ABSTRACT
We investigated the mechanism of resistance to demethylation inhibitors (DMI) in
Penicillium digitatum
by isolating the
CYP51
gene, which encodes the target enzyme (P450
14DM
) of DMI, from ...three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of
CYP51
in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced
CYP51
, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased
CYP51
expression. We also found that if the number of copies of the repeat was reduced to two, resistance and
CYP51
expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances
CYP51
expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.
Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and ...acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (delta pmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus
We have cloned a novel ABC transporter gene PMR5 from the phytopathogenic fungus Penicillium digitatum by RT-PCR using degenerate primers. The deduced amino acid sequence of PMR5 showed 37% identity ...to PMR1 from the same fungus, 71% identity to AtrB from Aspergillus nidulans, and 65% identity to BcatrB from Botrytis cinerea. Disruption mutants for PMR5 were generated in two independent P. digitatum strains and their phenotypes were characterized. These mutants displayed increased sensitivity to thiabendazole (a benzimidazole), benomyl (a benzimidazole), dithianon (a quinone), resveratrol (the phytoalexin of grape), and camptothecin (an alkaloid). Delta pmr1 disruption mutants were previously reported to show resistance to demethylation inhibitors (DMIs). These mutants were found also to display increased sensitivity to phloretin (the phytoanticipin of apples), camptothecin and oligomycin (an antibiotic). Transcription of PMR1 and PMR5 was strongly induced in response to several toxicants, including DMIs that specifically induced PMR1. In contrast, dithianon and resveratrol specifically induced PMR5 transcription. These findings indicate that expression of the two ABC transporter genes is regulated differently, and that they have complementary roles in multidrug resistance, with each having different substrate-specificities.
Template preparation is important in reverse-transcription polymerase chain reaction (RT-PCR)-based detection methods. A TissueLyser with tungsten carbide beads was used for simultaneous processing ...of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for RT reaction. A sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. It was also demonstrated that the combination use of random primers and oligo dT primer in an RT reaction was efficient for simultaneous cDNA synthesis of viral and viroid RNAs in plant extracts. These template preparation methods were used for the amplification of Grapevine leafroll-associated virus-1,-2, and -3; Grapevine virus A and B; Grapevine rupestris stem pitting-associated virus; Grapevine fleck virus; and Grapevine fanleaf virus. All these viruses tested in this study were reliably detected up to a 10
3-fold or higher dilution of the original extract. Besides, Hop stunt viroid and Grapevine yellow speckle viroid 1 were well amplified in the same manner as the template preparation and following PCR for virus detection. These methods would contribute to cost-effective testing of a large number of samples through the year and help to detect viral pathogens in grapevine.
Colletotrichum acutatum, the fungus causing anthracnose disease of various fruits and crops, has inherent resistance to benomyl. The mechanism underlying its resistance to benomyl remains unclear. We ...generated a benomyl-sensitive mutant CAT7-150 by transformation-mediated insertional mutagenesis. Subsequently, a
CaBEN1 gene was isolated from CAT7-150 by plasmid rescue and sequenced.
CaBEN1 encodes a protein of 818 amino acids containing a leucine zipper motif, and the amino acid sequence has significant similarity to hypothetical proteins found in
Gibberella zeae and other fungi. Disruption and complementation of the
CaBEN1 gene demonstrated that CaBEN1 was necessary for resistance to benzimidazole fungicides, but was not essential for mycelial growth. Northern analysis suggests that CaBEN1 has an important role in transcriptional activation of
CaTUB1 in response to benomyl. Benomyl resistance of
CaBEN1-disrupted transformant was recovered by overexpression of
CaTUB1. Our results clearly demonstrate that the benomyl resistance is due to enhanced expression of
CaTUB1, which is controlled by CaBEN1.
Elsinoë ampelina, the causal organism of grapevine anthracnose, can be easily grown in culture, yet its sporulation is poor and unstable in culture. In this study, we sought the optimum conditions ...for a simple method to stably generate conidia. We first examined the optimum period of incubation for young colonies grown on potato dextrose agar (PDA) in water. The resultant number of conidia showed a logarithmic increase, which slowed at about 8 to 10 h. This suggests that 8 to 10 h of preculture would provide a sufficient number of conidia under the culture conditions used. A high negative correlation between colony density on PDA and the number of resultant conidia existed: colonies grown at >2.5 colonies per cm2 produced few or no conidia, whereas those grown at <1.0 colony per cm2 stably produced as many as 2.9 x 10(6) conidia. The optimum condition for preculture was to incubate colonies grown for 6 days on PDA at a density of <1.0 colony per cm2. The conidia obtained by our method were pathogenic on the grape cultivar Rizamat.
In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes ...to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.
PDF
