Mechanism of Action of the Benzimidazole Fungicide onFusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule

Abstract

Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. β2-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α1-, α2-, and β2-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant β2-tubulin and reduced the maximum fluorescence intensity of 2 μM β2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α1-/β2-tubulins and α2-/β2-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α1-/β2-tubulins and α2-/β2-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α1-/β2-tubulins, whereas it had less effect on α2-/β2-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and β/α subunit interface on β/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.

Analysis of β-tubulin-carbendazim interaction reveals that binding site for MBC fungicides does not include residues involved in fungicide resistance

“…Therefore, resistance to these fungicides in phytopathogenic fungi has rapidly evolved through the selection of mutants in the target gene such as the typical mutations E198A or F200Y 30 . As previously described in Venturia nashicola , causal agent of pear scab, and grey mold Botrytis cinerea , was described that point mutations in β-tubulin genes reduced carbendazim biding to tubulin-like proteins 31 . However, the mechanism by which those mutations affect fungicide binding is unknown.…”

Cytological Effects of Herbicidal and Fungicidal Pesticides on Root Apex Meristem Cells of Triticum Aestivum

“…The most pronounced effect on the formation of lagging chromosomes was exerted by the fungicide TEB and the herbicide MET. This violation is associated with anomalies in the formation of the spindle of the division, since pesticides can disrupt the polymerization of tubulin [51,52]. It's known, that the herbicide butachlor act as potent spindle inhibitor.…”

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat

“…tritici genes and at comparing HIGS efficacy in wheat and barley powdery mildew. The B. graminis β2-tub gene, an essential housekeeping gene and a canonical fungicide target (Zhou et al, 2016;Vela-Corcía et al, 2018), was selected as target gene for this assay. There is no β1-tub gene in B. graminis, indicating that transcriptional knockdown of the single-copy β2-tub gene is highly relevant for assessing the impact of HIGS on fungal virulence.…”