Identification of ABC transporter genes of Fusarium graminearum with roles in azole tolerance and/or virulence

Abstract

Fusarium graminearum is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of F. graminearum representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of FgABC3 and FgABC4 belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of F. graminearum revealed that, irrespective of their chemotypes, deletion mutants of FgABC1 (ABC-C subfamily group V) and FgABC3 were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that FgABC3 may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, FgABC1 may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of F. graminearum.

Figure 1. Sensitivity to SBI class I fungicides.

For each deletion, colonial areas of two transformants of each genetic background were assessed on PDA amended with the indicated concentration of fungicides. Error bars represent SD. An asterisk indicates a significant difference between a mutant and the wild type. (A) PH-1 background, strains tested: 1) PH-1, 2) ΔFgABC1-PH.2, 3) ΔFgABC1-PH.3, 4) ΔFgABC2-PH.2, 5) ΔFgABC2-PH.7,6) ΔFgABC3-PH.1, 7) ΔFgABC3-PH.5, 8) ΔFgABC4-PH.4, 9) ΔFgABC4-PH.15; (B) NRRL 13383 background, strains tested: 1) NRRL 13383, 2) ΔFgABC1-NRRL.4, 3) ΔFgABC1-NRRL.7, 4) ΔFgABC2-NRRL.5, 5) ΔFgABC2-NRRL.8, 6) ΔFgABC3-NRRL.2, 7) ΔFgABC3-NRRL.8, 8) ΔFgABC4-NRRL.2, 9) ΔFgABC4-NRRL.3.

Figure 2. Transcript levels determined by RT-qPCR.

For each deletion, one transformant of the NRRL 13383 background was analysed. Columns show calculated initial fluorescence after normalisation with three reference genes. The analysed gene is indicated in the upper left corner of a given box. For each strain, RNA preparations were assayed originating from cultures amended with 5# indicate significant differences between the fungicide treatment and the control. Within each treatment, an asterisk indicates a significant difference between a mutant and the wild type.

Figure 3. Virulence on wheat heads.

For each deletion, symptom development of two transformants of each genetic background is compared to the respective wild type strain for up to 14-inoculated wheat heads. Error bars represent SE. A) PH-1 background, B) NRRL 13383 background.

Figure 4. Symptoms on infected cereals.

Photos show symptoms occurring at the end of the monitoring. One representative example is provided for each genotype. Mocks were treated with 0.02% Tween 20. A) PH-1 background, B) NRRL 13383 background.

Figure 5. Virulence on maize stems.

For each deletion, two transformants of each genetic background are compared to the respective wild type strain. Columns give symptomatic areas in maize stems that were harvested at 14) PH-1 background, B) NRRL 13383 background.

Figure 6. Virulence on barley heads.

For each deletion, two transformants of each genetic background are compared to the respective wild type strain. Columns show the percentage of symptomatic spikelets in spray-inoculated barley heads at 14 dpi. Error bars represent SE. A) PH-1 background, B) NRRL 13383 background.

Figure 7. Mycotoxin production.

For each deletion, one transformant of each genetic background is compared to the respective wild type strain. Columns show the concentration of a given substance in rice cultures determined by HPLC-MS/MS. Error bars represent SD. An asterisk indicates a significant difference between a mutant and the wild type. A) to C) PH-1 background, D) to F) NRRL 13383 background. A), E) DON; B) 15ADON; C), F) ZEN; D) NIV.