RESUMO
We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ascomicetos/fisiologia , Farmacorresistência Fúngica/fisiologia , Triticum/microbiologia , Transportadores de Cassetes de Ligação de ATP/genética , Ascomicetos/genética , Farmacorresistência Fúngica/genética , MutaçãoRESUMO
Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ascomicetos/genética , Sequência de Aminoácidos , Ascomicetos/efeitos dos fármacos , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Triticum/microbiologia , Xenobióticos/farmacologiaRESUMO
Two cellobiohydrolase-encoding genes, cbhA and cbhB, have been isolated from the filamentous fungus Aspergillus niger. The deduced amino acid sequence shows that CbhB has a modular structure consisting of a fungus-type cellulose-binding domain (CBD) and a catalytic domain separated by a Pro/Ser/Thr-rich linker peptide. CbhA consists only of a catalytic domain and lacks a CBD and linker peptide. Both proteins are homologous to fungal cellobiohydrolases in family 7 of the glycosyl hydrolases. Northern blot analysis showed that the transcription of the cbhA and cbhB genes is induced by D-xylose but not by sophorose and, in addition, requires the xylanolytic transcriptional activator XlnR.
Assuntos
Aspergillus niger/genética , Celulase/genética , Proteínas Fúngicas , Genes Fúngicos , Transativadores/fisiologia , Xilose/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Celulase/química , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase , Clonagem Molecular , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus Aspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of the xlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and beta-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including alpha-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA and eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
Assuntos
Aspergillus niger/genética , Celulase/genética , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Glucosidases/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Aspergillus niger/metabolismo , Celulase/biossíntese , Celulose/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos , Glucosidases/biossíntese , Mutação , Xilanos/metabolismoRESUMO
The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other alpha-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial alpha-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.