Dataset Information

Overexpression of the 14alpha-demethylase target gene (CYP51) mediates fungicide resistance in Blumeriella jaapii.

ABSTRACT: Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14alpha-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMI(R)) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMI(S)) isolates. Analysis of sequences upstream of CYP51 in 59 DMI(R) isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMI(S) isolates examined.

SUBMITTER: Ma Z

PROVIDER: S-EPMC1449055 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Overexpression of the 14alpha-demethylase target gene (CYP51) mediates fungicide resistance in Blumeriella jaapii.

Ma Zhonghua Z Proffer Tyre J TJ Jacobs Janette L JL Sundin George W GW

Applied and environmental microbiology 20060401 4

Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14alpha-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMI(R)) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared ...[more]

PMID: 16597960

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Project description:Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use.

Project description:Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14alpha-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 mug of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position -117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.

Dataset Information

Overexpression of the 14alpha-demethylase target gene (CYP51) mediates fungicide resistance in Blumeriella jaapii.

Publications

Overexpression of the 14alpha-demethylase target gene (CYP51) mediates fungicide resistance in Blumeriella jaapii.

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