In this study, we developed and validated a new 8 × 15 k Agilent microarray, and employed it to analyze gene expression in F. graminearum after treatment with tebuconazole. Our results demonstrate that this multiplex microarray is an effective and versatile tool to detect transcriptome responses at high sensitivity.
Due to its scientific and economic relevance, microarray technology has rapidly evolved into different platforms using short or long oligonucleotides provided by several commercial manufacturers, such as Affymetrix and Agilent Technologies. As studies using different microarray platforms indicated an overall good comparability , the choice of a platform is mainly governed by practical and cost considerations. Agilent's inkjet-like printing technology provides high flexibility for microarray design and allows convenient optimization in follow-up versions. In addition, multiplex formats were developed that carry several microarrays on a single slide, allowing for cost-effective transcription profiling. The chosen Agilent 8 × 15 k format is fully sufficient for covering the entire genome of F. graminearum, thus permitting to simultaneously perform eight independent expression profiling experiments. The employment of the one-color labeling technique recently introduced for the Agilent platform facilitates comparisons across microarrays and between groups of samples without compromising the quality of results .
To address the reliability of the results obtained by the new microarray, we selected 31 genes for determining their transcript levels by an independent experimental approach, i.e. qRT-PCR. For most of these genes, the log2 FC values from the qRT-PCRs were very close to those of the microarray experiments. The comparison of the entire data sets reveals a highly positive correlation (R = 0.95) which corroborates the results of a previous report that also examined these two experimental approaches .
The new 8 × 15 k Agilent microarray was used to analyze gene expression patterns in F. graminearum treated with tebuconazole. We assess the results in the context of microarray experiments that were performed earlier. On the one hand, results that are supported by those previously observed (e.g. in S. cerevisiae) highlight the capability of the new microarray to gain information relevant for better understanding the regulatory networks mediating azole responses. This will be especially helpful for studying novel fungicides whose mode-of-action should not only be analyzed in a model organism like S. cerevisiae but ideally also in the targeted pathogens, e.g. F. graminearum. Consistent with studies in other fungi [44–47] we found that the transcript levels of most of the genes encoding proteins involved in ergosterol-biosynthesis were increased by azole treatment. Furthermore, our functional enrichment analyses using GeneOntology and FunCat demonstrated that the ergosterol pathway ranks at the top among all functional categories containing genes with significantly enhanced transcript abundances. Interestingly, we found that within this category FGSG_04092 (= Cyp51A), which is one of the three genes encoding CYP51 in F. graminearum, exhibited the most significantly increased transcript levels of the entire study. In S. cerevisiae binding of azoles to CYP51 leads to ergosterol depletion, accumulation of a toxic aberrant sterol and compromised membrane rigidity . It is thus possible that a feed-back loop connecting transcriptional regulation of ergosterol biosynthesis and sterol levels exists in F. graminearum, as previously reported for S. cerevisiae . In yeast this link is mediated by UPC2 and ECM11 which regulate transcription of sterol biosynthetic genes by binding to a sterol regulatory element (SRE) in their promotors .
The fact that our microarray study uncovered the transcript levels of genes in the ergosterol biosynthesis pathway as highly significantly increased by tebuconazole treatment demonstrates its utility for fungicide research. The data presented here clearly indicate that microarray analysis can contribute to identify unknown mode-of-actions, as has previously been shown for the fungicide ciclopirox olamine in Candida albicans . Sixty percent of the up-regulated genes were found to encode proteins for iron uptake and metabolism suggesting that treated cells suffered from iron limitation. This was supported by physiological experiments showing that addition of Fe2+ or Fe3+ diminished the detrimental effect of ciclopirox olamine on germ tube formation.
Comparison of our results with published microarray data indicates that about one quarter of the genes up-regulated by tebuconazole and almost half of the down-regulated genes correspondingly responded when F. graminearum was exposed to two unrelated starvation stress conditions. This suggests that many of the genes exhibit a rather unspecific stress response, which may in part originate from defective nutrient supply resulting from the azole-mediated membrane perturbations. On the other hand, the majority of the genes with enhanced transcript abundances responded specifically to fungicide treatment, which is underlined by the fact that all Cyp51 variants only responded in our azole treatment study. Thus, the novel microarray is an excellent tool supporting identification of target genes. Microarray data of course need to be confirmed in subsequent studies, e.g. by targeted gene deletion or RNAi experiments, to address the function of individual genes to attenuate fungicide impact and the mechanisms by which this may be achieved.
Currently it is uncertain whether the CYP51A, B, and C proteins have specific functions in F. graminearum. In Aspergillus fumigatus transformants carrying individual deletions of Cyp51A and Cyp51B remained viable whereas the gene family as a whole was essential . However, in this human pathogen up to now only point mutations in Cyp51A but not in Cyp51B were discovered in clinical isolates and resistant strains generated in vitro [52–54]. This suggests that the functions of these proteins may not completely overlap.
Also ABC transporters were reported to be involved in azole stress responses and in the development of azole tolerance in several fungi as some of them mediate active efflux of fungicides and other xenobiotics . The subfamily most closely associated with drug resistance in the Saccharomycotina is PDR which have up to 10 members in yeasts . In contrast, the genome of F. graminearum harbors 19 PDR-type ABC transporters. An evolutionary expansion of the PDR subfamily was previously noticed for Pezizomycotina . In addition, the numbers of genes encoding MRP and MDR transporters (16 each) are clearly increased in F. graminearum as compared to S. cerevisiae (4 and 7, respectively).
Our microarray data provide valuable insight into the transcriptional responses of ABC transporter genes from F. graminearum, suggesting that several of them may attenuate the effects of tebuconazole action. Similarly, microarrays analyzing azole responses of C. albican s wild type strains showed that the transcript levels of the ABC transporter genes Cdr1 and Cdr2 were increased . Both genes were also up-regulated in in vitro azole-adapted strains and in clinically resistant isolates [57, 58]. Gene deletions in an azole-resistant isolate of C. albicans indicated that CDR1 is essential to mediate azole resistance whereas CDR2 seems to be less important . However, heterologous overexpression of both Cdr1 or Cdr2 conferred increased azole tolerance to S. cerevisiae . Another well-characterized ABC transporter is PDR5 of S. cerevisiae, which is exporting a wide range of xenobiotics, including azoles [60, 61]. Additional reports underline the involvement of ABC transporters in contributing to fungicide resistance also in plant pathogenic Pezizomycotina [62, 63]. Since most of this research has focused on PDR-type transporters, it will be interesting to analyze also the contribution of azole-responsive MDR- and MRP-type proteins in the future.
Transcription factors have been identified as additional elements in conferring fungicide resistance [64, 65]. Since the corresponding transcriptional networks vary to some degree when comparing species in the Saccharomycotina [64, 66] it is important to extend such analyses to Pezizomycotina, including toxigenic plant pathogens like F. graminearum. Among the genes with significantly increased transcript levels, we detected several similarities with genes encoding the transcriptional regulators Upc2 and Tac1 known to be involved in azole responses in S. cerevisiae and/or C. albicans. Also in C. albicans transcript levels of Upc2 and Tac1 increased in response to azole-stress [36, 67]. In both yeasts, UPC2 homologs are activators of genes encoding proteins for ergosterol-biosynthesis and sterol uptake [35, 67–69]. In C. albicans a disruption of Upc2 induced hypersusceptibility and its overexpression increased azole resistance . A G648D exchange in UPC2 created an allele that constitutively up-regulated Erg11 expression and thereby improved azole resistance. Similarly, Tac1 in C. albicans and Pdr1 in S. cerevisiae regulate the PDR-type ABC transporter genes Cdr1, Cdr2, and Pdr5, respectively [36, 71]. In these two yeasts transcription factors mediate the response to azoles by regulating the expression not only of genes for ergosterol biosynthesis but also of efflux transporters. Other putative regulators with significantly increased transcript levels were similar to CRZ1 from S. cerevisiae and C. albicans, a transcription factor that is involved in regulating cell wall integrity . This may reflect a more general response of the fungus in surviving the fungicide treatment. Apart from putative transcription factors with similarity to proteins reported to coordinate azole stress responses in S. cerevisiae and C. albicans we found additional azole-responsive transcription factors in F. graminearum. The response of these factors may reflect differences in transcriptional regulation of azole response that are either specific for F. graminearum or higher taxonomic levels.