RESUMO
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.
Assuntos
Ascomicetos/genética , Metabolismo dos Carboidratos , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas Luminescentes/genética , Transformação Genética , Triticum/microbiologia , Western Blotting , Fluorometria , Proteínas de Fluorescência Verde , Folhas de Planta/microbiologia , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Ascomicetos/genética , Fungos Mitospóricos/genética , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
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.
