Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

Heidrun Häweker,Stephan Rips,Hisashi Koiwa,Susanne Salomon,Yusuke Saijo,Delphine Chinchilla,Silke Robatzek,Antje Von Schaewen

doi:10.1074/jbc.m109.063073

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.

Highlights

The glycosylation of asparagine residues (N-glycosylation) is an essential, highly conserved co-translational modification of secreted proteins occurring in all eukaryotic cells
Glycosylation patterns are conserved between plant receptors, especially NX(S/T) glycosylation motifs located in the ␣-helical parts of
We investigated whether alteration of N-glycosylation in the endoplasmic reticulum (ER) and N-glycan modification in the Golgi apparatus affect pattern recognition receptors (PRRs) function and plant immunity

Summary

EXPERIMENTAL PROCEDURES

A. thaliana Mutants—tDNA insertion mutants (supplemental Fig. S1B) stt3a-1, stt3a-2, stt3b-1, hgl, cgl, fucTa, fucTb, and xylT have been described previously [4, 5, 8]. Arabidopsis wild type and mutant plants used for transient transfections were grown under sterile conditions on solidified medium for 4 – 6 weeks prior to preparation of leaf protoplasts. Insensitivity of stt3a-2 was specific for elf (and epistatic to other glycosylation mutants tested), and its response to flg was similar to wild type (Fig. 1A). This was true for the oxidative burst, an immediate early MAMP response, because elicitation with elf failed to trigger ROS production in the stt3a-2 mutant (supplemental Fig. 2). Transient transfection of Arabidopsis protoplasts Because EFR and FLS2 (with co-receptor BAK1) share downwas achieved by the polyethylene glycol method using 30 ␮g of stream components [14, 16], the elf18-insensitive but flg22-. Cross-linking was performed in vitro to protoplasts were imaged as described previously [8]

RESULTS

DISCUSSION