Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis

Abstract

Plant-pathogenic fungi hijack their hosts by secreting effector proteins. Effectors serve to suppress plant immune responses and modulate the host metabolism to benefit the pathogen. Smut fungi are biotrophic pathogens that also parasitize important cereals, including maize1. Symptom development is usually restricted to the plant inflorescences. Ustilago maydis is an exception in its ability to cause tumours in both inflorescences and leaves of maize, and in inducing anthocyanin biosynthesis through the secreted Tin2 effector2,3. How the unique lifestyle of U. maydis has evolved remains to be elucidated. Here we show that Tin2 in U. maydis has been neofunctionalized. We functionally compared Tin2 effectors of U. maydis and the related smut Sporisorium reilianum, which results in symptoms only in the inflorescences of maize and fails to induce anthocyanin. We show that Tin2 effectors from both fungi target distinct paralogues of a maize protein kinase, leading to stabilization and inhibition, respectively. An ancestral Tin2 effector functionally replaced the virulence function of S. reilianum Tin2 but failed to induce anthocyanin, and was unable to substitute for Tin2 in U. maydis. This shows that Tin2 in U. maydis has acquired a specialized function, probably connected to the distinct pathogenic lifestyle of this fungus.

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Fig. 1: S. reilianum encodes a Tin2-related virulence-promoting effector.
Fig. 2: Expression of U. maydis tin2 in S. reilianum and vice versa.
Fig. 3: Interaction of SrTin2 and UmTin2 with ZmTTK1, ZmTTK2 and ZmTTK3.
Fig. 4: Resurrected ancestral Tin2 protein functions like SrTin2.

Data availability

U. maydis and S. reilianum genes and encoding protein sequences are available at NCBI under the following accession numbers: Umtin2 (UMAG_05302), XP_011392015; Umppi (UMAG_03726), XP_011390187; Srtin2 (Sr10057), CBQ70078; Srppi (Sr11196), CBQ68333; Patin2 (PSAN_05779), XP_014654584; Pbtin2 (PSEUBRA_SCAF21g03481), XP_016292238; Ssctin2 (SSCI_00084), CDS81902; Srrtin2 (Srs_10057), SJX65729. For Z. mays genes, sequence data can be found at MaizeSequence.org (http://www.maizesequence.org) under the following accession numbers: ZmGAPDH, GRMZM2G046804; ZmTTK1, GRMZM2G448633; ZmTTK2, GRMZM2G068192; ZmTTK3, GRMZM2G088409. The two kinases used as the outgroup in Supplementary Fig. 4a are GRMZM2G015073 and GRMZM2G181002. Remaining data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We are very grateful to S. Reissmann and E. Stukenbrock for critical reading and constructive comments on the manuscript, to the entire group in Marburg for helpful discussions, to K. Münch for help with plant infections and to D.K. Gupta and R. Sharma for bioinformatics support. Our work was supported by generous funds from the Max Planck Society and by the LOEWE initiative of the government of Hesse in the framework of the Centre for Translational Biodiversity Genomics (TBG).

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S.T. designed the concept of the study and performed experiments. G.S. contributed to the bioinformatic analysis including protein sequence alignment, phylogenetic analysis and ancestral sequence reconstruction. N.R. and F.F. generated strains of U. maydis and S. reilianum and performed virulence assays. M.T. provided information on tin2 orthologues in smut fungi. R.K. directed the project. S.T. and R.K. wrote the manuscript with input from all co-authors.

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Correspondence to Regine Kahmann.

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Supplementary Figures 1–17, Supplementary Tables 1–4.

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Tanaka, S., Schweizer, G., Rössel, N. et al. Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis. Nat Microbiol 4, 251–257 (2019). https://doi.org/10.1038/s41564-018-0304-6

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