RESUMO
TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine beta-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.
Assuntos
Ascomicetos/genética , Elementos de DNA Transponíveis/genética , Células Eucarióticas/metabolismo , Poli A/genética , RNA Mensageiro/genética , 4-Hidroxifenilpiruvato Dioxigenase/genética , Ascomicetos/enzimologia , Sequência de Bases , Cistationina beta-Sintase/genética , DNA Bacteriano/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Magnaporthe/enzimologia , Magnaporthe/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Poli A/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes. The enzyme is unique in terms of catalyzing both decarboxylation and transamination. Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear. We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout. The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia. Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster. Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat. The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis. This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.
Assuntos
Ascomicetos/genética , Carboxiliases/genética , Filogenia , Sequência de Aminoácidos , Ácidos Aminoisobutíricos/metabolismo , Ascomicetos/enzimologia , Southern Blotting , Carboxiliases/metabolismo , Mapeamento Cromossômico , Primers do DNA , Dados de Sequência Molecular , Análise de Sequência de DNA , Transformação GenéticaRESUMO
TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.
Assuntos
Ascomicetos/genética , Engenharia Genética/métodos , Clonagem Molecular , Cosmídeos/genética , Elementos de DNA Transponíveis , Eletroporação , Marcação de Genes/métodos , Mutação , Análise de Sequência de DNA , Transformação Genética , Triticum/microbiologiaRESUMO
CBS1 from Magnaporthe grisea is a structural and functional homolog of the cystathionine beta-synthase (CBS) gene from Saccharomyces cerevisiae. Our studies indicated that M. grisea can utilize homocysteine and methionine through a CBS-independent pathway. The results also revealed responses of M. grisea to homocysteine that are reminiscent of human homocystinuria.
