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The endocytic cargo adaptor complex is required for cell-wall integrity via interacting with the sensor FgWsc2B in Fusarium graminearum

  • Luona Xu
  • Minhui Wang
  • Guangfei Tang
  • Zhonghua Ma
  • Wenyong ShaoEmail author
Original Article
  • 123 Downloads

Abstract

AP2 is a heterotetrameric clathrin adaptor complex that owns important roles in vesicle generation and cargo recognition. Cell-wall integrity (CWI) pathway is essential for fungal development, virulence, and adaptation to environment stresses. To date, the relationship between AP2 and CWI is largely unknown in phytopathogenic fungi. In this study, we identified the adaptor complex FgAP2 in Fusarium graminearum. The biological function analysis showed that FgAP2 complex contains FgAP2α, FgAP2β, FgAP2σ, and FgAP2μ, and the subunit FgAP2μ, which is required for hyphal growth, conidiation, CWI, and virulence. Yeast two-hybrid showed that FgAP2μ interacts with the CWI sensor FgWsc2B. Consistently, western blotting analysis revealed that FgWsc2B positively regulates phosphorylation of FgMgv1, the MAP kinase of CWI. Moreover, the FgWsc2B deletion mutant exhibited defects in hyphal growth, virulence, and response to CWI damaging agents. Taken together, our data indicated that FgAP2μ is involved in CWI and virulence via interacting with FgWsc2B in F. graminearum.

Keywords

F. graminearum Adaptor complex FgAP2μ Cell-wall integrity 

Notes

Acknowledgements

This research was supported by the National Key R & D Plan (2017YFC1600904) and China Agriculture Research System (CARS-3-29).

Supplementary material

294_2019_961_MOESM1_ESM.jpg (159 kb)
Figure S1. Identification of AP2 complex FgAP2 inF. graminearum. Phylogenetic analysis of AP2 complex orthologues from F. graminearum (FgAP2α, FgAP2β, FgAP2μ, FgAP2σ) and other fungal species including Magnaporthe oryzae (Mg), Aspergillus nidulans (An), Neurospora crassa (Nc) and Saccharomyces cerevisiae (Sc). The phylogenetic tree was constructed with the computer program MEGA using the neighbour-joining method. The bootstrap values from 1000 replications are indicated on the branches. (JPEG 160 kb)
294_2019_961_MOESM2_ESM.tif (6.4 mb)
Figure S2. Generation and identification of the FgAP2 complex deletion mutant ofF. graminearum. a Gene replacement strategy for FgAP2. The hygromycin resistance cassette (hph) is denoted by the large grey arrow. Primer pairs binding sites are indicated by arrows (see Table S1 for the primer sequences). b Identification of FgAP2 deletion mutants via the PCR method using different particular primer pairs. The 1-2, 3-4, 5-6 and 7-8 was amplified by ID primers of ΔFgAP2μ, ΔFgAP2α, ΔFgAP2β, and ΔFgAP2σ, respectively. c Identification of complemented strains via the PCR method using different particular primer pairs. The 1-3, 4-6, 7-9 and 10-12 was amplified by IN primers of ΔFgAP2μ, ΔFgAP2α, ΔFgAP2β, and ΔFgAP2σ, respectively. (TIFF 6549 kb)
294_2019_961_MOESM3_ESM.tif (3.9 mb)
Figure S3 The sensitivity of PH-1 and ΔFgAP2μ to CFW, Congo red, caffeine and SDS. (TIFF 3994 kb)
294_2019_961_MOESM4_ESM.tif (6.7 mb)
Figure S4 a Colony morphology of the PH-1, ΔFGSG_03574, ΔFGSG_03884 and ΔFGSG_10435 on potato dextrose agar (PDA), minimal medium (MM) and complete medium (CM) after 3 days of incubation at 25 °C. b The colony diameter of the above strains on PDA, CM and MM for 3 days at 25 °C in dark. Bars denote standard errors from three repeated experiments. (TIFF 6868 kb)
294_2019_961_MOESM5_ESM.tif (2.3 mb)
Figure S5 The amounts of DON produced by PH-1 and ΔFgAP2μ in infected wheat kernels after 20 days of inoculation. Line bars in each column denote standard errors of three replicated experiments. (TIFF 2310 kb)
294_2019_961_MOESM6_ESM.xlsx (20 kb)
Table S1 The primers used in this study (XLSX 21 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of BiotechnologyZhejiang UniversityHangzhouChina

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