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Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses.

Becher R, Weihmann F, Deising HB, Wirsel SG - BMC Genomics (2011)

Bottom Line: A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization.The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Agrar- und Ernährungswissenschaften, Naturwissenschaftliche Fakultät III, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann-Str. 3, D-06120 Halle (Saale), Germany.

ABSTRACT

Background: The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.

Results: We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.

Conclusions: The novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

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Molecular phylogeny of F. graminearum ABC transporters. The depicted phylogram was obtained by Neighbor-Joining using MEGA4 software and reflects the relationships between 54 amino acid sequences of putative ABC transporters that were extracted from the F. graminearum proteome. Results from bootstrapping with 1000 replicates are indicated when higher than 30%. Classification for subfamily uses a published nomenclature [26,27] and is indicated by colored boxes. ALDp, adrenoleukodystrophy protein; MDR, multi drug resistance proteins; MRP, multidrug resistance-associated proteins; PDR, pleiotropic drug resistance proteins; NBD, nucleotide-binding domain; TMS, transmembrane spanning domain.
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Figure 2: Molecular phylogeny of F. graminearum ABC transporters. The depicted phylogram was obtained by Neighbor-Joining using MEGA4 software and reflects the relationships between 54 amino acid sequences of putative ABC transporters that were extracted from the F. graminearum proteome. Results from bootstrapping with 1000 replicates are indicated when higher than 30%. Classification for subfamily uses a published nomenclature [26,27] and is indicated by colored boxes. ALDp, adrenoleukodystrophy protein; MDR, multi drug resistance proteins; MRP, multidrug resistance-associated proteins; PDR, pleiotropic drug resistance proteins; NBD, nucleotide-binding domain; TMS, transmembrane spanning domain.

Mentions: Previous studies on azole responses in other fungi had indicated a contribution of ABC transporters to drug tolerance [34]. Using FunCat and Blast2GO annotations, 54 putative ABC transporter proteins were identified in the F. graminearum proteome. We constructed a phylogenetic tree to examine their relationships especially in the context of subfamilies known from ABC transporters [26,27] (Figure 2). Thereby, we classified multidrug resistance proteins (MDR), multidrug resistance-related proteins (MRP), pleiotropic drug resistance proteins (PDR), and adrenoleukodystrophy protein (ALDp)-type ABC transporters, which were represented by 16, 16, 19, and 2 members, respectively (Figure 2). One putative ABC transporter gene, FGSG_08373, did not group with any of these subfamilies established first in the Saccharomycotina. Its domain organization corresponded to MDR-type proteins, but it had highest similarity to PDR-type proteins from C. albicans and S. cerevisiae. Typically, ABC transporters comprise two transmembrane and two nucleotide-binding domains. However, for the MDR- and PDR-types, half-sized proteins with only one copy of the domains are also known. In F. graminearum, six half-size MDR and three PDR-proteins exist (Figure 2). Two sequences (FGSG_08027 and FGSG_09611) seemed to miss parts of the corresponding proteins putatively as a result of erroneous ORF predictions.


Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses.

Becher R, Weihmann F, Deising HB, Wirsel SG - BMC Genomics (2011)

Molecular phylogeny of F. graminearum ABC transporters. The depicted phylogram was obtained by Neighbor-Joining using MEGA4 software and reflects the relationships between 54 amino acid sequences of putative ABC transporters that were extracted from the F. graminearum proteome. Results from bootstrapping with 1000 replicates are indicated when higher than 30%. Classification for subfamily uses a published nomenclature [26,27] and is indicated by colored boxes. ALDp, adrenoleukodystrophy protein; MDR, multi drug resistance proteins; MRP, multidrug resistance-associated proteins; PDR, pleiotropic drug resistance proteins; NBD, nucleotide-binding domain; TMS, transmembrane spanning domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037902&req=5

Figure 2: Molecular phylogeny of F. graminearum ABC transporters. The depicted phylogram was obtained by Neighbor-Joining using MEGA4 software and reflects the relationships between 54 amino acid sequences of putative ABC transporters that were extracted from the F. graminearum proteome. Results from bootstrapping with 1000 replicates are indicated when higher than 30%. Classification for subfamily uses a published nomenclature [26,27] and is indicated by colored boxes. ALDp, adrenoleukodystrophy protein; MDR, multi drug resistance proteins; MRP, multidrug resistance-associated proteins; PDR, pleiotropic drug resistance proteins; NBD, nucleotide-binding domain; TMS, transmembrane spanning domain.
Mentions: Previous studies on azole responses in other fungi had indicated a contribution of ABC transporters to drug tolerance [34]. Using FunCat and Blast2GO annotations, 54 putative ABC transporter proteins were identified in the F. graminearum proteome. We constructed a phylogenetic tree to examine their relationships especially in the context of subfamilies known from ABC transporters [26,27] (Figure 2). Thereby, we classified multidrug resistance proteins (MDR), multidrug resistance-related proteins (MRP), pleiotropic drug resistance proteins (PDR), and adrenoleukodystrophy protein (ALDp)-type ABC transporters, which were represented by 16, 16, 19, and 2 members, respectively (Figure 2). One putative ABC transporter gene, FGSG_08373, did not group with any of these subfamilies established first in the Saccharomycotina. Its domain organization corresponded to MDR-type proteins, but it had highest similarity to PDR-type proteins from C. albicans and S. cerevisiae. Typically, ABC transporters comprise two transmembrane and two nucleotide-binding domains. However, for the MDR- and PDR-types, half-sized proteins with only one copy of the domains are also known. In F. graminearum, six half-size MDR and three PDR-proteins exist (Figure 2). Two sequences (FGSG_08027 and FGSG_09611) seemed to miss parts of the corresponding proteins putatively as a result of erroneous ORF predictions.

Bottom Line: A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization.The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Agrar- und Ernährungswissenschaften, Naturwissenschaftliche Fakultät III, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann-Str. 3, D-06120 Halle (Saale), Germany.

ABSTRACT

Background: The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.

Results: We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.

Conclusions: The novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

Show MeSH
Related in: MedlinePlus