Cell death signaling in the Arabidopsis chitin receptor mutant cerk1-4

Field: Agricultural and Biological Sciences (Life Sciences)

Plant surface immunity is mediated by pattern recognition receptors (PRRs) that recognize con-served molecules derived from microorganisms. In Arabidopsis, CERK1 is the main component of the receptor complex that binds chitin and transduces the signal intracellularly (Miya et al., 2007; Petutschnig et al., 2010; Wan et al., 2008). The cerk1-4 mutant exhibits deregulated cell death upon infection with powdery mildew and during senescence (Petutschnig et al., 2014). This pheno-type is dependent on salicylic acid, yet chitin-induced responses remain unimpaired. Although in wild-type plants the CERK1 ectodomain is cleaved and released from the plasma membrane, this soluble fragment is undetectable in the cerk1-4 mutant. Remarkably, the cerk1-4 mutation leads to an amino acid substitution (L124F) in the inner part of the CERK1-4 ectodomain (Petutschnig et al., 2014). To broaden our understanding of the cell signaling in the cerk1-4 mutant and identify new components of this pathway, suppressor screens were conducted. In this work, two mutants that suppress the deregulated cell death of the cerk1-4 mutant were iden-tified through a mapping-by-sequencing approach. Complementation assays indicated that one of them is the catalytic subunit of the ER glucosidase II (GCSIIα) involved in the N-glycosylation pathway (Trombetta et al., 1996; Trombetta et al., 2001). The second suppressor is a mutant of the UDP-GLCNAC TRANSPORTER 1 (UGNT1) that provides N-acetylglucosamine into the Golgi (Ebert et al., 2018b). The identification of these suppressor mutants suggests the importance of complex or hybrid N-glycans for the cerk1-4 cell death signaling. Additional mutants of UGNT1 were evaluated and successfully suppressed the cerk1-4 phenotype. The initial step in the pathway toward complex N-glycans formation is performed by the N-acetylglucosaminyltransferase I (GnTI). Mutants of GnTI, known as complex glycan less mutants (cgl), suppress the cerk1-4 phe-notype, supporting that the maturation of N-glycans into complex or hybrid forms is crucial for the deregulated cell death response of cerk1-4. Complex glycans are further modified by the α-mannosidase II (GMII) and the N-acetylglucosaminyltransferase II (GnTII), among other enzymes in the medial Golgi. A mutant of GMII (hgl1) did not suppress the cell death phenotype of cerk1-4 and GnTII (gntII-2 and gntII-3) cerk1-4 double mutants show a cerk1-4 phenotype. This indicates that the α1,3-arm linked N-acetylglucosamine residue on glycoproteins is sufficient to induce cell death in cerk1-4. Notably, none of these mutants have a detrimental effect on CERK1-4. Wild-type-like membrane localization was observed for CERK1 and CERK1-4 that are deprived of complex N-glycans in cgl mutants. Moreover, chitin-induced MAPK activation and ROS pro-duction were not impaired in these mutants. This suggests that complex or hybrid N-glycans are not required for the localization of CERK1 and the mutant CERK1-4 and for CERK1-mediated immune responses. The cerk1-4-induced substitution L124F is embedded in the N-glycosylation motif N-X-S/T. Tar-geted mutagenesis of the N123 glycosylation site abolished cerk1-4-induced cell death, indicating that this site is critical for the phenotype. Furthermore, truncated versions of CERK1 and CERK1-4 were differentially cleaved by Endoglycosidase H. In addition, it was demonstrated that the lack of ectodomain in CERK1, the perception of synthet-ic peptides corresponding to the three LysM of CERK1 and the disturbance of the disulfide bridges of the CERK1 ectodomain do not induce a cerk1-4-like phenotype. Interestingly, it was demon-strated that MAMP treatment induces the uncontrolled cell death in cerk1-4. These findings suggest that complex N-glycans, specifically the first GlcNAc attached to N123 of CERK1-4, is indispensable for the exaggerated cell death in cerk1-4. Based on the results obtained in the current study, a model was suggested in which wild-type CERK1 is decorated on its aspara-gine N123 with oligomannosidic-type glycans, while the mutant CERK1-4 contains a complex N-glycan at that site. After shedding, CERK1-4 ECD is further cleaved into peptides and the CERK1-4-LysM2 derived peptides harboring the N123-linked complex glycans are suggested to be signal that trigger the resulting uncontrolled cell death.