Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

Helena Volk, Kristina Marton, Marko Flajsman, Sebastjan Radisek, Hui Tian, Ingo Hein, Črtomir Podlipnik, Bart P. H. J. Thomma, Katarina Kosmelj, Branka Javornik, Sabina Berne (Lead / Corresponding author)

Research output: Contribution to journalArticle

Abstract

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

Original languageEnglish
Pages (from-to)1378-1390
Number of pages13
JournalMolecular Plant-Microbe Interactions
Volume32
Issue number10
Early online date7 May 2019
DOIs
Publication statusPublished - Oct 2019

Fingerprint

Verticillium
Chitinases
Chitin
chitinase
chitin
binding proteins
Immunity
Carrier Proteins
Fungi
immunity
fungi
Humulus
carbohydrate binding
Mycoses
hops
Cell Wall
Carbohydrates
cell walls
circular dichroism spectroscopy
Xylem

Keywords

  • Fungal effectors
  • Fungus–plant interactions
  • Mechanisms of pathogenicity

Cite this

Volk, Helena ; Marton, Kristina ; Flajsman, Marko ; Radisek, Sebastjan ; Tian, Hui ; Hein, Ingo ; Podlipnik, Črtomir ; Thomma, Bart P. H. J. ; Kosmelj, Katarina ; Javornik, Branka ; Berne, Sabina. / Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity. In: Molecular Plant-Microbe Interactions. 2019 ; Vol. 32, No. 10. pp. 1378-1390.
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abstract = "During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.",
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author = "Helena Volk and Kristina Marton and Marko Flajsman and Sebastjan Radisek and Hui Tian and Ingo Hein and Črtomir Podlipnik and Thomma, {Bart P. H. J.} and Katarina Kosmelj and Branka Javornik and Sabina Berne",
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Volk, H, Marton, K, Flajsman, M, Radisek, S, Tian, H, Hein, I, Podlipnik, Č, Thomma, BPHJ, Kosmelj, K, Javornik, B & Berne, S 2019, 'Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity', Molecular Plant-Microbe Interactions, vol. 32, no. 10, pp. 1378-1390. https://doi.org/10.1094/MPMI-03-19-0079-R

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity. / Volk, Helena; Marton, Kristina; Flajsman, Marko; Radisek, Sebastjan; Tian, Hui; Hein, Ingo; Podlipnik, Črtomir; Thomma, Bart P. H. J.; Kosmelj, Katarina; Javornik, Branka; Berne, Sabina (Lead / Corresponding author).

In: Molecular Plant-Microbe Interactions, Vol. 32, No. 10, 10.2019, p. 1378-1390.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

AU - Volk, Helena

AU - Marton, Kristina

AU - Flajsman, Marko

AU - Radisek, Sebastjan

AU - Tian, Hui

AU - Hein, Ingo

AU - Podlipnik, Črtomir

AU - Thomma, Bart P. H. J.

AU - Kosmelj, Katarina

AU - Javornik, Branka

AU - Berne, Sabina

PY - 2019/10

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N2 - During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

AB - During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.

KW - Fungal effectors

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