N-glycosylation Enables Smut Fungal Nge1 Orthologs to Prevent the Escape of Maize Evolved-PMEIs

Cell wall integrity (CWI) is regulated by the coordinated activity of CW-modifying enzymes, including pectin methylesterases (PMEs) and their inhibitors (PMEIs). PMEs de-methylesterify pectins, making them more susceptible to degradation and loosening the CW, facilitating pathogen invasion. Conversely, PMEIs inhibit PMEs, reinforcing the CWI and enhancing plant defense. However, how biotrophic pathogens overcome PMEI-mediated defense remains unclear. Here, we report that smut fungal effectors have evolved to directly target host specific PMEIs, manipulating cell wall integrity to enhance virulence. N-glycosylated Effector 1 (Nge1) from Ustilago maydis selectively interact with PMEI45 and PMEI46, as well as the auto-inhibitory PRO-domains of PMEs. This interaction disrupts PMEI inhibition, liberating PME19 and PME20, which reduce pectin methylesterification and likely loosen the CW, promoting fungal invasion. Notably, the interaction between Nge1 and PMEI45, but not PMEI46, is N-glycosylation-dependent. Restoring glycosylation in a non-glycosylated Nge1 ortholog allows it to functionally replace U. maydis Nge1, suggesting that smut fungal effectors have evolved through glycan modifications to overcome host-adapted PMEIs that would otherwise escape non-glycosylated effectors and impede fungal infection. Our findings reveal bidirectional host-pathogen strategies in a co-evolutionary arms race to fine-tune molecular interactions in the extracellular space.