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The ASK1 gene regulates the sensitivity of Fusarium graminearum to carbendazim, conidiation and sexual production by combining with β2-tubulin

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Abstract

β-tubulin, a component of microtubules, is involved in a wide variety of roles in cell shape, motility, intracellular trafficking and regulating intracellular metabolism. It has been an important fungicide target to control plant pathogen, for example, Fusarium. However, the regulation of fungicide sensitivity by β-tubulin-interacting proteins is still unclear. Here, ASK1 was identified as a β-tubulin interacting protein. The ASK1 regulated the sensitivity of Fusarium to carbendazim (a benzimidazole carbamate fungicide), and multiple cellular processes, such as chromatin separation, conidiation and sexual production. Further, we found the point mutations at 50th and 198th of β2-tubulin which caused carbendazim resistance decreased the binding between β2-tubulin and ASK1, resulting in the deactivation of ASK1. ASK1, on the other hand, competed with carbendazim to bind to β2-tubulin. The point mutation F167Y in β2-tubulin broke the intermolecular H-bonds and salt bridges between β2-tubulin and ASK1, which reduced the competitive effect of ASK1 to carbendazim and resulted in the similar carbendazim sensitivities in F167Y-ΔASK1 and F167Y. These findings have powerful implications for efforts to understand the interaction among β2-tubulin, its interacting proteins and fungicide, as well as to discover and develop new fungicide against Fusarium.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31730072).

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Authors and Affiliations

  1. Key Laboratory of Pesticides, College of Plant Protection, Nanjing Agricultural University, Jiangsu Province, Nanjing, 210095, China

    Xiu-Shi Song, Xue-Mei Xiao, Kai-Xin Gu, Jing Gao, Shao-Chen Ding & Ming-Guo Zhou

  2. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, 210095, China

    Xiu-Shi Song & Ming-Guo Zhou

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  1. Xiu-Shi Song
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Correspondence to Ming-Guo Zhou.

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Electronic supplementary material

Table S1 Primers and sequences used in this study.

Fig. S1 PCR and Southern blot analyses of transformants. a A schematic diagram for homologous recombination between the replacement vectors and the ASK1 gene locus. i) Homologous recombination occurred between the replacement vectors and the target genes, resulting in the generation of ΔASK1, Y50C-ΔASK1, F167Y-ΔASK1 and E198K-ΔASK1 deletion mutant strains and ASK1C complementation strain. ii) The point mutant strains Y50C, F167Y and E198K were constructed by a replacement of β2-tubulin with its point mutant genes. b PCR analyzed three gene knock-out mutants and one complementation strains. Fragments of 955 bp (with primers FgASK1U-F/FgASK1U-R) and 840 bp (with primers FgASK1D-F/FgASK1D-R) were amplified from ΔASK1 mutant and 1179 bp (with primers FgASK1-F/FgASK1-R) was amplified from wild-type and ASK1C strains. c Southern blot analysis of the wild-type strain, mutant strains and reconstituted strains. The genomes of PH-1, ΔASK1, ASK1C, Y50C-ΔASK1, F167Y-ΔASK1 and E198K-ΔASK1 were digested with SpeI. The size of DNA fragment was shown on the side of the picture (WT, 3564 bp; ASK1-deletion mutants, 5655 bp; ASK1C, 4243 bp).

Fig. S2 Growth and virulence assay of ASK1 mutants and different strains. a A 5-mm mycelial plug was transferred from the margin of a 3-day-old colony to the center of a PDA plate and kept at 25 °C for 3 days. The experiment was repeated three times with the same patterns. b Virulence assay of wild-type, null mutants and complementation strains. Ten microliters of macroconidia (5 × 105 spores ml−1) was injected into the single floret of wheat cultivar Huaimai33 and kept moist in a plastic bag for 3 d. Infected spikelets were scored 14 d post inoculation at which point photographs were taken. Percentages of infected spikelets were calculated as means ± SD of 30 spikes inoculated with each strain. Different letters represent a significant difference at P < 0.05.

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Song, XS., Xiao, XM., Gu, KX. et al. The ASK1 gene regulates the sensitivity of Fusarium graminearum to carbendazim, conidiation and sexual production by combining with β2-tubulin. Curr Genet 67, 165–176 (2021). https://doi.org/10.1007/s00294-020-01120-9

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