RESUMO
A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.
Assuntos
Proteínas Fúngicas/fisiologia , Magnaporthe/enzimologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Alelos , Western Blotting , Núcleo Celular/metabolismo , Deleção de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Modelos Genéticos , Mutação , Fosforilação , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/químicaRESUMO
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
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.
