A sterol C-14 reductase encoded by FgERG24B is responsible for the intrinsic resistance of Fusarium graminearum to amine fungicides

Abstract

Fusarium graminearum, the causal agent of wheat head blight, shows intrinsic resistance to amine fungicides. It is commonly accepted that the amines target sterol C-14 reductase and sterol D 8 -D 7 isomerase of ergosterol biosynthesis, encoded by the genes ERG24 and ERG2, respectively. Analysis of the genome sequence of F. graminearum revealed that the fungus contains two paralogous FgERG24 genes (FgERG24A and FgERG24B), which are homologous to the ERG24 of Saccharomyces cerevisiae. In this study, we disrupted FgERG24A and FgERG24B in F. graminearum. Compared to the wild-type strain HN9-1, FgERG24A and FgERG24B deletion mutants did not show recognizable phenotypic changes in mycelial growth on potato dextrose agar or in virulence on wheat heads. HPLC analysis showed that the amount of ergosterol in FgERG24A or FgERG24B deletion mutants was not significantly different from that in the wild-type strain. These results indicate that neither of the two genes is essential for growth, pathogenicity or ergosterol biosynthesis in F. graminearum. FgERG24B deletion mutants exhibited significantly increased sensitivity to amine fungicides, including tridemorph, fenpropidin and spiroxamine, but not to non-amine fungicides. In contrast, FgERG24A deletion mutants did not show changed sensitivity to any amine tested. The resistance of the FgERG24B deletion mutant to amines was restored by genetic complementation of the mutant with wild-type FgERG24B. These results indicate that FgERG24B controls the intrinsic resistance of F. graminearum to amines. The finding of this study provides new insights into amine resistance in filamentous fungi.

Two FgLEU2 Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in Fusarium graminearum

“…We have characterized functions of three cyp51 genes encoding 14-αdemethylases and two ERG24 genes encoding sterol C-14 reductase of the ergosterol biosynthetic pathway, both enzymes are the targets of sterol biosynthesis inhibitors (SBIs). Targeted disruption of individual gene revealed different roles of these genes in mediating sensitivity to SBIs, however, deletion of single gene showed no effects on vegetative development or virulence [23,32]. In A .…”

“…FgLEU2A deletion vector pBS-leu2A-Del was constructed by inserting two flanking sequences of FgLEU2A gene into the left and right sides of the hygromycin resistance gene HPH in the pBS-HPH1 vector [23]. First, a 651-bp upstream flanking sequence fragment of FgLEU2A was amplified by using primer pair A1 + A2 (S1 Table) from the genomic DNA of PH-1, and was inserted into Kpn I- Xho I sites of the pBS-HPH1 vector to generate pBS-leu2A-up.…”

Acetohydroxyacid synthase FgIlv2 and FgIlv6 are involved in BCAA biosynthesis, mycelial and conidial morphogenesis and full virulence in Fusarium graminearum

“…To obtain FgILV2 deletion mutants, plasmid pBS-ilv2-Del was constructed. Two flanking sequences of FgILV2 were amplified from the genomic DNA of the wild-type PH-1 and inserted into the left and right sides of a hygromycin resistance gene ( HPH ) in the multiple cloning site of the plasmid pBS-HPH1 ( Supplementary Figure 2A ) 31 . Using primer pair A1 + A2 ( Table S1 ), a 991-bp upstream flanking sequence fragment of FgILV2 was amplified from PH-1 genomic DNA and inserted into Kpn I- Xho I sites of the pBS-HPH1 vector to generate plasmid pBS-ilv2-up.…”

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high‐osmolarity glycerol signaling pathways

“…A 50 g of aliquot of healthy wheat kernels was sterilized and inoculated with 10 mycelial plugs (5 mm in diameter) of each strain taken from the periphery of a 3-day-old colony of the wild-type PH-1, ΔFgMKK1 and ΔFgRLM1. After incubation at 25°C for 30 days, DON was extracted using a previously described protocol (Mirocha et al, 1998), and the amount of F. graminearum ergosterol in each sample was determined using a high-performance liquid chromatogra-phy (HPLC) method as described previously (Liu et al, 2011). The DON extracts were purified with PuriToxSR DON column TC-T200 (Trilogy Analytical Laboratory, Washington, MO, USA), and the amount of DON and ergosterol in each sample was determined by using a HPLC system Waters 1525 (Waters Co., Massachusetts, USA).…”