Abstract
Ubiquitinated biosynthetic and surface proteins destined for degradation are sorted into the lysosome/vacuole via the multivesicular body sorting pathway, which depends on the function of ESCRT machinery. Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases for wheat and barley worldwide. To better understand the role of ESCRT machinery in F. graminearum, we investigated the function of ESCRT-III accessory proteins FgVps60, FgDid2 and FgIst1 in this study. FgVps60–GFP, FgDid2–GFP and FgIst1–GFP are localized to punctate structures adjacent to the vacuolar membrane except for FgIst1–GFP that is also found in the nucleus. Then, the gene deletion mutants ΔFgvps60, ΔFgdid2 and ΔFgist1 displayed defective growth to a different extent. ΔFgvps60 and ΔFgdid2 but not ΔFgist1 also showed significant reduction in hydrophobicity on cell surface, conidiation, perithecia production and virulence. Interestingly, ΔFgist1 mutant produced a significantly higher level of DON while showing a minor reduction in pathogenicity. Microscopic analyses revealed that FgVps60 but not FgIst1 and FgDid2 is necessary for endocytosis. Moreover, spontaneous mutations were identified in the ΔFgvps60 mutant that partially rescued its defects in growth and conidiation. Taken together, we conclude that ESCRT-III accessory proteins play critical roles in growth, reproduction and plant infection in F. graminearum.
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Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 31601583 & 31670142), the FAFU international cooperation project (KXB16010A), the FAFU Outstanding Youth Scientific Research Project (xjq201625), the Graduate School Research Foundation (No. 324-1122yb030) and the Plant Protection College Foundation of Fujian Agriculture and Forestry University.
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Supplementary material 1 Identification of homologous ESCRT-III accessory proteins in F. graminearum. A Comparative analysis of orthologs of ESCRT-III accessory proteins between S. cerevisiae and F. graminearum. Alignment of amino acid sequences of B F. graminearum FgVps60 and S. cerevisiae ScVps60, C FgIst1 and ScIst1, D FgDid2 and ScDid2. (TIF 4533 KB)
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Supplementary material 2 Deletion of FgVPS60, FgIST1 and FgDID2 gene from F. graminearum. FgVPS60, FgIST1 and FgDID2 are marked large white arrows while hph gene is marked with black arrow. Primers used in this study is marked with small and black arrows. A Targeted gene deletion of F. graminearum FgVPS60. Southern blots of KpnI-digested genomic DNA from wild-type strain PH-1 and putative ΔFgvps60 transformants were hybridized with probe marked in left panel. A 2.2-kb band was detected in the wild type, while 2.7-kb bands were detected in the two independent ΔFgvps60 mutants (right panel). B Targeted gene deletion of F. graminearum FgIST1. Southern blots of XhoI-digested genomic DNA from wild-type strain PH-1 and putative ΔFgist1 transformants were hybridized with probe marked in left panel. A 2.9-kb band was detected in the wild type, while 4.2-kb bands were detected in the two independent ΔFgist1 mutants (right panel). C Targeted gene deletion of F. graminearum FgDID2. Southern blots of XhoI-digested genomic DNA from wild-type strain PH-1 and putative ΔFgdid2 transformants were hybridized with probe marked in left panel. A 1.0-kb band was detected in the wild type, while 2.7-kb bands were detected in the two independent ΔFgdid2 mutants (right panel). (TIF 529 KB)
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Supplementary material 3 Relative expression level of hydrophobin gene FGSG_03960 in PH-1 and the ESCRT-III accessory proteins deletion mutants. RNA samples were extracted from mycelia of the strains after inoculation in liquid CM medium for 3 days. The expression level in wild-type PH-1 was arbitrarily set to 1. Line bars in each column represent the standard deviation (SD) from three independent experiments. *p<0.05; **p<0.01; ***p<0.001; Student’s T test was used. (TIF 194 KB)
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Supplementary material 4 Ascospores release of PH-1, ΔFgist1 and ΔFgist1-com strains. Fascicles of asci observed by a microscope after squeezing the perithecia 14 days after self-cross of each strain on carrot plates. Scale bar = 50 μm. (TIF 2545 KB)
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Xie, Q., Chen, A., Zhang, Y. et al. ESCRT-III accessory proteins regulate fungal development and plant infection in Fusarium graminearum. Curr Genet 65, 1041–1055 (2019). https://doi.org/10.1007/s00294-019-00949-z
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DOI: https://doi.org/10.1007/s00294-019-00949-z