RESUMO
In order to isolate in planta-induced genes encoding putative pathogenicity factors of the late blight fungus Phytophthora infestans, a genomic library was differentially screened. For the differential hybridization, labeled first-strand cDNA synthesized on mRNA isolated from P. infestans-infected potato leaves and on mRNA isolated from the fungus grown in vitro were used as probes. This screening resulted in the isolation of the P. infestans calmodulin gene. The gene, designated calA, contains an open reading frame of 447 base pairs without introns and is unique in the P. infestans genome. The predicted amino acid sequence is 89.9-94.6% identical to calmodulins from higher eukaryotes, whereas the identity to calmodulins of higher fungi is significantly less (60.8-85.1%). Expression studies revealed that the P. infestans calA gene is constitutively expressed in in vitro grown mycelium. However, during pathogenesis on potato the level of P. infestans calmodulin mRNA is increased approximately fivefold.
Assuntos
Calmodulina/genética , Genes Bacterianos/genética , Phytophthora/genética , Doenças das Plantas/etiologia , Solanum tuberosum/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Calmodulina/biossíntese , Expressão Gênica , Dados de Sequência Molecular , Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.
Assuntos
Proteínas Fúngicas/genética , Regulação da Expressão Gênica/fisiologia , Phytophthora/genética , Ubiquitinas/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Sondas de DNA/genética , Dados de Sequência Molecular , Família Multigênica/genética , Phytophthora/fisiologia , Solanum tuberosum/genéticaRESUMO
Thiabendazole, 2-(4'-thiazolyl)benzimidazole, at 80 micrometer completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration. Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [14C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly. Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wide-type strain. This indicates that in this mutant the structure of the binding site is affected. The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.
Assuntos
Aspergillus nidulans/metabolismo , Glicoproteínas/metabolismo , Penicillium/metabolismo , Tiabendazol/farmacologia , Tubulina (Proteína)/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Benzimidazóis/metabolismo , Carbamatos/metabolismo , DNA/biossíntese , Fungicidas Industriais/metabolismo , Cinética , Mitose/efeitos dos fármacos , Penicillium/efeitos dos fármacos , Ligação Proteica , RNA/biossíntese , Especificidade da EspécieRESUMO
The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.
