# A GH81-type β-glucan-binding protein enhances colonization by mutualistic fungi in barley ## Summary Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other’s cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of β-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated β-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a β-glucan interactor. Mutation of *GBP1* and its only paralog, *GBP2*, in barley led to decreased colonization by the beneficial root endophytes *Serendipita indica* and *S. vermifera*, as well as the arbuscular mycorrhizal fungus *Rhizophagus irregularis*. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, *GBP* mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen *Bipolaris sorokiniana* and inhibited the penetration success of the obligate biotrophic leaf pathogen *Blumeria hordei*. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi—a role that has potentially been appropriated by barley-adapted pathogens. <img src=./_publication/1-s2.0-S0960982223014495-fx1_lrg.jpg width=50%> ## Original publication Wanke, A., van Boerdonk, S., Mahdi, L. K., Wawra, S., Neidert, M., Chandrasekar, B., Saake, P., Saur, I. M. L., Derbyshire, P., Holton, N., Menke, F. L. H., Brands, M., Pauly, M., Acosta, I. F., Zipfel, C., & Zuccaro, A. (2023). A GH81-type β-glucan-binding protein enhances colonization by mutualistic fungi in barley. Current biology : CB, 33(23), 5071–5084.e7. https://doi.org/10.1016/j.cub.2023.10.048 ## License Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. ## Data availability Lead Contact Further information and requests for resources, reagents, datasets, and protocols should be directed to and will be fulfilled by the lead contact, Alga Zuccaro (azuccaro@uni-koeln.de). Materials Availability Plasmids and barley lines generated in this study are available upon request and will be provided by the lead contact, Alga Zuccaro (azuccaro@uni-koeln.de). Data and Code Availability Proteomic data has been deposited at the ProteomeXchange Consortium via PRIDE partner repository (Perez-Riverol et al., 2019) and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. ## ARC structure ``` mermaid flowchart subgraph CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays end subgraph HordeumVulgare ROS_burst_assay_preparation germination growth_chamber end subgraph NicotianaBenthamiana greenhouse_conditions end subgraph HeterologousProteinProductionAndPurification Agrobacterium_growth Nicotiana_infiltration ground_material incubation purification end subgraph PlasmidConstructionForHeterologousProteinProduction amplification cloning site-directed_mutagenesis_PCR purification_and_E.coli_transfo Agrobacterium_transformation end subgraph ProteinPull-down biothinylation_of_laminarin extraction incubation pull-down SDS-PAGE end subgraph OxidativeBurstAssay preparation_of_plant_material ROS_burst_assay end subgraph MALDI-TOFAnalysis MALDI-TOF end subgraph EnzymaticCarbohydrateDigestionAndTLC EnzymaticCarbohydrateDigestion TLC end subgraph StainingForConfocalMicroscopy staining end subgraph CRISPRCas9-basedMutagenesis expression_cassettes plasmid_generation CRISPR_Cas9_mutagenesis end subgraph MultipleSequenceAlignment alignment end subgraph MS-MSAnalysisOfThePull-downProteins MS-MS_analysis end subgraph FungalMaterialGrowthConditionsAndBarleyColonizationAssays R.irregularis R.irregularis_staining Si_Sv_Bs B.hordei B.hordei_staining end subgraph QuantificationAndStatisticalAnalyses end subgraph qRT-PCR RT-PCR end Wanke-2023 --> CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays Wanke-2023 --> HordeumVulgare Wanke-2023 --> NicotianaBenthamiana ROS_burst_assay_preparation --3--> germination germination --3--> growth_chamber growth_chamber --3--> preparation_of_plant_material growth_chamber --1--> R.irregularis growth_chamber --1--> Si_Sv_Bs growth_chamber --1--> B.hordei greenhouse_conditions --1--> preparation_of_plant_material Agrobacterium_growth --3--> Nicotiana_infiltration Nicotiana_infiltration --1--> ground_material ground_material --1--> incubation incubation --1--> purification purification --2--> EnzymaticCarbohydrateDigestion amplification --1--> cloning cloning --1--> site-directed_mutagenesis_PCR cloning --1--> Agrobacterium_transformation site-directed_mutagenesis_PCR --1--> purification_and_E.coli_transfo purification_and_E.coli_transfo --1--> Agrobacterium_transformation Agrobacterium_transformation --2--> Agrobacterium_growth biothinylation_of_laminarin --1--> extraction extraction --1--> incubation incubation --6--> pull-down pull-down --6--> SDS-PAGE SDS-PAGE --5--> MS-MS_analysis preparation_of_plant_material --6--> ROS_burst_assay EnzymaticCarbohydrateDigestion --28--> MALDI-TOF EnzymaticCarbohydrateDigestion --28--> TLC expression_cassettes --3--> plasmid_generation plasmid_generation --1--> CRISPR_Cas9_mutagenesis CRISPR_Cas9_mutagenesis --2--> ROS_burst_assay_preparation R.irregularis --3--> R.irregularis_staining Si_Sv_Bs --3--> staining Si_Sv_Bs --9--> RT-PCR B.hordei --3--> B.hordei_staining ```