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Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens

Nature Plants volume 5, pages 1167–1176 (2019)Cite this article

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Abstract

Soil-borne fungal pathogens that cause crop disease are major threats to agriculture worldwide. Here, we identified a secretory polysaccharide deacetylase (PDA1) from the soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, that facilitates virulence through direct deacetylation of chitin oligomers whose N-acetyl group contributes to host lysine motif (LysM)-containing receptor perception for ligand-triggered immunity. Polysaccharide deacetylases are widely present in fungi, bacteria, insects and marine invertebrates and have been reported to possess diverse functions in developmental processes rather than virulence. A phylogenetics analysis of more than 5,000 fungal proteins with conserved polysaccharide deacetylase domains showed that the V. dahliae PDA1-containing subtree includes a large number of proteins from the Verticillium genus as well as the Fusarium genus, another group of characterized soil-borne fungal pathogens, suggesting that soil-borne fungal pathogens have adopted chitin deacetylation as a major virulence strategy. We showed that a Fusarium PDA1 is required for virulence in cotton plants. This study reveals a substantial virulence function role of polysaccharide deacetylases in pathogenic fungi and demonstrates a subtle mechanism whereby deacetylation of chitin oligomers converts them to ligand-inactive chitosan, representing a common strategy of preventing chitin-triggered host immunity by soil-borne fungal pathogens.

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Fig. 1: VdPDA1 is a secretory protein and is required for full virulence in cotton plants.
Fig. 2: VdPDA1 is a chitin deacetylase.
Fig. 3: VdPDA1-mediated deacetylation of chitin oligomers impedes chitin-triggered immunity in cotton plants.
Fig. 4: Phylogenetic analysis of fungal polysaccharide deacetylase family proteins and virulence function analysis of FovPDA1.

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All data generated or analysed during this study are included in this published article and its Supplementary Information.

References

  1. De Coninck, B., Timmermans, P., Vos, C., Cammue, B. P. A. & Kazan, K. What lies beneath: belowground defense strategies in plants. Trends Plant Sci. 20, 91–101 (2015).

    PubMed  Google Scholar 

  2. Möller, M. & Stukenbrock, E. H. Evolution and genome architecture in fungal plant pathogens. Nat. Rev. Microbiol. 15, 756–771 (2017).

    PubMed  Google Scholar 

  3. Gao, F. et al. A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity. PLoS ONE 5, e15319 (2010).

    PubMed  PubMed Central  Google Scholar 

  4. Klosterman, S. J. et al. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog. 7, e1002137 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sesma, A. & Osbourn, A. E. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature 431, 582–586 (2004).

    CAS  PubMed  Google Scholar 

  6. Zhao, Y.-L., Zhou, T.-T. & Guo, H.-S. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS–Ca2+ signaling is required for plant infection by Verticillium dahliae. PLoS Pathog. 12, e1005793 (2016).

    PubMed  PubMed Central  Google Scholar 

  7. Zhou, T.-T., Zhao, Y.-L. & Guo, H.-S. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae. PLoS Pathog. 13, e1006275 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. Mosquera, G., Giraldo, M. C., Khang, C. H., Coughlan, S. & Valent, B. Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1–4 as Biotrophy-associated secreted proteins in rice blast disease. Plant Cell 21, 1273–1290 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang, L. et al. The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections. New Phytologist 215, 368–381 (2017).

    CAS  PubMed  Google Scholar 

  10. Qin, J. et al. The plant-specific transcription factors CBP60g and SARD1 are targeted by a Verticillium secretory protein VdSCP41 to modulate immunity. eLife 7, e34902 (2018).

    Google Scholar 

  11. Zhang, T. et al. Host-induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahliae. Mol. Plant 9, 939–942 (2016).

    CAS  PubMed  Google Scholar 

  12. Zhang, T. et al. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat. Plants 2, 16153 (2016).

    CAS  PubMed  Google Scholar 

  13. Akamatsu, A. et al. An OsCEBiP/OsCERK1–OsRacGEF1–OsRac1 module is an essential early component of chitin-induced rice immunity. Cell Host Microbe 13, 465–476 (2013).

    CAS  PubMed  Google Scholar 

  14. Hayafune, M. et al. Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization. Proc. Natl Acad. Sci. USA 111, E404–E413 (2014).

    CAS  PubMed  Google Scholar 

  15. Liu, T. et al. Chitin-induced dimerization activates a plant immune receptor. Science 336, 1160–1164 (2012).

    CAS  PubMed  Google Scholar 

  16. Cao, Y. et al. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife 3, e03766 (2014).

    PubMed Central  Google Scholar 

  17. van den Burg, H. A., Harrison, S. J., Joosten, M. H., Vervoort, J. & de Wit, P. J. Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Mol. Plant Microbe Interact. 19, 1420–1430 (2006).

    PubMed  Google Scholar 

  18. Marshall, R. et al. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat. Plant Physiol. 156, 756–769 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. de Jonge, R. et al. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329, 953–955 (2010).

    PubMed  Google Scholar 

  20. Mentlak, T. A. et al. Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease. Plant Cell 24, 322–335 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Sanchez-Vallet, A. et al. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. eLife 2, e00790 (2013).

    PubMed  PubMed Central  Google Scholar 

  22. Takahara, H. et al. Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity. New Phytologist 211, 1323–1337 (2016).

    CAS  PubMed  Google Scholar 

  23. Fiorin, G. L. et al. Suppression of plant immunity by fungal chitinase-like effectors. Curr. Biol. 28, 3023–3030 (2018).

    CAS  PubMed  Google Scholar 

  24. Yang, C. et al. Binding of the Magnaporthe oryzae chitinase MoChia1 by a rice tetratricopeptide repeat protein allows free chitin to trigger immune responses. Plant Cell 31, 172–188 (2019).

    CAS  PubMed  Google Scholar 

  25. Han, L. B. et al. The cotton apoplastic protein CRR1 stabilizes chitinase 28 to facilitate defense against the fungal pathogen Verticillium dahliae. Plant Cell 31, 520–536 (2019).

    CAS  PubMed  Google Scholar 

  26. Kombrink, A. et al. Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts. Mol. Plant Pathol. 18, 596–608 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhao, Y., Park, R. D. & Muzzarelli, R. A. Chitin deacetylases: properties and applications. Mar. Drugs 8, 24–46 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhou, B. J., Jia, P. S., Gao, F. & Guo, H. S. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae. Mol. Plant Microbe Interact. 25, 964–975 (2012).

    CAS  PubMed  Google Scholar 

  29. Hayakawa, Y., Ishikawa, E., Shoji, J. Y., Nakano, H. & Kitamoto, K. Septum-directed secretion in the filamentous fungus Aspergillus oryzae. Mol. Microbiol. 81, 40–55 (2011).

    CAS  PubMed  Google Scholar 

  30. Qi, M. et al. A small cysteine-rich protein from the Asian soybean rust fungus, Phakopsora pachyrhizi, suppresses plant immunity. PLoS Pathog. 12, e1005827 (2016).

    PubMed  PubMed Central  Google Scholar 

  31. Blair, D. E. et al. Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum. Biochemistry 45, 9416–9426 (2006).

    CAS  PubMed  Google Scholar 

  32. Kaku, H. et al. Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc. Natl Acad. Sci. USA 103, 11086–11091 (2006).

    CAS  PubMed  Google Scholar 

  33. Böhm, H., Albert, I., Fan, L., Reinhard, A. & Nürnberger, T. Immune receptor complexes at the plant cell surface. Curr. Opin. Plant Biol. 20, 47–54 (2014).

    PubMed  Google Scholar 

  34. Bozsoki, Z. et al. Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception. Proc. Natl Acad. Sci. USA 114, E8118–E8127 (2017).

    CAS  PubMed  Google Scholar 

  35. Shimizu, T. et al. Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J. 64, 204–214 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Geoghegan, I. A. & Gurr, S. J. Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae. Cell Microbiol. 19, e12743 (2017).

    PubMed Central  Google Scholar 

  37. de Jonge, R. et al. Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing. Proc. Natl Acad. Sci. USA 109, 5110–5115 (2012).

    PubMed  Google Scholar 

  38. Ulloa, M. et al. Inheritance and QTL mapping of Fusarium wilt race 4 resistance in cotton. Theor. Appl. Genet. 126, 1405–1418 (2013).

    CAS  PubMed  Google Scholar 

  39. Hurlburt, N. K., Chen, L. H., Stergiopoulos, I. & Fisher, A. J. Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein. PLoS Pathog. 14, e1007263 (2018).

    PubMed  PubMed Central  Google Scholar 

  40. Sanchez-Vallet, A., Mesters, J. R. & Thomma, B. P. The battle for chitin recognition in plant–microbe interactions. FEMS Microbiol. Rev. 39, 171–183 (2015).

    CAS  PubMed  Google Scholar 

  41. Geoghegan, I., Steinberg, G. & Gurr, S. The role of the fungal cell wall in the infection of plants. Trends Microbiol. 25, 957–967 (2017).

    CAS  PubMed  Google Scholar 

  42. Upadhya, R. et al. Cryptococcus neoformans Cda1 and its chitin deacetylase activity are required for fungal pathogenesis. mBio 9, e02087-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  43. El Gueddari, N. E., Rauchhaus, U., Moerschbacher, B. M. & Deising, H. B. Developmentally regulated conversion of surface-exposed chitin to chitosan in cell walls of plant pathogenic fungi. New Phytologist 156, 103–112 (2002).

    CAS  Google Scholar 

  44. Cord-Landwehr, S., Melcher, R. L., Kolkenbrock, S. & Moerschbacher, B. M. A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells. Sci. Rep. 6, 38018 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Berbee, M. L. The phylogeny of plant and animal pathogens in the Ascomycota. Physiological Mol. Plant Pathol. 59, 165–187 (2001).

    CAS  Google Scholar 

  46. Mauricio, U. et al. Inheritance and QTL mapping of Fusarium wilt race 4 resistance in cotton. Theor. Appl. Genet. 126, 1405–1418 (2013).

    Google Scholar 

  47. Wang, S., Xing, H., Hua, C., Guo, H. S. & Zhang, J. An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption method for Verticillium dahliae. Phytopathology 106, 645–652 (2016).

    CAS  PubMed  Google Scholar 

  48. van Esse, H. P., Bolton, M. D., Stergiopoulos, I., de Wit, P. J. G. M. & Thomma, B. P. H. J. The Chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor. Mol. Plant Microbe Interaction 20, 1092–1101 (2007).

    Google Scholar 

  49. Katoh, K. & Standley, D. M. A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics 32, 1933–1942 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank X. Zhang for assistance with bioinformatics analysis, J. Wang for assistance with co-immunoprecipitation, E. Wang for chitooctaose (A8), B. Thomma for the JR2 strain and C. Hua for helpful discussion. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11040500 to H.-S.G.), National Natural Science Foundation of China (31730078 to H.-S.G., 31460451 to F.G and 31560494 to J.H.).

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Author notes

  1. These authors contributed equally: Feng Gao, Bo-Sen Zhang.

Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Feng Gao, Bo-Sen Zhang, Jian-Hua Zhao, Sheng Wang, Jie Zhang & Hui-Shan Guo

  2. College of Agriculture, Shihezi University, Shihezi, China

    Feng Gao, Bo-Sen Zhang, Jia-Feng Huang & Pei-Song Jia

  3. Key Laboratory at Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Shihezi, China

    Jia-Feng Huang & Pei-Song Jia

  4. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jian-Min Zhou

  5. CAS Center for Excellence in Biotic Interactions, University of the Chinese Academy of Sciences, Beijing, China

    Hui-Shan Guo

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Contributions

H.-S.G. and F.G. designed experiments. F.G. and B.-S.Z. performed experiments. J.-H.Z. performed phylogenetic analysis. J.-F.H. P.-S.J. and S.W. provided technical support. H.-S.G., F.G., J.Z. and J.-M.Z. analysed data and discussed the results. H.-S.G. and F.G. wrote the paper.

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Correspondence to Hui-Shan Guo.

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Extended data

Extended Data Fig. 1 Identification of the virulence gene VdPDA1 of V. 1ahlia.

a, Identification of a pathogenicity-deficient T-DNA mutant. The T-DNA mutant had markedly reduced virulence in cotton plants. Disease symptoms of cotton plants infected with V592 and T-DNA mutant strains at 30 days postincubation (dpi). The disease grades were evaluated with three replicates of 36 plants for each inoculum. Asterisk indicates the significant difference of the disease grades between wild-type V592 and Vd∆pda1 mutant infection (P< 0.05; t-test, two-sided, mean ± SD, mean values are shown). Disease grades on cotton leaves were classified into five levels of severity of disease symptoms during fungal invasion: 0 = no visible wilting or yellowing symptoms, 1 = one or two cotyledons wilted or dropped off, 2 and 3 = one or two true leaves wilted or dropped off, 4 = all of the leaves dropped off or the whole plant died. b, The T-DNA mutant shows normal colony morphology and microsclerotium formation similar to those of the wild-type V592 strain on potato dextrose agar (PDA) plates. c, The T-DNA mutant contains a single copy of the T-DNA integrated in the promoter of VdPDA identified by the TAIL-PCR approach 3 and DNA gel blotting. Genomic DNA extracted from the T-DNA mutant strain was digested by the restriction enzymes as indicated. A T-DNA-specific sequence was used as a probe. d, VdPDA1 is highly expressed in mycelia in wild type V592 and greatly reduced in those of the T-DNA mutant. The expression pattern of VdPDA1 was detected by RNA gel blotting using a 32P- labeled VdPDA1-specific sequence as a probe. Methylene blue- stained rRNA is shown as loading controls. Three times the experiments in b,c,d were repeated independently with similar results.

Source data

Extended Data Fig. 2 The genomic structure of VdPDA1 and the conserved PDA motifs.

a, The protein primary structure of VdPDA1. VdPDA1 encodes a 19-amino acid signal peptide at the N-terminus and a polysaccharide deacetylase (PDA) homology domain that contains five conserved motifs that form the active enzyme site. b, Alignment of the five conserved motifs and the chitin deacetylase (CDA)-typical metal-binding amino acids (arrows in red) of the PDA domain of V. 2ahlia, Fusarium oxysporum f. sp. Vasinfectum and the characterized PDAs or CDAs from other fungal species. GenBank accession nos.:V.dahliae PDA1 (this work, NW_009276934), F. oxysporum f. sp.vasinfectum PDA(JH657946), Mucor rouxii CDA (Z19109), Flammulina velutipes FV-PDA (AB250372), Cryptococcus neoformans PDA (AJ414580), Schizophyllum commune CDA (AF271216), Saccharomyces cerevisiae CDA (AY557948) and C. lindemuthianum CDA (AY633657).

Extended Data Fig. 3 Detection of the VdΔpda1 knockout mutant.

a, Schematic description of the homologous recombination event involved in the targeted replacement of VdPDA1 with the TrpC::hpt resistance cassette for generation of the Vd∆pda1 mutant as previously described 47. Conformation of the knockout mutant for VdPDA1 by DNA gel blotting using a VdPDA1-specific DNA probe. Three times these experiments were repeated independently with similar results. b, The disease grades of the wild-type V592, Vd∆pda1 and Vd∆pda1/VdPDA1 complemented strains were evaluated. Asterisk indicates the significant difference of the disease grades between wild-type V592 and Vd∆pda1 mutant infection (mean ± SD; mean values are shown, n=108 biologically independent plants, P<0.05; t-test two-sided) as described in Supplementary Fig. 1b. Disease symptoms of cotton plants are shown in Fig. 1c.

Source data

Extended Data Fig. 4 VdPDA1 is not required for V. 3ahlia initial infection in cotton and does not function as an elicitor molecule.

a, The Vd∆pda1 mutant penetrated the cellophane membrane. The Vd∆pls1 mutant that is incapable of forming an infectious structure 3 was used as a negative control. Photographs show fungus grown on a cellophane membrane laid on minimal medium (M0) (above, at 7 dpi) and after removal of the cellophane membrane (below). b, Hyphae recovered from stem sections of different inoculated cotton plants at 7 days postculture. c, Reduced fungal biomass in Vd∆pda1-infected cotton plants compared with V592-infected ones at 15 dpi. The values were quantitative real time (qPCR) of fungal internal transcribed spacer DNA relative to cotton GhUBQ7 (GeneBank: DQ116441) DNA. (mean ± SD; n=9 biologically independent samples, mean values are shown, *P< 0.05, one- way ANOVA). d, Expression of VdPDA1, but not VdPDA3, was rapidly induced at early time points during cotton infection by RT-qPCR. The value of VdPDA1 mRNA relative to elf1-α at 12 hours postinfection (hpi) was arbitrarity to 1. Different letters (capital letters for VdPDA1 and lower-case letters for VdPDA3) indicate significant differences (mean ± SD; n =9 biologically independent samples, mean values are shown, P< 0.05, one-way ANOVA). e, VdPDA1 does not function as an elicitor molecule. VdPDA1-Myc was transiently expressed in Nicotiana benthamiana and cotton leaves. VdNLP1-Myc 4 and Myc-GFP was used as controls. Neither necrosis nor cotton resistance-related genes 4 were induced by VdPDA1-Myc. Photographs were taken 3 days postagroinfiltration (dpa). Western blot conformed protein production. RT-qPCR was performed with total RNA extracted at 2 dpa. The value of Myc-GFP relative to cotton β-tubulin was arbitrarity to 1. (n =9 biologically independent plants, mean ± SD, mean values are shown, *P<0.05, one-way ANOVA). f, VdPDA1 is a secretory protein. The VdPDA1-GFP-expressing V. dahliae strain was incubated on M0 medium overlaid with cellophane for induction of infectious structure and hyphal neck, where the VdSep5 ring was organized for delivery of secretory proteins 5. VdPDA1-GFP was observed at the hyphal septa (arrowheads) and surrounding the VdSep5-RFP ring observed with CLSM microscopy. Numbers indicate the distance from the center of the hyphopodium where the first column (0 μm) shows the beginning of a continuous z series. The VdSep5-RFP ring is at the hyphopodium pore (3 μm). Images were taken at 8 dpi. Bar = 5 μm. Three times the experiments in a,b,f were repeated independently with similar results.

Source data

Extended Data Fig. 5 VdPDA1 could not protect fungal hyphae from cotton ChiB- mediated hydrolysis.

a, Cotton apoplastic crude extract is capable of digesting chitin oligomers. Chitin oligomerchitohexaose with six GlcNAc moieties (A6) was incubated with crude apoplastic extract from cotton seedlings for different times as indicated and validated by MALDI-TOF-MS analysis. The data show that cotton apoplastic crude extract is capable of digesting A6 into the short-chain chitin oligomerschitotetraose (A4) and chitotriose (A3), indicating that cotton apoplastic crude extract contains active basic chitinases (ChiB). At least three times these experiments were repeated independently with similar results. b, VdPDA1 could not protect against ChiB-mediated fungal hyphae hydrolysis. V. 4ahlia hyphae were pretreated with H2O or 15 mM VdPDA1-StrepII followed by addition of cotton crude extract (ChiB). Mg3LysM, known to protect against ChiB18, was used as a positive control. Micrographs of V. 4ahlia were taken 24 hours after incubation with ChiB. Images are representative of three independent experiments. Scale bars=90 μm.

Extended Data Fig. 6 Detection of VdPDA1 chitin deacetylation activity under different conditions.

a, VdPDA1-mediated deacetylation ofchitopentaose (A5) and chitotetraose (A4) by MALDI-TOF-MS spectrum analysis. Purified VdPDA1-StrepII (15 μM) incubated with 1 mM A5 or A4 at 37 °C in 50 mM Tris-HCl buffer (pH 8.0) for 30 min, using 10 mg/ml 2,5-dihydroxybenzoic acid in 2:1 acetonitrile water as the matrix. All of the labeled peaks are single charged ions of chitin or chitosanoligomers labeled with 3- aminoquinoline (3-AMQ) on the reducing end as Na+ or K+ adducts. A: N-acetyl-D-glucosamine; D: D-glucosamine. The completely deacetylated products D5 and D4 were detected. b, VdPDA1 has higher deacetylase activities on longer-chain chitin oligomers. Chitin oligomers with 2 to 6 GlcNAc moieties (A2 to A6) (with final concentration 1 mg/ml) were treated with 15 μM VdPDA1 for 30 min at 37 °C. The deacetylase activity was calculated based on the amount of released acetate. Different letters indicate significant differences at P< 0.05 (mean ± SD; n =9 biologically independent samples, one-way ANOVA). c, VdPDA1 is incapable of deacetylation of insoluble colloid chitin. Colloid chitin were treated with VdPDA1-StrepII (15 μM) for 10 to 30 min (upper panel); deacetylated D8 to D6 was detected when the reaction was also mixed with cotton apoplastic extract (ChiB) and incubated for 10 min. d, VdPDA1 is an extracellular chitin deacetylase. Purified VdPDA1-StrepII was incubated with A6 at different pH values, and the amount of released acetate (mean ± SD; n = 9 biologically independent samples) was measured. The highest activity of VdPDA1 is in the alkaline range, implicating an extracellular chitin deacetylase. Three times the experiments in a,c were repeated independently with similar results.

Source data

Extended Data Fig. 7 VdPDA1-mediated deacetylation of chitin oligomers impedes chitin-triggered chitin receptor oligomerization and dimerization in Arabidopsis.

a, VdPDA1-mediated deacetylation of A6 impeded A6- induced heterodimerization between AtLYK5 and AtCERK1. Flag-tagged AtCERK1 and HA-tagged AtLYK5 under each native promoter were coexpressed in wild-type Arabidopsis protoplasts. Protoplasts with (+) or without (-) the treatment with 1 μM A6, or D6, A≤2D≥4, or A≤4D≥2 (products of A6 incubated with VdPDA1 at different conditions as shown in Fig. 2) or VdPDA1 as a negative control for 15 min were harvested. Coimmunoprecipitation was performed using an anti-HA antibody and subjected to immunoblot analysis with an anti-Flag or an anti-HA antibody as indicated. b, VdPDA1- mediated deacetylation of chitin impeded chitin-induced homodimerization and phosphorylation of AtCERK1. HA- tagged AtCERK1 and Flag-tagged AtCERK1 under the AtCERK1 native promoter were coexpressed in wild-type Arabidopsis protoplasts. Protoplasts with (+) or without (-) the treatment with chitin or chitin preincubated with VdPDA1 for 30 min were harvested. Incubation with VdPDA1 was used as a negative control. Coimmunoprecipitation was performed using an anti-HA antibody and subjected to immunoblot analysis with an anti-Flag or an anti-HA antibody as indicated. Chitin induced a band shift of the AtCERK1 protein, indicating its phosphorylation. Three times the experiments in a,b were repeated independently with similar results. c, AtCERK1 is responsible for the reduced virulence in the absence of VdPDA1. Disease symptoms of wild-type (Col-0) and cerk1 mutant Arabidopsis plants infected with V592, mutant or complemented strains at 17 dpi. The disease grades were evaluated with three replicates of 32 plants for each inoculum. Asterisk indicates the significant difference of the disease grades between wild-type V592 and Vd∆pda1 mutant infection (mean ± SD, mean values are shown; P<0.05; t-test two-sided). d, Vd∆pda1 mutant infection induced a greater defense response in Col-0 plants compare with V592 infection. RT- qPCR with total RNA extracted from Col-0 plant roots inoculated with V592 or Vd∆pda1 mutant at different time points as indicted; adenosine tRNA methylthiotransferase gene (AT4G33380) was used as an internal control. The levels of the genes in mock inoculation plant roots were set to 1. Asterisks indicate significant differences (n = 9 biologically independent samples, mean ± SD, mean values are shown, P<0.05, one-way ANOVA).

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Extended Data Fig. 8 VdPDA1 homologue genes, VdPDA2 and VdPDA3, are not required for virulence.

a, Most fungi containing CE4_CICDA_like domain proteins in the VdPDA1-clade (Fig. 4a) belong to the order Hypocreales (Sordariomycetes, Ascomycota). The fungal phylogenetic tree in the rectangle is derived from Grigoriev et al.,(2014). The numbers in brackets represent the numbers of fungal species in the VdPDA1-clade. b, Conformation of the knockout mutation for VdPDA1, VdPDA2 and VdPDA3 by DNA gel blotting using VdPDA-specific DNA probes. Vd∆pda1, Vd∆pda2 and Vd∆pda3 were generated by using homologous recombination methods as described in Supplementary Fig. 1e. c, Vd∆pda2 and Vd∆pda3 but not Vd∆pda1 reduced the production of spores. The numbers of conidia were counted at various time points in comparison with wild-type strain V592 after each fungal strain was cultured on Czapek medium for 5 days. Different letters indicate significant differences at P< 0.05 (mean ± SD; n =9 biologically independent samples. One-way ANOVA followed by Tukey’s multiple comparisons test). d, Vd∆pda1, Vd∆pda2 and Vd∆pda3 knockout mutants showed normal colony morphology and microsclerotium formation on potato dextrose agar (PDA) plates. The Vd∆pda1 knockout mutant showed slightly denser aerial hyphae growth than wild-type V592. Vd∆pda2 and Vd∆pda3 mutant strains exhibited virulence similar to that of wild-type V592 during infection in cotton plants. I Localization of VdPDA2-GFP fusion protein in spores, especially in germinating spores (arrow). Three times the experiments in b.d.e were repeated independently with similar results.

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Extended Data Fig. 9 VdPDA1 of the V. 7ahlia tomato isolate JR2 strain (VdPDA1JR2) is required forvirulence in tomato plants.

a, Conformation of VdPDA1JR2 gene knockout in the VdΔpda1JR2 mutant strain. The VdΔpda1JR2 mutant was created by using homologous recombination methods as described in Supplementary Fig. 3a. Wild-type JR2 and VdΔpda1JR2 mutant strains were grown on potato dextrose agar (PDA) plates. The VdΔpda1JR2 knockout mutant shows normal colony morphology and microsclerotium formation. Genomic DNA extracted from JR2 and VdΔpda1JR2 knockout mutant strains was examined by DNA gel blotting. A 32P-labeled VdPDA1JR2 or HPT gene-specific DNA probe was used. b, The VdΔpda1JR2 knockout mutant clearly reduced virulence in tomato plants. Wild-type JR2 infection shows typical disease symptoms in tomato plants with plant stunting and leaf wilting. Photographs were taken at 25 days postincubation (dpi). Three times these experiments in a,b were repeated independently with similar results.

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Extended Data Fig. 10 Fusarium oxysporum f. sp. Vasinfectum PDA1 (FovPDA1) is a chitin deacetylase.

a, Purification of Fov-produced FovPDA1-StrepII. b, FovPDA1-mediated deacetylation ofchitohexaose (A6). Purified FovPDA1-StrepII was incubated with 1 mM chitohexaose (A6) for 30 min, and the completely deacetylated product D6 was detected. c, The FovΔpda1 knockout mutant shows normal colony morphology similar to that of wild-type Fov. FovΔpda1 knockout mutants were examined by DNA gel blotting. A 32P-labeled FovPDA1 or HPT gene-specific DNA probe was used. Three times these experiments in a,b,c were repeated independently with similar results.

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Gao, F., Zhang, BS., Zhao, JH. et al. Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens. Nat. Plants 5, 1167–1176 (2019). https://doi.org/10.1038/s41477-019-0527-4

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