plantas , Epistasis Genética , Fungicidas Industriales/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Polinización/efectos de los fármacos , Polinización/genética , Proteínas Quinasas/fisiología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Triticum/fisiologíaRESUMEN
The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Botrytis/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Fungicidas Industriales/farmacología , Vitis/microbiología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Botrytis/genética , Productos Agrícolas/microbiología , Vino/microbiología , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola.
Asunto(s)
Acetatos/farmacología , Ascomicetos/efectos de los fármacos , Ascomicetos/metabolismo , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/farmacología , Iminas/farmacología , Proteínas de Transporte de Membrana/metabolismo , Enfermedades de las Plantas/microbiología , Triticum/microbiología , Acetatos/metabolismo , Sustitución de Aminoácidos , Ascomicetos/genética , Ascomicetos/aislamiento & purificación , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Proteínas Fúngicas/genética , Fungicidas Industriales/metabolismo , Iminas/metabolismo , Proteínas de Transporte de Membrana/genética , Metacrilatos/metabolismo , Metacrilatos/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Estrobilurinas , Triticum/efectos de los fármacosRESUMEN
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Plantones/microbiología , Triticum/microbiología , Transportadoras de Casetes de Unión a ATP/genética , Inhibidores de Captación Adrenérgica/farmacología , Amitriptilina/farmacología , Antidiarreicos/farmacología , Ascomicetos/crecimiento & desarrollo , Antagonistas de Dopamina/farmacología , Sinergismo Farmacológico , Proteínas Fúngicas , Loperamida/farmacología , Pruebas de Sensibilidad Microbiana , Enfermedades de las Plantas/microbiología , Promazina/farmacología , Triazoles/farmacologíaRESUMEN
The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Ascomicetos/fisiología , Homeostasis/fisiología , Hierro/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Ascomicetos/genética , Farmacorresistencia Fúngica/genética , Farmacorresistencia Fúngica/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Enfermedades de las Plantas/microbiologíaRESUMEN
MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.
Asunto(s)
Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Proteínas Fúngicas/fisiología , Fungicidas Industriales/farmacología , Proteínas de Transporte de Membrana/fisiología , Triticum/microbiología , Ascomicetos/genética , Ascomicetos/metabolismo , Farmacorresistencia Fúngica Múltiple/genética , Etiquetas de Secuencia Expresada , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Cetoconazol/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Miconazol/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Mutación/genética , Mutación/fisiología , Perileno/análogos & derivados , Perileno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Triazoles/farmacologíaRESUMEN
The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.
Asunto(s)
Ascomicetos/genética , Genes Fúngicos , Proteínas Quinasas Activadas por Mitógenos/genética , Ascomicetos/enzimología , Ascomicetos/crecimiento & desarrollo , Ascomicetos/patogenicidad , Farmacorresistencia Fúngica/genética , Etiquetas de Secuencia Expresada , Proteínas Quinasas Activadas por Mitógenos/fisiología , Datos de Secuencia Molecular , Mutación , Presión Osmótica , Triticum/microbiología , Virulencia/genéticaRESUMEN
Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.
Asunto(s)
Ascomicetos/efectos de los fármacos , Aspergillus nidulans/efectos de los fármacos , Botrytis/efectos de los fármacos , Farmacorresistencia Fúngica Múltiple/fisiología , Proteínas Fúngicas/fisiología , Fungicidas Industriales/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Agricultura , Ascomicetos/patogenicidad , Ascomicetos/fisiología , Aspergillus nidulans/fisiología , Evolución Biológica , Botrytis/fisiología , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Enfermedades de las PlantasRESUMEN
Molecular mechanisms that account for variation in base-line sensitivity to azole fungicides were examined in a collection of twenty field isolates, collected in France and Germany, of the wheat pathogen Mycosphaerella graminicola (Fuckel) Schroeter. The isolates tested represent the wide baseline sensitivity to the azole fungicide tebuconazole described previously. The isolates were cross-sensitive to other azoles tested, such as cyproconazole and ketoconazole, but not to unrelated chemicals like cycloheximide, kresoxim-methyl or rhodamine 6G. Progenies from a genetic cross between an isolate with an intermediate and a high sensitivity to azoles displayed a continuous range of phenotypes with respect to cyproconazole sensitivity, indicating that variation in azole sensitivity in this haploid organism is polygenic. The basal level of expression of the ATP-binding cassette transporter genes MgAtr1-MgAtr5 from Mgraminicola significantly varied amongst the isolates tested, but no clear increase in the transcript level of a particular MgAtr gene was found in the less sensitive isolates. Cyproconazole strongly induced expression of MgAtr4, but no correlation between expression levels of this gene and azole sensitivity was observed. One isolate with intermediate sensitivity to azoles over-expressed CYP51, encoding cytochrome P450 sterol 14alpha-demethylase from M graminicola. Isolates with a low or high sensitivity to azoles were tested for accumulation of cyproconazole, but no clear correlation between reduced accumulation of the fungicide in mycelium and sensitivity to azoles was observed. Therefore, differences in accumulation cannot account exclusively for the variation in base-line sensitivity of the isolates to azoles. The results indicate that multiple mechanisms account for differences in base-line sensitivity to azoles in field isolates of M graminicola.
Asunto(s)
Ascomicetos/efectos de los fármacos , Azoles/toxicidad , Fungicidas Industriales/toxicidad , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Ascomicetos/genética , Radioisótopos de Carbono , Cruzamientos Genéticos , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Cetoconazol/metabolismo , Cetoconazol/toxicidad , Pruebas de Sensibilidad Microbiana/métodos , Triazoles/metabolismo , Triazoles/toxicidadRESUMEN
The role in virulence of the ATP-binding cassette (ABC) transporters MgAtr1, MgAtr2, MgAtr3, MgAtr4, and MgAtr5 from Mycosphaerella graminicola was analyzed by gene disruption or replacement on seedlings of the susceptible wheat cultivar Obelisk. Disruption strains of MgAtr1 and MgAtr2 and replacement strains of MgAtr3 and MgAtr5 displayed the same phenotype as control strains, while virulence of the MgAtr4 disruption strains was significantly reduced. This reduction in virulence was independent of the wheat cultivar used. Histopathological analysis of the infection process revealed that MgAtr4 disruption strains colonize substomatal cavities less efficiently and display reduced intercellular growth in the apoplast of wheat leaves. In vitro growth experiments in different media showed no fitness penalty associated with the disruption of MgAtr4. Expression analysis demonstrated that transcripts of the constitutively expressed gene CYP51 encoding the fungal-specific cytochrome P450 sterol 14alpha-demethylase from M. graminicola were not detectable in interaction RNA from wheat infected with MgAtr4 disruption strains, thus confirming the reduced intercellular growth of these strains. The results indicate that MgAtr4 is a virulence factor of M. graminicola during pathogenesis on wheat and may function in protection against fungitoxic compounds present around the substomatal cavities of wheat leaves. MgAtr4 is the first virulence factor cloned from this important plant pathogen.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Hongos/genética , Hojas de la Planta/microbiología , Triticum/microbiología , Factores de Virulencia/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/patogenicidad , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/microbiología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Triticum/genética , Triticum/crecimiento & desarrollo , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.
Asunto(s)
Botrytis/efectos de los fármacos , Fungicidas Industriales/farmacología , Imidazoles/farmacología , Proteínas de Transporte de Membrana/metabolismo , Sinergistas de Plaguicidas/farmacología , Botrytis/genética , Calmodulina/antagonistas & inhibidores , Clorpromazina/farmacología , Sinergismo Farmacológico , Fungicidas Industriales/química , Fungicidas Industriales/metabolismo , Genotipo , Imidazoles/química , Imidazoles/metabolismo , Estructura Molecular , Sinergistas de Plaguicidas/química , Sinergistas de Plaguicidas/metabolismoRESUMEN
In natural environments, microorganisms are exposed to a wide variety of antibiotic compounds produced by competing organisms. Target organisms have evolved various mechanisms of natural resistance to these metabolites. In this study, the role of ATP-binding cassette (ABC) transporters in interactions between the plant-pathogenic fungus Botrytis cinerea and antibiotic-producing Pseudomonas bacteria was investigated in detail. We discovered that 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and phenazine-1-carboxamide (PCN), broad-spectrum antibiotics produced by Pseudomonas spp., induced expression of several ABC transporter genes in B. cinerea. Phenazines strongly induced expression of BcatrB, and deltaBcatrB mutants were significantly more sensitive to these antibiotics than their parental strain. Treatment of B. cinerea germlings with PCN strongly affected the accumulation of [14C]fludioxonil, a phenylpyrrole fungicide known to be transported by BcatrB, indicating that phenazines also are transported by BcatrB. Pseudomonas strains producing phenazines displayed a stronger antagonistic activity in vitro toward ABcatrB mutants than to the parental B. cinerea strain. On tomato leaves, phenazine-producing Pseudomonas strains were significantly more effective in reducing gray mold symptoms incited by a ABcatrB mutant than by the parental strain. We conclude that the ABC transporter BcatrB provides protection to B. cinerea in phenazine-mediated interactions with Pseudomonas spp. Collectively, these results indicate that fungal ABC transporters can play an important role in antibiotic-mediated interactions between bacteria and fungi in plant-associated environments. The implications of these findings for the implementation and sustainability of crop protection by antagonistic microorganisms are discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Botrytis/crecimiento & desarrollo , Proteínas Fúngicas/fisiología , Pseudomonas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Antifúngicos/metabolismo , Antifúngicos/farmacología , Botrytis/efectos de los fármacos , Radioisótopos de Carbono , Dioxoles/farmacología , Resistencia a Múltiples Medicamentos , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Mutación , Fenazinas/metabolismo , Fenazinas/farmacología , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Floroglucinol/farmacología , Enfermedades de las Plantas/microbiología , Pirroles/farmacologíaRESUMEN
Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14alpha-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Farmacorresistencia Fúngica Múltiple/genética , Transportadoras de Casetes de Unión a ATP/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Cicloheximida/farmacología , Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Rodaminas/farmacología , Triazoles/farmacologíaRESUMEN
Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.
Asunto(s)
Botrytis/efectos de los fármacos , Camptotecina/farmacología , Farmacorresistencia Fúngica/fisiología , Perileno/análogos & derivados , Perileno/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico Activo , Northern Blotting , Southern Blotting , Botrytis/genética , Botrytis/metabolismo , Clonación Molecular , Fungicidas Industriales/farmacología , Expresión Génica , Genes Fúngicos , Pruebas de Sensibilidad Microbiana , Mutación , Fenotipo , Especificidad por SustratoRESUMEN
Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.