Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme

Resistance to sterol 14 alpha-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14 alpha-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.     

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

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.     

Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136^th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other.     

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms—multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates—were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.     

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.     

The cytochrome P450 lanosterol 14alpha-demethylase gene is a demethylation inhibitor fungicide resistance determinant in Monilinia fructicola field isolates from Georgia

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.     

The Cytochrome P450 Lanosterol 14α-Demethylase Gene Is a Demethylation Inhibitor Fungicide Resistance Determinant in Monilinia fructicola Field Isolates from Georgia

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 14α-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 μg 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.     

Obtusifoliol 14alpha-demethylase (CYP51) antisense Arabidopsis shows slow growth and long life.

Obtusifoliol 14alpha-demethylase is a plant orthologue of sterol 14alpha-demethylase (CYP51) essential in sterol biosynthesis. We have prepared CYP51 antisense Arabidopsis in order to shed light on the sterol and steroid hormone biosynthesis in plants. Arabidopsis putative CYP51 cDNA (AtCYP51) was obtained from Arabidopsis expressed sequence tag (EST) library and its function was examined in a yeast lanosterol 14alpha-demethylase (Erg11) deficient mutant. A recombinant AtCYP51 protein fused with a yeast Erg11 signal-anchor peptide was able to complement the erg11 mutation, which confirmed AtCYP51 to be a functional sterol 14alpha-demethylase. AtCYP51 was then used to generate transgenic Arabidopsis by transforming with pBI vector harboring AtCYP51 in the antisense direction under CaMV35S promoter. The resulting transgenic plants were decreased in accumulation of AtCYP51 mRNA and increased in the amount of endogenous obtusifoliol. They showed a semidwarf phenotype in the early growth stage and a longer life span than control plants. This newly found phenotype is different from previously characterized brassinosteroid (BR)-deficient campesterol biosynthesis mutants.     

Overexpression of the 14α-Demethylase Target Gene (CYP51) Mediates Fungicide Resistance in Blumeriella jaapii

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 14α-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.     

A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance

Higher plants possess large multigene families encoding secreted class III peroxidase (Prx) proteins. In barley, two Prx cDNAs encoding HvPrx07 and HvPrx08 have been isolated and characterized to some extent with respect to a resistance-mediating function upon attack by the powdery-mildew fungus Blumeria graminis f.sp. hordei ( Bgh ). Here we present evidence for the tissue-specific accumulation of a new Prx mRNA, HvPrx40 , in Bgh -attacked epidermis of barley ( Hordeum vulgare ). The encoded protein is predicted to be secreted into the apoplastic space of epidermal cells due to the absence of a C-terminal extension, which distinguishes it from other Prx proteins reported to accumulate in leaf epidermis. Transient overexpression of HvPrx40 enhanced the resistance of wheat ( Triticum aestivum ) and barley against Blumeria graminis f.sp. tritici (wheat powdery mildew) and Bgh , respectively. These findings were complemented by transient-induced gene silencing showing hypersusceptibility of barley leaf epidermal cells to Bgh . The local accumulation of oxidized 3,3-diaminobenzidine that reflects H₂O₂ production at sites of attempted fungal penetration was not reduced in HvPrx40 -silenced cells, suggesting a role of this peroxidase other than the production of reactive oxygen species.     

Paralogous cyp51 genes in Fusarium graminearum mediate differential sensitivity to sterol demethylation inhibitors

Analysis of the genome sequence of Fusarium graminearum revealed three paralogous cyp51 genes (designated cyp51A, -B, and -C) encoding 14-α demethylases in this fungus. Targeted gene disruption showed that the cyp51A, -B or -C disruption mutants were morphologically indistinguishable from the parent isolate on potato dextrose agar medium, which indicates that none of these genes is essential for mycelial growth. The sensitivity of cyp51A deletion mutants to seven sterol demethylation inhibitor (DMI) fungicides increased significantly compared to the parent strain, while sensitivity of cyp51C deletion mutants increased to some but not all DMIs. No change in DMI sensitivity was observed for cyp51B deletion mutants. The parental phenotypes of cyp51A and cyp51C deletion mutants were completely restored by genetic complementation with the wild-type cyp51A and cyp51C genes, respectively. The sensitivity of F. graminearum isolates increased significantly when subjected in vitro to a mixture of DMI fungicides triadimefon and tebuconazole as compared to the individual components. These results indicate that different DMI fungicides target different CYP51 proteins in F. graminearum and that a mixture of DMI fungicides can result in synergistic effects. Our findings have directly implications on chemical management strategies of plant diseases caused by Fusarium species.     

Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.     

Expression and homology modeling of sterol 14alpha-demethylase from Penicillium digitatum

Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.     

Nested allele-specific PCR primers distinguish genetic groups of Uncinula necator.

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5. 8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.