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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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Comparison of differentially expressed genes exposed to different stress conditions. Venn diagrams illustrate results from comparative analyses of gene sets found differentially expressed by azole treatment, carbon, or nitrogen starvation [10]. A. Comparisons of gene sets showing significantly increased transcript levels. Nine of 596 genes found in the current tebuconazole study did not match a probe set on the Affymetrix GeneChip. B. Comparisons of genes sets showing significantly decreased transcript levels. Three out of 462 genes did not match a probe set on the Affymetrix GeneChip.
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Figure 3: Comparison of differentially expressed genes exposed to different stress conditions. Venn diagrams illustrate results from comparative analyses of gene sets found differentially expressed by azole treatment, carbon, or nitrogen starvation [10]. A. Comparisons of gene sets showing significantly increased transcript levels. Nine of 596 genes found in the current tebuconazole study did not match a probe set on the Affymetrix GeneChip. B. Comparisons of genes sets showing significantly decreased transcript levels. Three out of 462 genes did not match a probe set on the Affymetrix GeneChip.

Mentions: We compared our microarray data to those of a study that was previously published for F. graminearum [10] to assess how many of the genes responding to tebuconazole would also respond to other types of stress. We applied the same statistical procedures described above to the published microarray data that examined two nutritional stress conditions, i.e. carbon and nitrogen starvation. Based on the q value plots obtained by the EDGE software we used q < 0.01 as the cut-off for statistical significance for these data sets. In addition, transcript levels in the published data needed to be changed at least twofold (log2 FC ≥ 1 and ≤ -1) by the respective treatment to be considered significant. Resulting gene sets were used for subtractions (Figure 3). Transcript abundances from the majority, i.e. 457 (76.7%) of the above described 596 genes were specifically increased after the fungicide treatment whereas 139 (23.3%) genes responded also in at least one of the other two stress conditions (Figure 3 A; Additional file 6). Some of those belonged to one of the three groups of proteins discussed above. FGSG_02346 (homolog of the S. cerevisiae gene Erg24), FGSG_09381 (homolog of S.c. Erg9) and FGSG_10424 (homolog of S.c. Erg19) belonged to the ergosterol biosynthesis pathway, whereas for ABC transporters and transcription factors we correspondingly identified three genes (FGSG_02786, FGSG_06771, FGSG_08312), and five genes (FGSG_01293, FGSG_01307, FGSG_01936, FGSG_06324, FGSG_10639), respectively. However, functional enrichment analysis with the remaining 457 genes whose transcripts were specifically increased by tebuconazole still indicated a statistically significant enrichment of genes involved in steroid metabolism (data not shown).


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)

Comparison of differentially expressed genes exposed to different stress conditions. Venn diagrams illustrate results from comparative analyses of gene sets found differentially expressed by azole treatment, carbon, or nitrogen starvation [10]. A. Comparisons of gene sets showing significantly increased transcript levels. Nine of 596 genes found in the current tebuconazole study did not match a probe set on the Affymetrix GeneChip. B. Comparisons of genes sets showing significantly decreased transcript levels. Three out of 462 genes did not match a probe set on the Affymetrix GeneChip.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparison of differentially expressed genes exposed to different stress conditions. Venn diagrams illustrate results from comparative analyses of gene sets found differentially expressed by azole treatment, carbon, or nitrogen starvation [10]. A. Comparisons of gene sets showing significantly increased transcript levels. Nine of 596 genes found in the current tebuconazole study did not match a probe set on the Affymetrix GeneChip. B. Comparisons of genes sets showing significantly decreased transcript levels. Three out of 462 genes did not match a probe set on the Affymetrix GeneChip.
Mentions: We compared our microarray data to those of a study that was previously published for F. graminearum [10] to assess how many of the genes responding to tebuconazole would also respond to other types of stress. We applied the same statistical procedures described above to the published microarray data that examined two nutritional stress conditions, i.e. carbon and nitrogen starvation. Based on the q value plots obtained by the EDGE software we used q < 0.01 as the cut-off for statistical significance for these data sets. In addition, transcript levels in the published data needed to be changed at least twofold (log2 FC ≥ 1 and ≤ -1) by the respective treatment to be considered significant. Resulting gene sets were used for subtractions (Figure 3). Transcript abundances from the majority, i.e. 457 (76.7%) of the above described 596 genes were specifically increased after the fungicide treatment whereas 139 (23.3%) genes responded also in at least one of the other two stress conditions (Figure 3 A; Additional file 6). Some of those belonged to one of the three groups of proteins discussed above. FGSG_02346 (homolog of the S. cerevisiae gene Erg24), FGSG_09381 (homolog of S.c. Erg9) and FGSG_10424 (homolog of S.c. Erg19) belonged to the ergosterol biosynthesis pathway, whereas for ABC transporters and transcription factors we correspondingly identified three genes (FGSG_02786, FGSG_06771, FGSG_08312), and five genes (FGSG_01293, FGSG_01307, FGSG_01936, FGSG_06324, FGSG_10639), respectively. However, functional enrichment analysis with the remaining 457 genes whose transcripts were specifically increased by tebuconazole still indicated a statistically significant enrichment of genes involved in steroid metabolism (data not shown).

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