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Summary    The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin‐mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in  Laccaria bicolor  for ECM formation with  Populus tremula  ×  Populus tremuloides .  We combine transcriptomics of cell‐wall‐related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic  L. bicolor  to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation.  Immunolocalisation identified remodelling of pectin towards de‐esterified HG during ECM formation, which was accompanied by increased  LbPME1  expression and PME activity. Overexpression or RNAi of the ECM‐induced  LbPME1  in transgenic  L. bicolor  lines led to reduced ECM formation. Hartig Nets formed with  LbPME1  RNAi lines were shallower, whereas those formed with  LbPME1  overexpressors were deeper.  This suggests that  LbPME1  plays a role in ECM formation potentially through HG de‐esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation.

Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula × Populus tremuloides