A mutation in Asparagine-Linked Glycosylation 12 ( ALG12 ) leads to receptor misglycosylation and attenuated responses to multiple microbial elicitors

Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identiﬁed an Arabidopsis thaliana ‘changed calcium elevation 1’ ( cce1 ) mutant with attenuated calcium response to the bacterial ﬂagellin ﬂg22 peptide and several other elicitors. Whole-genome resequencing revealed a mutation in as-paragine-linked glycosylation 12 that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an a -1,6 linkage to the dolichol-PP-oligosaccharide N -glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N -glycosylation is required for proper functioning of client proteins.

In addition to preformed physical barriers, pattern-triggered immunity (PTI) is one of the first lines of defence of plants against pathogen attack. This is initiated by the recognition of conserved molecular features of pathogens/microbes, so-called microbe-associated molecular patterns (MAMPs) [1,2]. MAMP recognition by cognate receptors leads to the activation of signalling cascades resulting in plant defence reactions mediated through transcriptional reprogramming of defence gene expression, as well as changes in antimicrobial metabolite and stress hormone levels. MAMPs are recognised by surface-localised receptor-like kinases (RLKs) or receptor-like proteins (RLPs). In Arabidopsis thaliana, the leucine-rich repeat (LRR) domain kinases, Flagellin-sensing 2 (FLS2) and elongation factor thermo unstable (EF-Tu) receptor (EFR), detect bacterial flagellin [3,4] and EF-Tu protein [5,6], respectively. The Lysin motif (LysM) domain containing RLKs Lysin motif receptor kinase 5 (LYK5) and Chitin elicitor receptor kinase 1 (CERK1) detects the fungal MAMP chitin, whereas the lectin-SD-type RLK lipooligosaccharide-specific reduced elicitation is involved in the detection of bacterial medium-chain 3hydroxy fatty acids that are derived from lipopolysaccharide [7,8]. The LRR receptors Pep1-receptor 1/2 (PEPR1/2) detect the endogenous damage signal AtPep1, which is processed and released during pathogen attack [9][10][11][12][13]. In order to coordinate a precise defence output, RLKs and RLPs often form complexes with coreceptors and/or auxiliary proteins. For many LRR-type RLKs or RLPs, the coreceptor is Brassinosteroid-insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) or related members from the somatic embryogenesis receptor kinase (SERK) family and/or suppressor of BIR1-1, whereas CERK1 is the coreceptor of LysM domain containing sensor proteins such as LYK5 or LYM1/3 (see [14] and references therein). Once activated, the receptor complex will initiate propagation of the immune signal. This is achieved by the phosphorylation and thus activation of receptor-like cytoplasmic kinases (RLCKs), which will in turn phosphorylate downstream targets. For example, after flagellin recognition, the BAK1-interacting Botrytis-induced kinase 1 (BIK1; an RLCK) is phosphorylated by BAK1, phosphorylates FLS2 and dissociates from the complex to phosphorylate/activate the NADPH oxidase respiratory burst oxidase homologue D (RBOHD), thereby increasing reactive oxygen species (ROS) synthesis [15][16][17]. Furthermore, the RLCKs have been shown to directly activate components of mitogen-activated protein kinase (MAPK) cascades, which form an important signalling branch in PTI [18,19].
Whereas signal transmission starts as a series of phosphorylation events at the receptor complex, a variety of intracellular second messengers (e.g. nitric oxide, ROS and ion currents) are indispensable to generate a timely and adequate defence output. Calcium influx into the cytosol is one of the fastest reactions after MAMP recognition [20,21] and is generated at the plasma membrane by the influx of extracellular calcium through channels formed by the cyclic nucleotide-gated channel 2/4 complexes that are also activated by BIK1-mediated phosphorylation [22]. The latter explains earlier observations of RLCKs being involved in shaping the calcium signature [23]. Apart from their role in PTI, calcium signals are known to be involved in responses to abiotic stresses as well as in the relay of developmental signals. Plants have evolved a complicated network of components involved in generating, terminating and decoding calcium signals. Intracellularly, changes in calcium levels are sensed by calcium sensors. For instance, PTI-associated calcium bursts activate signalling cascades through calcium sensors such as the calcium-dependent protein kinases (CPKs), which are required for transcriptional reprogramming of immunity-related genes and may act by directly phosphorylating transcription factors [24]. Additionally, it was shown that calcium is involved in the regulation of RBOHD in defence-related ROS production: While RBOHD is phosphorylated by BIK1, it is also regulated through direct phosphorylation by CPK5, which is activated upon rising intracellular calcium levels [25]. Furthermore, RBOHD itself is also a calcium sensor and is able to detect changes in calcium concentration via EF-hand motifs [26]. Interestingly, elevated calcium levels during tissue damage can activate the metacaspase that processes Propep1 precursor into active AtPep1 peptides [27] and thus serve to amplify immune signalling [28]. These findings illustrate the importance of calcium signals in multiple stages of early PTI signalling. In order to improve understanding of plant calcium signalling in a PTI context, we screened a mutant population for plants with a 'changed calcium elevation' (cce) response to MAMPs and this mainly uncovered components of the receptor complexes [29]. In similar screens based on ROS as a read-out, mutants with deficient receptor biogenesis were isolated [30]. These findings underscore a tight functional association between the receptor complex and the generation of calcium/ROS signals.
These reports also pinpoint membranes as the site for the generation of PTI-related signals: Signals are initiated at membrane-localised receptor complexes, ion fluxes are mediated through opening of transmembrane channels, and second messengers feeding back into calcium/ROS and other signalling pathways are generated at membrane-localised protein complexes. The biogenesis of transmembrane proteins and secreted proteins is a tightly controlled pathway conserved over the whole domain of eukaryotes via the endoplasmic reticulum (ER)-based quality control (ERQC) system [31]. This involves the transfer of complex glycan structures onto the asparagine residues within the N-linked glycosylation consensus sequence NxS/T (x = any amino acid except P, S and T) [32]. The glycans that are initially transferred in N-linked glycosylation share a conserved core structure of Glc 3 Man 9 GlcNAc 2 with a branched structure, consisting of lower A, middle B and upper C arms (see Fig. 3A). They are assembled in a stepwise manner on dolichol lipid anchors at the ER membrane, starting at the cytosolic side with synthesis of the mannose (Man) residues on the A arm. The glycan and its anchor are then flipped through the ER membrane, and synthesis continues at the lumenal face by addition of the Man residues to the B and C arms and finally the addition of three glucose (Glc) residues on the A arm. N- acetylglucosamine (GlcNAc), Man and Glc residues are assembled by glycosyltransferases named asparagine-linked glycosylation (ALG). The resulting glycan is then transferred as a whole onto Asn residues in secreted and transmembrane proteins by the oligosaccharyltransferase (OST) complex during their translation [32,33]. The glycans of the A arm can be bound by the ER-based lectin chaperones of the ERQC pathway calnexin (CNX) and calreticulin (CRT). Binding and release of CNX and CRT are dependent on the conformation of the bound protein and are regulated by the addition or trimming of Glc residues attached to the N-linked glycan. After achieving its native conformation, the protein is released from the CNX/CRT pathway by trimming of the Glc residues and exported to the Golgi apparatus, where N-linked glycans undergo additional modifications. When a protein fails to achieve its native conformation in a given period, it undergoes Man cleavage in the C arm and thus increases the probability of interaction with lectins that remove proteins from the CNX/CRT cycle. Proteins are then retro-translocated to the cytosol and will undergo ER-associated degradation (ERAD) [34]. Mutations in ALG genes or in subunits of the OST complex cause drastic phenotypes in animals, but are partially tolerated in plants [35]. For instance, the alg mutants that showed reduced ROS or calcium response after MAMP treatment were mostly normallooking plants [36][37][38]. In fact, an alg12 mutant partially reverts the dwarf phenotype caused by mutation in the brassinosteroid receptor, BRI1 [39]. Recently, a systematic examination of multiple mutants in the ALG pathway showed its requirement for the cell death pathway triggered by silencing of BAK1 and a related homologue, SERK4 [40]. All these reports highlight the importance of proper glycosylation of ALG pathway client proteins for ERQC.
We previously isolated the cce1 mutant that does not show any dramatic growth phenotype but exhibits a broad-spectrum reduced calcium response to numerous MAMPs [29]. Here, we show that cce1 is a novel allele of ALG12, which encodes the a-1,6-mannosyltransferase responsible for adding the initial Man to the C arm of the glycan tree. ERQC is thus essential for proper biogenesis and functioning of MAMP receptors.
For the calcium, MAPK and qPCR assays, seeds were surface-sterilised and stratified for 2 days at 4°C and subsequently grown in liquid MS medium for 8-10 days under long-day conditions (16-h light, 8-h dark, 21°C) as described in Ref. [41].

Protoplast transfection, protein isolation, western blotting and endoglycosidase-based glycan analysis
Protoplasts were generated from adult (5-week-old) plants grown on soil at 22°C under short-day conditions (8-h light, 16-h dark, 22°C) and transfected according to the method described in Ref. [34]. After an overnight incubation (18°C, in the dark), protoplasts were harvested by centrifugation and total proteins were isolated from protoplast pellets with extraction buffer (25 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% (w/v) Nonidet P-40 and Serva Protease Inhibitor Mix P) by vigorous vortexing. After addition of 109 glycoprotein denaturation buffer (supplied with the NEB enzymes) to each sample (final conc. 19), samples were boiled for 10 min at 100°C and then cooled on ice. Subsequently, all samples were split into three aliquots, one was kept on ice as the 'untreated' control, and the other two were digested with Endo Hf (NEB GmbH, Frankfurt (Main), Germany) or Peptide:N-glycosidase F (PNGase F; NEB Inc.), respectively. Digestions were initiated by further addition of the corresponding reaction buffers (to 19) and 2 U of Endo Hf or PNGase F and incubation at 37°C for 1 h. Additionally, for the PNGase F digest, 1% NP40 (final concentration) was included. The digests were stopped by mixing with standard SDS/PAGE sample loading buffer and separated on 8% standard SDS/PAGE. Gels were blotted onto Porablot NCL nitrocellulose membranes (Macherey-Nagel), and proteins were detected by western blot using Living ColorsÒ GFP Monoclonal Antibody (Takara Bio Europe, Saint-Germain-en-Laye, France) and anti-mouse IgG-HRP (Sigma-Aldrich, Munich, Germany) according to the manufacturer's protocols.

Confocal microscopy
Microscopic analysis of fluorescence-tagged receptor and coreceptors expressed in mesophyll protoplasts was performed as described previously [38]. Briefly, after an overnight incubation (~16 h) period to enable protein expression, protoplasts were observed on a LSM 710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany), with the following excitation settings (GFP: 488 nm; YFP: 514 nm). Acquired images (ZEN 2012 software; Carl Zeiss) were processed with IMAGEJ software (National Institute of Health, Bethesda, MD, USA).

Statistical analysis
Statistical analyses were performed with the GraphPad Prism 5 (San Diego, CA, USA) software package with the tests specified in the respective figure legends.

Results
cce1 has a broad-spectrum calcium signalling phenotype (similar to cce2) but is not allelic to cce2 The 'cce' mutant, cce1, was reported to exhibit reduced response to a number of elicitors (but note that the actual calcium traces were not shown) [29]. Here, we validated the attenuated calcium response to several peptide elicitors (flg22, elf18 and pep1) or chitin as compared to the parental line, pMAQ2. By integrating the 'area under the curve (AUC)' as quantification of the total response, the cce1 mutant shows a~7-20% reduction in its calcium response to the tested elicitors (Fig. 1). This indicates a perturbation of multiple signalling pathways, as not only the FLS2/EFR/PEPR1-BAK1 receptor complexes involved in peptide MAMP recognition, but also the CERK1/LYK5 complexes involved in the sensing of chitin are disturbed. To exclude a general effect on calcium influxes, we tested an abiotic stress (200 mM NaCl treatment), where the salt stress-induced calcium response was not reduced, but rather mildly enhanced, in the cce1 mutant (Fig. S1). Hence, the reduced calcium response seems to be specific to the tested biotic signals. This broadspectrum cce1 phenotype to multiple biotic stress signals resembles that of a previously characterised cce2 mutant [38] and suggests that like cce2, the cce1 mutation(s) may affect a global function relevant for the different receptors.
To check for epistasis or allelism, cce1 was previously crossed with bak1-4 or cce2, where the attenuated flg22-induced calcium response of the cce1 mutant was restored to wild-type level in the F1 generation [29], suggesting that it is not allelic to the BAK1 coreceptor or the CCE2 gene, respectively. cce2 and the allelic cce3 are different mutants of ALG3, a mannosyltransferase involved in ER quality control via Nlinked glycosylation [38] (see Fig. 3A for scheme of ALG pathway). As any perturbation of this pathway will affect all transmembrane proteins harbouring Nlinked glycosylation sites in their exoplasmic domains, most receptors will be globally affected. Thus, we wondered whether cce1 may be mutated in the same pathway as cce2/cce3 and generated a cce1cce2 double mutant to test this hypothesis. When treated with flg22, the cce1cce2 double mutant exhibited a calcium phenotype similar to the cce1 or cce2 single mutations. From the AUC analysis, the mutants possess 83-89% of the wild-type calcium response but are not statistically different between themselves ( Fig. 2A).
Mitogen-activated protein kinase activation is one of the earliest signalling events downstream of calcium influx and can be visualised by a western blot-based detection of the phosphorylated MAPK proteoforms. Three phospho-MAPK bands are typically detected after flg22 elicitation. Using the Odyssey quantitative western blot analysis system (LI-COR Bioscience), we calculated the total signal intensities of all three bands (at 15 min postelicitation). Compared to the pMAQ2 parental plants, flg22-induced MAPK activation is attenuated in cce1, cce2 and cce1cce2 double mutant (Fig. 2B). Similar to the calcium response, there is no  (Fig. 2B). These results suggest that the two mutations may indeed affect the same pathway.
cce1 is a novel allele of ALG12 Whole-genome resequencing was performed on the cce1 mutant, and 35 nonsynonymous mutations were identified (Table S1). Among the candidates, a putative SNP in a mannosyltransferase gene (AT1G02145) was detected, which was validated by resequencing and CAPS (cleaved amplified polymorphic sequence) analysis (see Materials and methods). This C-to-T mutation leads to a T70I exchange in the encoded ALG12 protein (Fig. 3A, B). A mutant of ALG12 had been previously isolated in an 'EMS-mutagenised brassinosteroid receptor 1 suppressor (ebs)' screen, with the SNP in the ebs4-3 mutant leading to an E38K exchange located in a predicted ER lumen-localised protein loop [35]. As illustrated by the alignment with several plant orthologues and the human ALG12 (Fig. 3B), the T70I exchange caused by the cce1 mutation is located in this same luminal loop, which is highly conserved between plants, fungi and animals. Fortuitously, a human T67M exchange at the corresponding orthologous T70 position has been identified in patients with congenital disorder of glycosylation (CDG), a severe disease with multisystem defects [44]. Since the human ALG12 T67M allele failed to complement alg12 in yeast, the ALG12 T70I allele isolated here is likely to also encode mannosyltransferase with reduced or impaired activity.
In order to validate the causal relationship of the T70I mutation in ALG12 and the calcium phenotype  no homozygous alg12-T was recovered from the segregating GABI-Kat line, indicating that homozygous alg12-T may be lethal. Thus, cce1 was crossed to a heterozygous alg12-T line. Out of 20 F1 plants screened, only one heterozygous cce1 alg12-T plant was recovered, thus supporting the notion of reduced fitness of plants bearing the alg12-T allele. The segregating F2 population of this heterozygous cce1 alg12-T plant was genotyped for both the cce1 SNP and the T-DNA insertion in ALG12. Only homozygous cce1 individuals or heterozygous cce1 alg12-T plants were recovered (Fig. 3C). The calcium response to flg22 treatment in this pool of F2 segregants from the cce1 alg12-T heterozygous line is indistinguishable from that of the cce1 mutant (Fig. 3D). Thus, from this allelism analysis, alg12-T does not complement cce1 and supports the notion that alg12 is the causal mutation for the cce1 phenotype.

Receptor localisation is unaltered in cce1
Previous studies have suggested that mutations in genes of the ALG pathway lead to a reduction in ERQC stringency, allowing mutated and malformed proteins to reach the plasma membrane, which would otherwise be targeted for ERAD [35]. To check whether receptors are correctly targeted to the plasma membrane in cce1 and cce1cce2 double mutant lines, we analysed protoplasts transiently expressing fluorescent protein-tagged FLS2, EFR, PEPR1 or CERK1 receptors. Most of the fluorescence signals were present in the cell periphery, suggesting correct targeting of receptors to the plasma membrane (Fig. 4A). We had previously reported that whereas cce2 and cce3 harbour slightly less endogenous FLS2 than the parental line [38], this was not the case for cce1 [29]. Here, anti-GFP antibodies were used to check protein levels of the various GFP-or YFP-tagged receptor proteins in the cce background, but unfortunately, inconsistent transfection efficiencies between samples precluded this comparison. However, most of the receptors expressed in the cce1, cce2 or cce1cce2 double mutant background show a faster migration pattern in the SDS/ PAGE (Fig. 4B). An exception is CERK1 that showed little-to-no difference in the cce1 background when compared to the pMAQ2, but as reported previously, enhanced mobility of CERK1 can be discerned in the cce2 background [38] or also here, with a shorter exposure of the western blot (last panel on the right of Fig. 4B). Thus, the observation of receptors with altered sizes is in line with ALG12 mutation leading to misglycosylation. where n = 32). The per cent AUC compared to pMAQ2 is shown (**P < 0.01, ***P < 0.001, one-way ANOVA). (B) Flg22-induced MAPK activation was monitored using an antibody that recognises phosphorylated MAPKs (a-pTEpY). Leaf material was harvested at the indicated time points after infiltration of flg22 (5 nM) and processed for quantitative western blotting with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Linearity of the assay was validated using a serial dilution (see Fig. S2). Total signal intensity of all three immunoreactive MAPK bands was normalised against the total proteins (Revert 700 Total Stain; LI-COR) and used as an estimation of total flg22-induced MAPK activation (at 15 min). Three independent biological replicates were processed, and all values were normalised against pMAQ2 (with the average signals set as 100 %). Alphabets mark the distinct statistically significant groups (one-way ANOVA). Mean (horizontal line) and SD (error bars) are included in the scatter plot.

Receptor glycosylation is compromised in cce1
Levels of the endogenous ALG12 mRNAs are not compromised in the cce1 or cce1cce2 double mutants (Fig. 5A). Hence, the cce1 phenotype is not due to reduced expression at the transcript level. If the hypomorphic T70I cce1 mutation results in a less efficient mannosyltransferase activity of ALG12, then glycosylated proteins with extracellular domains should consequently exhibit aberrant sizes, which is consistent with enhanced electrophoretic mobility for FLS2, EFR and PEPR1 when expressed in the cce1, cce2 and the cce1cce2 double mutant background. For CERK1, the poor gel resolution did not permit clear conclusion on size differences in cce1 although it is indeed smaller in the cce2 background (Fig 4B and  [38]).
To compare the receptor glycosylation status in the cce mutants, we selected FLS2-YFP as a representative for further analysis. Deglycosylation assays were performed on membrane proteins prepared from the different genotypes. Upon treatment with PNGase F (an amidase that cleaves the linkage between GlcNAc and the Asn), YFP-tagged FLS2 expressed in the different lines mostly ran as a species of approximately 190 kDa, suggesting that the observed differences in running behaviour (compared to the wild-type) are indeed caused by different glycan structures. The partial cleavage of FLS2 expressed in the mutants may reflect poorer accessibility of PNGase F due to differentially glycosylated receptors.
By contrast, receptors expressed in cce2 and the cce1cce2 double mutant were completely resistant to To genotype the offspring from a cce1 alg12-T heterozygous F1 plant (highlighted by box), PCR was performed using the primers indicated in (B). Top panels: For CAPS analysis, the PCR products were digested with ApoI, resulting in 481 and 307 bp fragments for wild-type sequence or 454, 307 and 29 bp fragments for the cce1 sequence (note that the 29 bp fragment is not visible on this gel). Bottom panels: Gels showing the absence/presence of amplicons diagnostic for the alg12 T-DNA insertion. Note that no plants homozygous for the T-DNA insertion could be identified. The representative gels shown are taken from a single experiment with n = 24. Two further experiments were performed and the pooled results used to compute the observed segregation ratio. (D) Calcium elevation in response to flg22 treatment (1 µM) in the F2 segregants described in panel C (Note that this represents a mix of cce1 homozygotes and cce1alg12-T heterozygotes). Error bars represent the 95% confidence interval (n = 36 for pMAQ2 or cce1 and 93 for the F2 pool).

Discussion
We identified a calcium response mutant, cce1, with attenuated response to several MAMPs and damageassociated molecular patterns (DAMPs), which acts in the same genetic pathway as ALG3. The corresponding cce1 mutation was identified to reside in ALG12, which encodes the a-1,6-mannosyltransferase that acts two steps downstream of ALG3 in the N-linked glycosylation pathway (see Fig. 3A). Mutations in ALG12 (designated as ebs4) have previously been described in a brassinolide signalling context for biogenesis of the BRI1 receptor. As shown in sequence alignment (Fig 3B), the E38K mutation that abrogates ALG12 ebs4-3 function [35] lies in the same ER luminal protein loop as the T70I cce1 exchange (which is equivalent to the human CDG disease-associated T67M CDG exchange [44]). Thus, this protein loop is likely to be important for its function. As the cce1 mutation did not lead to lowered ALG12 T70I mRNA levels, the phenotype can be attributed to impaired protein function leading to the production of aberrant glycan structures.
Crosses of cce1 with a T-DNA insertion line for ALG12 (alg12-T) support the interpretation that alg12 accounts for the observed calcium signalling phenotype. The genetic analysis was complicated by the failure to isolate homozygous alg12-T plants, suggesting the T-DNA insertion likely creates a null and lethal allele. Indeed, besides not obtaining homozygous alg12-T plants in the offspring from the heterozygous cce1 alg12-T line, a non-Mendelian segregation pattern was observed. Contrary to the expected 1 : 2 ratio of cce1 homozygotes and cce1 alg12-T heterozygotes (if alg12-T homozygotes were nonviable), an approximate 2 : 1 ratio (i.e. 46 cce1 homozygotes:28 cce1 alg12-T heterozygotes) was obtained (Fig. 3C). This ratio inversion is indicative of alg12-T heterozygous plants having reduced fitness (that may lead to conditional lethality). Analogously, this is in agreement with the severe developmental defects and psychomotor retardation manifested in a patient heterozygous for the ALG12 T67M allele and a second ALG12 R146Q allele [44]. Nevertheless, since another ALG12 ebs4-1 aberrant splicing allele that results in premature translational termination was previously identified [35], null alleles of alg12 may not necessarily be lethal and we cannot fully exclude that lethality of the alg12-T allele may be caused by a closely linked gene. Similarly, the cce1 mutant contains additional mutations that may contribute to the calcium response. For instance, it has seven putative nonsynonymous SNPs on the same chromosome as ALG12 (Table S1), albeit the closest is 1 million bp away. Thus, although the allelism analysis involving the F2 population of the cce1xalg12-T cross is good genetic evidence that the altered flg22-induced calcium phenotype is due to ALG12, further validation should be obtained in the future by genetic complementation specifically with the ALG12 locus.
In accordance with hampered mannosyltransferase activity, receptors expressed in mutant-derived protoplasts show a different separation pattern in SDS/ PAGE, most likely caused by occupancy of glycosylation sites with aberrant glycans. Consistent with the proposed function of ALG12 and previous glycan analyses on alg12, receptors expressed in cce1 background contain high-Man glycans that can be partially removed by EndoH f in contrast to receptors expressed in alg3 lines. Remarkably, the proposed glycan structures in alg12 and alg3 only differ in the presence of two Man residues on the B-arm of the glycan but still result in differential cleavage by Endo H f . As receptors expressed in the mutant lines mostly migrated as single species, cce1 appears to lead to complete residence of glycosylation sites, albeit with aberrant glycans, as described previously [46]. This is in contrast to alg phenotypes in yeast, where malformed glycans are transferred with less efficiency by the OST, leading to incomplete glycosylation site occupancy [33].
Receptor localisation was unaltered in cce1, which is in accordance with the phenotypes in ebs4, which renders ERQC less stringent for misglycosylated receptors [35]. We had previously found that receptor levels of FLS2 were not reduced in cce1 compared to wild-type plants, whereas receptor levels were reduced in cce2 [29]. We, however, cannot determine whether this translates into different mechanisms behind the observed mutant phenotypes or whether lacking ERQC will always lead to faulty calcium signalling independently from receptor levels. In this connection, we could not clearly clarify if the CERK1 receptors are differentially glycosylated in cce1 although it is underglycosylated in the cce2 background (Fig 4B), thus questioning if the minor reduction in calcium response to chitin is indeed linked to compromised receptor function in cce1. Notably, the chitin-induced response in cce1 showed a loss of the 'shoulder region' of the calcium response curve (Fig 1D), which is reminiscent to that seen in the rbohD mutant or after inhibition of ROS production by treatment with the NADPH oxidase inhibitor, diphenyleneiodonium [20]. Thus, although glycosylation of plant RBOHD has not been reported, it is tempting to speculate that RBOHD misglycosylation may contribute to the altered calcium response in cce1. By analogy, human NOX1 NADPH oxidase is N-glycosylated at N162 and N236 but their mutations have no impact on surface localisation [47]. Taken together, other N-linked glycosylation client proteins that are relevant for the pathway, for example BAK1 [40] or potentially also the calcium channels, will influence calcium response in most alg mutants. Along the same line of argument, receptor biogenesis has long been described as an important factor in proper response of plants to external stimuli. Due to the conserved role of N-linked glycosylation in ERQC, glycosylation mutants have been discovered in screens for various signalling pathways in plants. Consequentially, calcium signalling is affected by ERQC pathway mutants as are other signalling pathways that rely on exoplasmic or transmembrane glycosylated protein components. The affected pathways are diverse and so are the consequences of mutations in N-linked glycosylation. Whereas mutations in ALG3 have been shown to lead to reduced numbers of receptors in the plasma membrane [29,38], ALG12 mutations have been linked to a less stringent ERQC allowing for mutated receptors to reach the plasma membrane which would otherwise be trapped in the ER [35,39,48]. In human cell lines, faulty N-linked glycosylation was shown to alter re-internalisation kinetics of a G protein-coupled receptor [44]. In plant N-glycosylation mutants, the EFR receptor was shown to lose ligand binding and display reduced plasma membrane accumulation and interaction with its coreceptor BAK1 [49]. The manifold possible consequences of lacking glycosylation and the often cryptic phenotypes impede the identification of causal relations between mis-or underglycosylation and the observed phenotypes. This is aggravated by the fact that different proteins show different reactions for similar glycan aberrations and even different glycosylation sites on the same protein fulfil different functions. In order to uncover the functional relevance of lacking glycosylation, components of the receptor complexes and downstream signalling components need to be studied separately.
In summary, when the findings in this report are taken in context with our cce screen [29,38], the recurrent isolation of components of receptor complexes or elements required for proper receptor biogenesis when using calcium response as a read-out during genetic screens shows that quantitative calcium measurement is a good proxy for function of receptors and surface components.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig S1. Calcium response to osmotic (NaCl) stress is not reduced in the cce1 mutant. Fig. S2. Linearity of signal intensities of activated MAPK phospho-forms quantified using the Odyssey Western blot system. Table S1. (A) List of SNPs found in cce1, which lead to non-synonomous substitution. (B) #Complete list of single nucleotide polymorphisms (SNPs) found in cce1 after Illumina whole genome resequencing.