Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

Abstract

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.

Specialized Roles of the Conserved Subunit OST3/6 of the Oligosaccharyltransferase Complex in Innate Immunity and Tolerance to Abiotic Stresses

“…2A). In contrast, seedling growth inhibition mediated by flg22 that is recognized by the related LRR-RK FLAGELLIN SENSING2 (FLS2; Gómez-Gómez and Boller, 2000) was similar to wild-type plants (data not shown), showing that ost3/6-1 is sensitive to flg22 treatment similar to efr-1 and the previously characterized stt3a-2 mutant Häweker et al, 2010). Consistent with this findings, the oxidative burst triggered by elf18 was almost abolished (Fig.…”

“…Since EFR is heavily glycosylated Saijo et al, 2009;Häweker et al, 2010), we hypothesized that N-glycosylation of EFR is altered in the ost3/6-1 mutant, potentially leading to altered accumulation and/or function of EFR. We generated ost3/6-1 efr-1 double mutants expressing GFP-tagged EFR under the control of the native promoter.…”

“…STT3A-deficient plants are viable but display a protein underglycosylation defect that affects the biogenesis of heavily glycosylated proteins, such as the pattern recognition receptor EF-TU RECEPTOR (EFR; Nekrasov et al, 2009;Saijo et al, 2009;Häweker et al, 2010). In contrast, STT3B deficiency does not lead to an obvious underglycosylation defect, and EFR function is not compromised (Koiwa et al, 2003;Nekrasov et al, 2009;Häweker et al, 2010). However, Arabidopsis stt3a stt3b double knockout plants are gametophytic lethal (Koiwa et al, 2003).…”

“…In Arabidopsis (Arabidopsis thaliana), two proteins, termed STT3A and STT3B, with homology to the yeast and mammalian catalytic subunits have been identified (Koiwa et al, 2003). STT3A-deficient plants are viable but display a protein underglycosylation defect that affects the biogenesis of heavily glycosylated proteins, such as the pattern recognition receptor EF-TU RECEPTOR (EFR; Nekrasov et al, 2009;Saijo et al, 2009;Häweker et al, 2010). In contrast, STT3B deficiency does not lead to an obvious underglycosylation defect, and EFR function is not compromised (Koiwa et al, 2003;Nekrasov et al, 2009;Häweker et al, 2010).…”

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

“…However, further deletions at the N terminus as in EFR⌬5 or further truncation from its C-terminal side resulted in proteins lacking affinity for elf peptides. Recent reports have shown that accumulation of functional EFR strongly depends on quality control steps and glycosylation processes in the secretory pathway of the plant (27,28). The constructs used in our experiments all contained an N-terminal signal sequence, and they led to accumulation of C-terminally tagged proteins with the size of appropriately glycosylated EFR derivatives ( Fig.…”