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The putative deubiquitinating enzyme MoUbp4 is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae

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Abstract

Ubiquitination is a key regulatory mechanism that affects numerous important biological processes, including cellular differentiation and pathogenesis in eukaryotic cells. Attachment of proteins to ubiquitin is reversed by specialized proteases, deubiquitinating enzymes (DUBs), which are essential for precursor processing, maintaining ubiquitin homeostasis and promoting protein degradation by recycling ubiquitins. Here, we report the identification of a novel non-pathogenic T-DNA-tagged mutant T612 of Magnaporthe oryzae with a single insertion in the second exon of MoUBP4, which encodes a putative ubiquitin carboxyl-terminal hydrolase. Targeted gene deletion mutants of MoUBP4 are significantly reduced in mycelial growth, conidiation, and increased in tolerance to SDS and CR (Congo red) cell-wall damage. The ΔMoubp4 mutants are blocked in penetration and invasive growth, which results in the loss of pathogenicity. Many conidia produced by the ΔMoubp4 mutants are unable to form appressoria and mobilization and degradation of glycogen and lipid droplets are significantly delayed. Moreover, immunohybridization analysis revealed that total protein ubiquitination levels of the null mutants were significantly increased, indicating that MoUbp4 functions as a deubiquitination enzyme. Taken together, we conclude that MoUbp4 is required for deubiquitination, infection-related morphogenesis and pathogenicity in M. oryzae.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (Grant Nos. 31570135 and 31770153) to ZW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. Yawei Que and Zhe Xu contribution to this work.

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  1. State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, People’s Republic of China

    Yawei Que, Zhe Xu, Chunyan Wang, Wuyun Lv, Xiaofeng Yue, Lin Xu, Shuai Tang, Han Dai & Zhengyi Wang

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Contributions

Conceived and designed the experiments: YQ, ZX, and ZW. Performed the experiments: YQ, ZX, CW, WL, XY, LX, ST, HD, and ZW. Analyzed the data: YQ, ZX, and ZW. Wrote the paper: YQ, ZX, and ZW.

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Correspondence to Zhengyi Wang.

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294_2019_1049_MOESM1_ESM.docx

Supplementary material 1 Figure S1. Phylogenetic analysis of MoUbp4 with the homologues from other fungal species. Phylogenetic tree of MoUbp4 and its homologs from other fungal species was constructed by the neighbor-joining method using the MEGA version 5.0 program. Numbers at the nodes in the rooted tree represent bootstrapping value on 1000 replications. GenBank accession numbers are presented after fungal species names. The percentage in bracket indicates the sequence identity between MoUbp4 with its homologues in other species. The bar indicates 0.2 distance units (DOCX 15 kb)

294_2019_1049_MOESM2_ESM.tif

Supplementary material 2 Figure S2. Construction of the MoUBP4 gene deletion vector and generation of the targeted gene deletion mutants. (A) Construction of the vector pUBP4-KO and targeted gene replacement of MoUBP4. P, PstI. (B) PCR analysis of the wild-type strain (Guy11), ΔMoubp4 mutants (-1, -17, -24) and an ecotopic transformant (ECT-5) by primer pairs of UBP4yqdx-F/UBP4yqdx-R (top panel) and UBP4yq-F/UBP4yq-F (bottom). (C) Southern blot analysis. Genomic DNAs of different strains were digested with PstI and hybridized with a 998 bp probe amplified with the primers Probe-F and Probe-R (TIFF 27857 kb)

294_2019_1049_MOESM3_ESM.tif

Supplementary material 3 Figure S3. MoUBP4 is not required for the sexual reproduction in M. oryzae. (A) Sexual reproduction assay. The wild-type Guy11 (MAT1-2) and ΔMoubp4 mutants were crossed with a standard tester strain, TH3 (MAT1-1) to allow perithecium production. By 30 days following inoculation, numerous perithecia and ascospores at the junctions of both the crosses of Guy11 × TH3 and ΔMoubp4 × TH3, indicating that MoUBP4 is not required for development of fruiting bodies by M. oryzae. Arrows indicate perithecia. (B) Microscopic observation of ascospore development. Scale bar = 10 μm (TIFF 30307 kb)

294_2019_1049_MOESM4_ESM.tif

Supplementary material 4 Figure S4. Deletion of MoUBP4 does not result in alteration of appressorium turgor pressure in M. oryzae. Statistical analysis of collapsed appressoria (24 h) of the ΔMoubp4 mutants after incubation for 15 min in 1, 2 or 3 M glycerol solutions. Error bars represent standard deviation (TIFF 26663 kb)

294_2019_1049_MOESM5_ESM.tif

Supplementary material 5 Figure S5. MoUbp4 interacts with MoBro1 in M. oryzae. Physical interaction among MoUbp4, MoBro1 and MoSnf7 was detected by Y2H assays. The yeast transformants were assayed for growth on SD/-Ade/-His/-Trp/-Leu medium. Positive control, pGBKT7-53/pGADT7. Negative control, pGBKT7-lam/pGADT7 (TIFF 28334 kb)

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Que, Y., Xu, Z., Wang, C. et al. The putative deubiquitinating enzyme MoUbp4 is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae. Curr Genet 66, 561–576 (2020). https://doi.org/10.1007/s00294-019-01049-8

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