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1.
Rev Iberoam Micol ; 17(1): S43-6, 2000 Mar.
Artigo em Espanhol | MEDLINE | ID: mdl-15762781

RESUMO

Botrytis cinereais an important plant pathogenic fungi with a wide host range, which can make use of different infection mechanisms. Although genetic variation for resistance to B. cinereahas been observed within some species, no gene-for-gene relationship has been found. The development of resistant genotypes is, therefore, complicated. Any attempt to develop control strategies makes it necessary a detailed knowledge of both the fungal infection mechanisms and the plant defence mechanisms. The application of different experimental approaches allows the analysis of the infection process in different hosts, the description of the elements that participate in each stage of the process and the identification of those pathogenicity factors which are essential for the establishment of the interaction. The characterisation of the latter will provide information about key elements of the infection process as the basis for the development of effective, long term and environmentally friendly control strategis.

2.
Curr Microbiol ; 39(5): 259-64, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10489434

RESUMO

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.


Assuntos
Mucor/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Phycomyces/genética , Carotenoides/metabolismo , DNA Complementar , Expressão Gênica , Genes Fúngicos , Mucor/enzimologia , Phycomyces/enzimologia , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transformação Genética
3.
Appl Environ Microbiol ; 65(8): 3335-40, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10427016

RESUMO

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.


Assuntos
Fusarium/genética , Fusarium/isolamento & purificação , Sequência de Bases , Primers do DNA/genética , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Fabaceae/microbiologia , Fusarium/patogenicidade , Variação Genética , Doenças das Plantas/microbiologia , Plantas Medicinais , Polimorfismo de Fragmento de Restrição , Espanha
4.
Mol Gen Genet ; 253(6): 734-44, 1997 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-9079885

RESUMO

By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.


Assuntos
Regulação Fúngica da Expressão Gênica , Luz , Oxirredutases/genética , Phycomyces/enzimologia , Vitamina A/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Difenilamina/farmacologia , Escherichia coli , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Dados de Sequência Molecular , Phycomyces/efeitos dos fármacos , Phycomyces/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos
5.
Plant Mol Biol ; 32(5): 947-57, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8980545

RESUMO

Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. In order to isolate Botrytis cinerea genes whose expression is induced during its interaction with tomato, a comparative analysis of the expression pattern of the fungus in planta with its expression pattern during in vitro culture was performed by differential display of mRNA (DDRT-PCR). Discrimination of fungal genes induced in planta from plant defense genes induced in response to the pathogen was attempted by including in this comparative analysis the expression patterns of healthy tomato leaves and of tomato leaves infected with two different pathogens, either Rhytophthora infestans or tobacco necrosis virus (TNV). Using a limited set of primer combinations, three B. cinerea cDNA fragments, ddB-2, ddB-5 and ddB-47, were isolated representing fungal genes whose expression is enhanced in planta. Northern blot analysis showed that the transcripts detected with the cDNA clones ddB-2 and ddB-5 accumulated at detectable levels only at late time points during the interaction. The cDNA clone ddB-47 detected two different sizes of transcripts displaying distinct, transient expression patterns during the interaction. Sequence analysis and database searches revealed no significant homology to any known sequence. These results show that the differential display procedure possesses enough sensitivity to be applied to the detection of fungal genes induced during a plant-pathogen interaction. Additionally, four cDNA fragments were isolated representing tomato genes induced in response to the infection caused by B. cinerea, but not by P. infestans.


Assuntos
Expressão Gênica , Genes Fúngicos , Fungos Mitospóricos/genética , Reação em Cadeia da Polimerase , Solanum lycopersicum/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Fragmentação do DNA , DNA Complementar , DNA Fúngico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Transcrição Gênica
6.
Mol Gen Genet ; 248(2): 126-35, 1995 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-7651335

RESUMO

Pyrimidine auxotrophs of Mucor circinelloides were isolated after mutagenesis with nitrosoguanidine and selected for resistance to 5-fluoroorotate. These mutants were genetically and biochemically characterized and found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase) activity or in orotate phosphoribosyl transferase (OPRTase) activity. Different circular DNA molecules containing the homologous pyrG gene were used to transform a representative OMPdecase-deficient strain to uracil prototrophy. Southern analysis, as well as mitotic stability analysis of the transformants, showed that the transforming DNA is always maintained extrachromosomally. The smallest fragment tested that retained both the capacity to complement the pyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kb M. circinelloides genomic DNA fragment. This fragment consists of the pyrG coding region flanked by 606 nucleotides at the 5' and 330 nucleotides at the 3' ends, respectively. Sequence analysis reveals that it does not share any element in common with another M. circinelloides genomic DNA fragment which also promotes autonomous replication in this organism, except those related to transcription. Furthermore, it differs from elements which have been shown to be involved in autonomous replication in other fungal systems. An equivalent plasmid harbouring the heterologous Phycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication in M. circinelloides may be driven by elements normally present in nuclear coding genes.


Assuntos
Genes Fúngicos , Mucor/enzimologia , Orotidina-5'-Fosfato Descarboxilase/genética , Transformação Genética , Sequência de Bases , Southern Blotting , DNA Circular/genética , Mitose/genética , Dados de Sequência Molecular , Mucor/genética , Mutação/genética , Orotato Fosforribosiltransferase/genética , Pirimidinas/metabolismo , Análise de Sequência
7.
Gene ; 116(1): 59-67, 1992 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-1628845

RESUMO

A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.


Assuntos
Mucor/enzimologia , Orotidina-5'-Fosfato Descarboxilase/genética , Transformação Genética/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Teste de Complementação Genética , Íntrons/genética , Dados de Sequência Molecular , Mucor/genética , Mutação/genética , Orotidina-5'-Fosfato Descarboxilase/química , Plasmídeos/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
8.
Curr Genet ; 21(3): 215-23, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1563047

RESUMO

The leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus alpha-IPM (isopropylmalate) isomerase.


Assuntos
Genes Fúngicos , Técnicas Genéticas , Hidroliases/genética , Mucor/genética , Phycomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Biblioteca Genômica , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Transformação Genética
9.
Mol Gen Genet ; 224(2): 269-78, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2277645

RESUMO

The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA". The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.


Assuntos
Genes Fúngicos , Orotidina-5'-Fosfato Descarboxilase/genética , Phycomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Fungos/genética , Humanos , Íntrons , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Phycomyces/enzimologia , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
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