The Drug H+ Antiporter FgQdr2 Is Essential for Multiple Drug Resistance, Ion Homeostasis, and Pathogenicity in Fusarium graminearum

Abstract

Increased emergence of drug resistance and DON pollution pose a severe problem in Fusarium head blight (FHB) control. While the H⁺ antiporter (DHA) family plays crucial roles in drug resistance, the characterization of DHA transporters has not been systematically studied in pathogenetic fungi. In this study, a systematic gene deletion analysis of all putative DHA transporter genes was carried out in Fusarium graminearum, and one DHA1 transporter FgQdr2 was found to be involved in multiple drug resistance, ion homeostasis, and virulence. Further exploration showed that FgQdr2 is mainly localized in the cell membrane; its expression under normal growth conditions is comparatively low, but sufficient for the regulation of drug efflux. Additionally, investigation of its physiological substrates demonstrated that FgQdr2 is essential for the transport of K⁺, Na⁺, Cu²⁺, and the regulation of the membrane proton gradient. For its roles in the FHB disease cycle, FgQdr2 is associated with fungal infection via regulating the biosynthesis of virulence factor deoxynivalenol (DON), the scavenging of the phytoalexin, as well as both asexual and sexual reproduction in F. graminearum. Overall, the results of this study reveal the crucial roles of FgQdr2 in multiple drug resistance, ion homeostasis, and pathogenicity, which advance the understanding of the DHA transporters in pathogenetic fungi.

Keywords: Fusarium head blight; H+ antiporter (DHA); multiple drug resistance; virulence.

Figure 1

FgQdr2 is required for multiple drug resistance and mycelial growth in F. graminearum. (A) Sensitivity of each strain to 9 commonly used fungicides. ΔFGSG_09737 (ΔFgQdr2) exhibited increased sensitivity to prochloraz, tebuconazole, fludioxonil, and iprodione, and ΔFGSG_08749 showed increased sensitivity to prochloraz. Each strain was cultured on PDA, and PDA amended with 0.1 ppm prochloraz, 0.25 ppm tebuconazole, 5 ppm tridemorph, 20 ppm Spiroxamine, 0.5 ppm carbendazim, 0.1 ppm fludioxonil, 10 ppm iprodione, 0.2 ppm phenamacril, and 0.02 ppm pydiflumetofen. The images were taken after 3 d incubation at 25 °C, and the mycelial growth inhibition was calculated for each strain. (B) The mycelial growth inhibition of each strain under the above drug treatments. (C) Colony morphology of PH-1 and 34 DHA transporter deletion mutants. Colony morphology was observed after culture on PDA for 3 d. (D) Colony diameters of PH-1 and 34 DHA transporter deletion mutants. For (B,D), mean and standard deviation were estimated with data from three independent biological replicates (n = 3). Different letters indicate significant differences based on ANOVA analysis followed by Turkey’s multiple comparisons test (p < 0.05).

Figure 2

The overexpression, mutation of phosphorylation sites, and deletion of the LOOP region did not alter the regulation of FgQdr2 in drug resistance. (A) Overexpressed FgQdr2 is localized along the plasma membrane. Bar = 10 μm. (B,C) Sensitivity of each strain to fungicide stress, colony morphology (B) and mycelial growth inhibition (C) were photographed and calculated after 3 d incubation at 25 °C, respectively. (D) The quantitative reverse transcription PCR (qRT-PCR) revealed that the transcription level of FgQDR2 was not upregulated under the above drug treatments. The stain was first cultured in yeast extract peptone dextrose (YEPD) for 24 h, then transferred to YEPD supplied with (treated) or without (non-treated) the corresponding fungicide. The FgQDR2 transcription was determined after another 1 h culture. The expression level of FgQDR2 in the non-treated strain was referred to as 1, and the FgACTIN gene was used as the internal control for normalization. (E) Analysis of the phosphorylation sites within the LOOP substrate binding region of Qdr2. The phosphorylation site of FgQdr2 was predicted using NetPhos 3.1 Server. The LOOP region of FgQdr2 was predicted using the Swiss-Model Repository program. For (C,D), mean and standard deviation were estimated with data from three independent biological replicates (n = 3). Different letters indicate significant differences based on ANOVA analysis, followed by Turkey’s multiple comparisons test (p < 0.05).

Figure 3

FgQdr2 is involved in the transport of potassium, sodium, and copper, as well as the regulation of membrane proton gradient. (A) Sensitivity of each strain to the corresponding stress agents. ΔFgQdr2 exhibited increased sensitivity to K⁺ Na⁺, Cu²⁺ osmotic stresses, and displayed similar sensitivity to the divalent cation chelator ethylene diamine tetra-acetic acid (EDTA), compared with the wild type. Each strain was cultured on MM, with or without the corresponding stress agents. The images were taken after 3 d incubation at 25 °C, and the mycelial growth inhibition was calculated for each strain. (B) The mycelial growth inhibition of each strain under the above drug treatments. (C) The qRT-PCR revealed that the transcription level of FgQDR2 was increased under K⁺ and H⁺ inductions. The stain was first cultured in yeast extract peptone dextrose (YEPD) for 24 h, then transferred to YEPD supplied with (treated) or without (non-treated) the corresponding ion stress agents. The expression level of FgQDR2 in the non-treated strain was referred to as 1, and the FgACTIN gene was used as the internal control for normalization. For (B,C), mean and standard deviation were estimated with data from three independent biological replicates (n = 3). Different letters indicate significant differences based on ANOVA analysis, followed by Turkey’s multiple comparisons test (p < 0.05).

Figure 4

FgQdr2 is important for the virulence of F. graminearum. Virulence of each strain on wheat heads (A), coleoptiles (B), and corn silks (C). The mutant ΔFgQdr2 showed significantly reduced virulence on the above wheat tissues. The mock treatments indicate the corresponding host tissues inoculated with sterile water as a negative control.

Figure 5

FgQdr2 is required for DON production and phytoalexin extrusion in F. graminearum. (A) DON content determination of each strain. ΔFgQdr2 showed significant deficiency in DON biosynthesis. (B) The gene expression level of FgTRI genes in each strain. The expression level of each FgTRI in PH-1 in TBI with putrescine was referred to as 1, and the FgACTIN gene was used as the internal control for normalization. (C) Sensitivity of each strain to phytoalexin BOA and 2-AP. ΔFgQdr2 showed increased sensitivity towards the two phytoalexins shown above. (D) Hyphal growth inhibition rate under with the above phytoalexins. In (A–C), mean and standard deviation were estimated with data from three independent biological replicates (n = 3). Different letters indicate significant differences based on ANOVA analysis, followed by Turkey’s multiple comparisons test (p < 0.05).

Figure 6

The deletion of FgQDR2 resulted in defects in sexual and asexual reproduction in F. graminearum. (A) The spores of the wild-type and ΔFgQdr2 were stained with calcium fluorescent white (CFW). Bar = 10 μm. (B) The number of conidia produced by each strain. (C) The number of spore septa in each strain. A total of 200 conidia were examined for each strain. (D) The spore length in each strain. A total of 200 conidia were examined for each strain. (E) Deletion of FgQDR2 leads to sexual reproduction defects in F. graminearum.