F240 of β₂-Tubulin Explains why Fusarium graminearum is Less Sensitive to Carbendazim than Botrytis cinerea

Abstract

β-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for β-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of β-tubulin amino acid sequences showed that position 240 of β-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fgβ-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC) value of MBC against the FgβF240L mutant of F. graminearum is 0.047 μg/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC value of MBC against the BcβL"240"F (actually position 232) mutant of Botrytis cinerea was 0.44 μg/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 μg/ml), microtubules were clearly visible in Fgβ-enhanced green fluorescent protein (EGFP) but not in FgβF240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fgβ-tubulin and MBC and increased the binding affinity of Fgβ-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgβ-tubulin and MBC.

Sexual specific functions of Tub1 beta‐tubulins require stage‐specific RNA processing and expression in Fusarium graminearum

“…Therefore, it is not clear what confers functional difference between TUB1 and TUB2 during sexual reproduction and why Tub1 betatubulins have the stage-specific function during ascosporogenesis. Interestingly, various mutations conferring resistance to carbendazim fungicides targeting at fungal beta-tubulins have been identified in the TUB2 gene but not in the TUB1 gene in field isolates (Chen et al, 2009;Begaye et al, 2011;Zhu et al, 2018). Like in other fungi, resistant mutations in TUB2 of F. graminearum often occur at amino acid residues F167, E198 and F200 that form a carbendazim-binding pocket in beta-tubulins (Qiu et al, 2011).…”

Genome-Wide Identification and Characterization of Fusarium graminearum-Responsive lncRNAs in Triticum aestivum

“…T. aestivum seeds were grown in a light incubator at 25 °C for two and a half days, under a photoperiod of 16 h light and 8 h dark, then the tips of the coleoptile were cut off quickly with aseptic scissors for F. graminearum inoculation. A 3-acetyldeoxy-nivalenol (3ADON) chemotype strain F. graminearum 2021 [ 30 , 31 ], isolated from a field experiencing an FHB epidemic in Zhejiang Province, China, was used as the parental strain in this study. F. graminearum 2021 were cultivated on potato dextrose agar (PDA) medium for 3–4 days, and then, high-activity hyphae at the edge of the colony were collected and shook in 3% sterile mung bean broth at 25 °C at 175 rpm for 3 days.…”

The Dis1/Stu2/XMAP215 Family Gene FgStu2 Is Involved in Vegetative Growth, Morphology, Sexual and Asexual Reproduction, Pathogenicity and DON Production of Fusarium graminearum

“…The growth rate was tested as previously described (Zhu et al, 2018). In brief, 5 mm mycelial plugs taken from the margin of a 3-day-old colony of wild-type strain PH1 and FgStu2 silent mutants were transferred into PDA plates with or without 30 µM ZEA and incubated at 25 • C. After three days cultivation, the colony diameters of wild-type strain and FgStu2-Si mutants were significantly different.…”

“…Radial growth was used to test the sensitivity of the wild-type strain PH-1 and FgStu2 mutants to MBC as previously described (Zhu et al, 2018). Colony diameter was measured after 3 days for PH1; after 7 days for FgStu2-Si mutants.…”

“…In order to assess the ability of asexual reproduction of FgStu2-Si mutants, we observed the conidial morphology and tested the number of conidial production of FgStu2-Si mutant as previously described (Zhu et al, 2018). For observe the septa and nuclei, fresh spores were stained with calcofluor white (CFW) and 4 ,6-diamidino-2-phenylindole (DAPI) and examined with a LEICA TCS SP8 confocal laser-scanning microscope (LEICA).…”