Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1
© Ranf et al.; licensee BioMed Central. 2014
Received: 6 August 2014
Accepted: 8 December 2014
Published: 19 December 2014
Plant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.
We characterized here the “changed calcium elevation 5” (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.
Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components.
During their infection attempt, microbes activate intracellular signaling cascades in their potential host. Specific pattern-recognition receptors (PRRs) from the host recognize conserved microbe-associated molecular patterns (MAMPs) or certain signature molecules resulting from tissue damage, often designated as damage-associated molecular patterns (DAMPs) . PRRs are typically receptor-like kinases (RLKs), such as FLS2 (Flagellin-Sensing 2), EFR (Elongation Factor Tu Receptor) or PEPR1/PEPR2 (AtPep-Receptor 1/2). These recognize the MAMPs, flg22 (N-terminal flagellin-derived peptide), elf18 (N-terminal fragment of Elongation Factor Tu) and the DAMP, AtPep1, respectively . Upon binding of the respective ligand -, FLS2, PEPR1/PEPR2 or EFR hetero-oligomerize with BAK1 (BRI1-Associated Kinase 1), a kinase originally found as an interactor of the brassinosteroid hormone receptor, BRI1 . Recent structural studies indicate that BAK1 is also in direct contact with the C-terminal part of the FLS2-bound flg22, and may thus be considered a co-receptor . Accordingly, bak1 mutants are impaired in responses to these MAMPs/DAMPs ,,. Thus, BAK1 acts as protein partner (or co-receptor?) for multiple pathways in plant immunity and development . On the other hand, signaling induced by other MAMPs, such as chitin is independent of BAK1 . This difference may be a consequence of the different structure of the potential receptor(s) required for perceiving chitin, CERK1 (Chitin Elicitor Receptor Kinase 1), a LysM-containing RLK in Arabidopsis - as compared to the LRR-type RLKs such as FLS2, EFR or PEPR1/R2.
Among the earliest signaling events after MAMP/DAMP perception are ion fluxes across the plasma membrane including influx of calcium into the cytosol ,-. The elevation of cytosolic calcium is detected by a number of calcium-binding “decoder” proteins such as calmodulins or calcium-dependent protein kinases (CPKs) or Calcineurin B-like (CBL) proteins and their partners, CBL-interacting protein kinases (CIPKs) to further transmit the signal ,. Calcium, as a signaling molecule, is a prerequisite for most downstream responses elicited by MAMPs/DAMPs. For instance, production of reactive oxygen species (ROS) by the NADPH oxidase, RBOHD, in Arabidopsis  is a calcium-dependent process stimulated by direct binding of calcium to EF-hands in the N-terminus of RBOHD. Furthermore, the calcium-dependent protein kinase 5 (CPK5) phosphorylates RBOHD to promote its activity . Activation of mitogen-activated protein kinases (MAPKs) also requires calcium since depletion of extracellular calcium or inhibition of calcium channels block MAMP-induced MAPK activation ,.
The importance of calcium for plant immunity is also indirectly supported by the observation that phytopathogenic bacteria secrete extracellular polysaccharides to sequester apoplastic calcium and attenuate host MAMP signaling . However, much of plant calcium signaling remains to be discovered, in particular, the steps between perception of MAMPs/DAMPs and generation of the calcium signals. We used an apoaequorin-expressing transgenic Arabidopsis thaliana line to investigate MAMP signaling events in whole seedlings . Aequorin is a calcium sensitive reporter for measuring changes in cellular calcium levels . Upon binding calcium, it oxidizes the bound coelenterazine prosthetic group into excited coelenteramide, which emits blue light at 469 nm. The so-called L/Lmax ratio of the aequorin-generated luminescence (L) to the total remaining aequorin (Lmax) is used as an estimate of relative calcium levels. With the appropriate calibration parameters, it is also possible to convert the L/Lmax values into absolute cytosolic calcium concentrations .
We previously demonstrated that the aequorin-based measurement is amenable to high throughput screening and used it to isolate mutants with a “changed calcium elevation (cce)” phenotype after flg22 elicitation. The first sets of identified cce mutants were the FLS2 receptor and its partner kinase, BAK1 . These mutants represent proof-of-principle of the suitability of the screen in finding signaling components between ligand recognition and calcium flux. This current work reports the characterization of the cce5 mutant and the identification of the receptor-like cytoplasmic kinase (RLCK), PBS1-like 1 (PBL1) being the CCE5 gene, where PBS1 stands for avrPphB sensitive 1, an RLCK targeted by the Pseudomonas syringae pv. phaseolicola protease avrPphB . The analysis of mutants of three related RLCKs revealed an additional requirement of Botrytis-induced kinase 1 (BIK1) for the MAMP/DAMP-induced calcium elevation.
The changed calcium elevation 5 (cce5) mutant is affected in early signaling
Differential MAMP/DAMP response of the cce5 mutants is reminiscent of a BAK1-dependent response, but CCE5 is not BAK1
CCE5encodes the receptor-like kinase, PBL1
PBL1 or PBS1-like 1 belongs to subfamily VII of RLCKs (Additional file 1: Figure S3) that include the founding member avrPphB-susceptible 1 (PBS1)  and the Botrytis-induced kinase 1 (BIK1) ,. To further validate that CCE5 is PBL1, a T-DNA insertion mutant of PBL1 was isolated. For comparison, T-DNA mutants of related members of this family of RLCKs shown to be involved in PTI (BIK1, PBS1, PBL2) , were also obtained. The T-DNA mutants were crossed with the cytosolic aequorin-expressing (pMAQ2 in Col-0 background) transgenic line. However, silencing of the aequorin reporter was observed in some crosses, and in these cases (i.e. for pbl2 and pbs1), an independently generated line with the apoaequorin expression driven by the UBIQUITIN10 promoter (pUBQ-AEQ in Col-0 background) was used for crossing.
Differential downstream responses in the pbl1 and/or bik1mutants
Kinase activities and proper localization of RLCKs determine downstream signaling
To test if the kinase activities have been affected, we immunoprecipitated the proteins with anti-HA antibodies and incubated the immunoprecipitates in the presence of radioactive ATP to enable autophosphorylation. After separation on a SDS-polyacrylamide gel, radioactive signals corresponding to the proteins could be seen for the wild type and the G2A mutated PBL1 and BIK1, suggesting that these are still active kinases (Figure 8C). Notably, the wild type BIK1 or PBL1 autophosphorylation signals are weak before flg22 treatment (highlighted with asterisks in Figure 8C, left panel). However, compared to the wild type PBL1 protein, there was no (or strongly reduced) autophosphosphorylation of the G70D, A97V or R172Q mutated PBL1 variants (Figure 8C). Taken together, these three cce5 mutations led to the loss of PBL1 kinase activity, while the mis-localization of PBL1G2A and BIK1G2A proteins prevented the in vivo phosphorylation of these kinases after flg22 signaling.
Specific RLCKs required for calcium signaling and MAMP/DAMP signaling
Using both forward and reverse genetics as well as complementation studies, we identified the CCE5 gene as encoding the receptor-like cytoplasmic kinase, PBL1, and with the cce5 mutants, we isolated five new pbl1 alleles. We further showed that another RLCK, BIK1, but not PBS1 and PBL2, is required for the MAMP/DAMP-induced calcium elevation pathway. This is in agreement with a recent report that flg22-induced calcium flux is compromised in bik1 . Using these new pbl1 alleles and T-DNA insertion mutants, a MAMP-mediated root growth inhibition assay confirmed the requirement of PBL1 for downstream signaling leading to growth arrest. However, the bik1 mutation had no apparent effect on flg22-mediated growth arrest. Thus, despite both pbl1 and bik1 mutants showing a reduced MAMP/DAMP-induced calcium elevation, downstream growth arrest effects differ. One explanation is that PBL1 and BIK1 are not simply redundant but also have distinct signaling roles, which is reinforced by other studies showing both overlapping and distinct requirements for PBL1 and BIK1 in MAMP-induced ROS production, callose deposition, gene expression and pathogen resistance ,,,. This notion is, in fact, evident from the different calcium signatures in the pbl1 and bik1 mutants (see Figure 5A). A second possibility is that a signaling critical threshold of cytoplasmic calcium level is not crucial or causal for determining the degree of growth inhibition (or other MAMP-induced responses), which would imply a more important signaling role of PBL1, compared to BIK1, in mediating the possible trade-offs between growth and defense activation. It is possible that the inverse roles of BIK1 as a positive regulator of defense but a negative regulator of brassinosteroid signaling  contribute to this growth differences. Recently, another RLCK, PBL27, was found to be the preferential substrate (as compared to BIK1) of the CERK1 chitin receptor. By contrast, BAK1, which is already known to phosphorylate BIK1, hardly phosphorylated PBL27. PBL27 also appears to be non-essential for flg22 signaling . Thus, depending on the ligand, different RLKs or RLCKs are recruited for signaling. These findings support the distinction of BAK1 requirement for flg22, elf18 and AtPep1 signaling to that of chitin ,. Our data on PBL1 requirement for optimal calcium signaling induced by flg22, elf18 and AtPep1, but not chitin (Figure 5), fits into this pattern. In conclusion, there appears to be a differential requirement for members of the RLCK family downstream of the receptors for distinct MAMP/DAMP signaling.
Phosphorylation is essential for signal relay
The recruitment (and/or exchange) of various RLKs and RLCKs at the plasma membrane after MAMP/DAMP perception is indicative of the roles of phosphorylation cascades in early signaling. Prior to stimulation, FLS2 and BIK1 are already in a protein complex  and BAK1 appears to be also in complex with BAK1-interacting RLKs (BIRs) . Within minutes after flg22 stimulation, FLS2 recruits BAK1  to phosphorylate BIK1. Activated BIK1, in turn, cross-phosphorylates FLS2 and BAK1 . BAK1 also cross-phosphorylates FLS2 but apparently at different residues as BIK1 . Based on the autophosphorylation assay (Figure 8C), the kinase activities of BIK1 and PBL1 appear to be higher in the flg22-treated protoplasts. The lower activities of BIK1/PBL1 in the untreated protoplasts may imply repression by some other components (eg. phosphatases) prior to elicitation. Along this idea, it is noteworthy that the N-terminal myristoylation BIK1G2A and PBL1G2A mutants are routinely recovered with higher autophosphorylation levels. One may speculate that mis-localization of PBL1G2A and BIK1G2A prevent contact with phosphatases that are presumably present in the FLS2-BIK1 (or PBL1) protein complex to restrict defense signaling. Indeed, Ser/Thr protein phosphatase type 2A (PP2A) has been shown to associate with BAK1 and control the activation of PRR complexes ; and whether the same or similar PP2As negatively regulate BIK1 or PBL1 remains to be demonstrated. Recently, it was shown that both BAK1 and BIK1 are dual-specific kinases that modify both serine/threonine as well as tyrosine residues . The complex series of phosphorylation between PRRs, BAK1, BIK1 and PBL1 are important as mutations abrogating activities of any of these kinases block signaling. As shown by mobility shifts in gel electrophoresis, the PBL1 protein, encoded by the CCE5 gene, is apparently phosphorylated in vivo after MAMP elicitation . The loss (or reduction) of the kinase activities of the cce5-derived protein variants reported here (Figure 8C) corresponds to changes of important residues of the kinase domain. The G70D and A97V mutations are found in the ATP binding region (i.e. the kinase subdomains I and II, respectively) while R172Q is N-terminal to the active center within kinase subdomain VI (c.f. Figure 4 and Figure 8C). Taken together with data from literature, the reduced calcium signaling of our newly isolated cce5/pbl1 alleles shows that kinase activities of PBL1 (and all the other recruited RLKS/RLCKs) are vital for early MAMP/DAMP signaling.
Is downstream MAPK activation affected in the pbl1 and bik1mutants?
Downstream of PBL1/BIK1 phosphorylation is calcium elevation, which, in turn, has been shown through pharmacological inhibitor studies to be required for downstream MAPK activation . However, despite an attenuation of calcium elevation, there was no reduction in MAPK activation by flg22, elf18 or AtPep1 in the pbl1 and bik1 T-DNA mutants as well as pbl1bik1 double mutant when compared to their Col-0 (pMAQ2) background line (Figure 7). This observation is in agreement with previous reports ,,. On the other hand, we observed a reduction/delay in elf18-induced MAPK activation in the cce5 mutants (Figure 1C), while for flg22, a weak effect could be observed when a lower concentration of the flg22 peptide was used. A possible explanation for this discrepancy may be that the mutated or truncated CCE5 proteins (with inhibitory properties) are expressed by the cce5 alleles as opposed to the (presumably) lack of proteins in the T-DNA insertion mutants. Alternatively, the different genetic background of the mutants may also play a role. In all studies where no difference in MAPK activation was observed, the mutations were in Col-0, while the cce5 (pbl1) mutants have a C24 background. We previously reported that the C24 accession has higher levels of FLS2 receptor while, on the basis of public gene expression profiling data, the opposite is true for the EFR receptor . Several SNPs were also detected within the FLS2 and EFR genes of HVA1 (C24) , which may further contribute to the different sensitivities to flg22 and elf18. Together, this may explain the observed reduced elf18-induced MAPK activation in the cce5 mutants but only a dose-dependent flg22-induced MAPK response. Along this notion, Zhang et al.  also reported that callose deposition in the pbl1 mutant was normal upon flg22 treatment but reduced when treated with elf18 . Thus, the differential MAMP receptor levels and/or yet unknown alterations in other signaling components between genotypes may determine the sensitivity of the system in signal relay from PBL1 to the MAPKs and other downstream events. Additionally, the RLCK, RIPK, phosphorylates RIN4 and in analogy to the recognition of the RIN4 phosphorylation by the RPM1 resistance protein as compared to recognition of RIN4 cleavage by RPS2 , one could speculate that the MAMP-induced RLCK phosphorylation may also be differentially recognized by the differing configuration of resistance protein spectrum between Arabidopsis accessions.
Localization to the membrane is a prerequisite for PBL1 and BIK1 function
Besides its kinase activities, localization of PBL1 and BIK1 appears to be important for complementation of the cce phenotype. BIK1, as a GFP fusion protein, has been shown to be plasma membrane-localized using heterologous expression in onion epidermal cells . However, to our knowledge, it has never been experimentally determined whether this is due to targeting or recruitment by other proteins. CASTAWAY, an RLCK required for organ abscission, showed reduced plasma membrane localization when a G2A mutation was introduced for the putative myristoylation site. Myristoylation is often associated with palmitoylation to enhance membrane interactions. However, no further decrease of CASTAWAY localization was observed even when the neighboring palmitoylation site (C4S) was additionally mutated . Myristoylation is thought to provide the initial but weak interaction with the plasma membrane; stabilization of this membrane localization may be further strengthened through other modifications or interactions with resident plasma membrane-localized components . We now show that the G2A mutation of the putative myristoylation site of PBL1/BIK1 prevented signaling (i.e. no in vivo phosphorylation after MAMP treatment, Figure 8) although kinase (autophosphorylation) function is apparently intact. Furthermore, the G2A variant did not complement the pbl1 mutation (Figure 9). In a similar manner, despite being cleaved by the avrPphB cysteine protease, the “active” PBS1 fragment that is recognized must be retained at the plasma membrane for RPS5 activation . In this case, plasma membrane targeting is mediated by S-acylation of a cysteine residue in the N-terminus of PBS1. Thus, not only the function of the PBS1 protein but also the recognition of its perturbation (i.e. ETI) requires correct membrane localization. Taken together, localization of the RLCKs to the proper cell compartment is crucial for function.
Disabling RLCK functions during pathogenesis blocks defense signaling
PBS1 is the founding member of the PBL (PBS1-like) group. The Pseudomonas avrPphB effector, a bacterial virulence protein injected into host cells, cleaves PBS1 via its cysteine protease activity . Subsequently, it was discovered that avrPphB can cleave at least 10 other PBS1-like RLCKs . Their cleavage/removal represents a virulence function of avrPphB and suggests that these PBLs/RLCKs act in resistance mechanisms against bacteria. Support for this notion is provided by studies involving the Xanthomonas XopAC effector, which appears to target multiple RLCKs . Unlike avrPphB, XopAC does not cleave but uridylates BIK1 and RIPK in susceptible plants . This transfer of uridine 5′-monophosphate to conserved phosphorylation sites in the activation loop of BIK1 and RIPK, prevents phosphorylation, thereby reducing their kinase activities and consequently inhibiting downstream defense signaling. In accordance to the arms-race hypothesis, XopAC appears to be a major avirulence factor for recognition in resistant plants such as the Arabidopsis Col-0 accession. Furthermore, there is more growth of Xanthomonas campestris pv. campestris expressing XopAC in the pbl2 background. This suggests that the RLCK, PBL2 is required for the XopAC-triggered immunity . These observations of various pathogen effectors targeting RLCKs are in line with the presumed importance of RLCKs in defense signaling. However, although avrPphB cleaves multiple PBLs/RLCKs , only PBS1 cleavage is recognized by RPS5 ,. We report here that PBL1 and BIK1, but neither PBL2 nor PBS1, are required for MAMP/DAMP-induced calcium signaling (Figure 5). This raises the question of “why avrPphB would target PBS1” if PBS1 is not important for MAMP-induced calcium signaling. In fact, there is no evidence so far for any importance of PBS1 in pathogen resistance. As proposed by Zhang et al. , one idea is that PBS1 may be a “decoy”  evolved to recognize perturbation of the real targets of the pathogen effectors. In this case, PBL1 and BIK1 would be such real avrPphB targets, which are relevant for cross-phosphorylation of the receptor complex components and the subsequent triggering of calcium and downstream defense signaling.
In summary, we showed the requirement for the two RLCKs, PBL1 and BIK1, in MAMP/DAMP-induced calcium signaling, and speculated on possible genotype variations that may differentially contribute to downstream signaling events. There are many more RLCK genes in the genome (Additional file 1: Figure S3) and it remains to be investigated whether these also affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. The large number of available RLCKs is presumably mirrored by an even wider repertoire of their downstream substrates. Besides phosphorylating receptor complex components, BIK1 was recently shown to target the NADPH oxidase, RBOHD, to control oxidative burst in a calcium-independent manner ,. Thus, a future challenge would be to identify the substrates of these various RLCKs and elucidate their role in cellular signaling.
Plant lines and cultivation conditions
The Arabidopsis thaliana lines pMAQ2 in Col-0 background and HVA1 in C24 background were obtained from M. and H. Knight . These lines express the apoaequorin gene under the control of the cauliflower mosaic virus 35S promoter. In the case of HVA1, the aequorin is targeted as a pyrophosphatase (H + −PPase)-apoaequorin fusion protein to the cytoplasmic-face of the tonoplast in the so-called vacuolar microdomain (vmd); thus enabling measurements of calcium changes at this vacuolar vicinity. For the backcross shown in Figure 2, the HVA1 (C24) line was first retransformed with pMAQ2 construct to obtain a line expressing additionally cytosolic apoaequorin (designated HVA1-P). This HVA1-P line was then crossed to the cce5 mutants. T-DNA lines used in this study are listed in the Additional file 1: Table S1). Plants for ROS assays were grown on soil in climate chambers under short day conditions (8 h light, 16 h dark cycles). For calcium and MAPK assays, seeds were surface-sterilized, stratified at 4°C for >2 d and grown in liquid MS under long day conditions (16 h light, 8 h dark cycles) as described .
Seed sterilization, growth of seedlings and other experimental set-up for the calcium measurements in a 96-well plate format was performed as described .
ROS, MAPK and growth inhibition assays
Detection of early MAMP-triggered responses such as MAPK activation and reactive oxygen species (ROS) accumulation was performed as described . As a late response to MAMPs, growth inhibition assay was performed as described . Briefly, seedlings were grown vertically on ATS agar plates with or without 1 μM flg22 for 14 days. To distinguish between growth differences due to treatment versus genotype effects, two-way ANOVA was performed on log2-transformed root length data (genotype vs. treatment; p < 0.001; R statistical package) . For a more compact and simplified overview, data in Figure 6 were depicted as percent growth inhibition compared to control.
Transient expression in protoplasts, immunoprecipitation and autophosphorylation
Transient expression in Arabidopsis protoplasts was performed as described . For each sample, 1 ml of protoplasts (~2 x 105 protoplasts ml−1) was transformed; of which 300 μl were kept for western blot analysis of protein expression. The remaining 700 μl were used for immunoprecipitation. Proteins were extracted from the transfected protoplasts as described except that the extraction buffer was supplemented with 1% Triton X-100 . The proteins were incubated with anti-HA (Covance) and protein-G-sepharose (for at least 2 h, 4°C). Washing of the beads was performed as in Lee et al. , with centrifugation in between washes to pellet the sepharose beads. Finally, sepharose beads with the immunoprecipitated proteins were resuspended in 20 μl of kinase buffer (20 mM Hepes pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM DTT); 5 μl was kept for a western blot to confirm recovery of the HA-tagged proteins. γ32P-ATP (3000 Ci mmol−1) (0.1 μl) was added to the remaining 15 μl and incubated at 30°C for 1 h to initiate autophosphorylation. Five μl of 4xSDS-loading buffer was added to the beads, incubated at 95°C for 5 min, and 12 μl loaded on a 10% SDS-PAGE. After electrophoresis, the gel was dried, exposed overnight and analyzed by Phosphorimaging.
An F2 population was generated from the cross of cce5-1 (C24) with the Arabidopsis ecotype Landsberg erecta (Ler-0). F2 plants containing the aequorin transgene (i.e. showing coelenterazine-dependent luminescence) were selected and selfed. DNA was isolated from leaves of F2 plants or from pooled F3 seedlings, and 3 to 5 markers (SNPs, INDELs) of each chromosome were genotyped. Calcium measurements were performed with the F3 seedlings and the segregation of the phenotype used to infer if the corresponding F2 parent is heterozygous or homozygous for the cce5 mutation.
Molecular cloning, plant transformation and complementation
For complementation analysis, a genomic fragment covering the PBL1-ORF and 2 kb upstream cis-regulatory region was amplified by PCR using Phusion® Hot Start High-Fidelity DNA polymerase (Thermo Scientific) with primers PBL1-Prom/-STOP and cloned into pENTR™/D-TOPO according to manufacturer’s instructions. Mutation of the N-myristoylation site (G2A) was performed using the QuikChangeII-Kit (Stratagene) with primers PBL1-NMSmut-F/-R according to manufacturer’s instructions. Clones were verified by sequencing and transferred via LR reaction into destination vector pGWB1 to obtain pPBL1::PBL1 and pPBL1::PBL1(G2A). After transfer of the constructs into Agrobacterium tumefaciens (GV3101), Arabidopsis pbl1 mutant plants were transformed by floral-dip transformation. Transgenic plants were selected on hygromycin-containing plates and crossed with pbl1-AEQ to introduce the apoaequorin transgene. For transient expression in protoplasts, PBL1- and BIK1-ORFs were amplified from cDNA obtained from Col-0 or the indicated cce5 alleles by PCR using Phusion® Hot Start High-Fidelity DNA polymerase (Thermo Scientific) with primers PBL1-START/-NoSTOP and BIK1-START/-NoSTOP and cloned into pENTRTM/D-TOPO according to manufacturer’s instructions. Mutation of the N-myristoylation site (G2A) was introduced using primers PBL1-STARTmut and BIK1-STARTmut. Clones were verified by sequencing and transferred via LR reaction into destination vector pUGW14 to obtain p35S::PBL1-3xHA, p35S::BIK1-3xHA, p35S::PBL1(G2A)-3xHA and p35S::BIK1(G2A)-3xHA.
The aequorin-ORF was amplified from plasmid pMAQ2 by PCR using Phusion® Hot Start High-Fidelity DNA polymerase (Thermo Scientific) with primers AEQ-START/-STOP, cloned into pENTRTM/D-TOPO according to manufacturer’s instructions, verified by sequencing and transferred via LR reaction into destination vector pUB-DEST to obtain pUBQ10::AEQ. After transfer of the construct into Agrobacterium tumefaciens (GV3101), Arabidopsis Col-0 plants were transformed by floral-dip transformation and transgenic plants were selected by spraying with BASTA® (glufosinat-ammonium; Bayer). All primers used for cloning are listed in the Additional file 1: Table S3).
Availability of supporting data
All the supporting data are available within the article or as additional files. The phylogenetic tree (Additional file 1: Figure S3) has been deposited in treebase (ID: 16757) and the data will be available at the following URL: http://purl.org/phylo/treebase/phylows/study/TB2:S16757.
This work is financed by the German Research Foundation, through the priority “SPP1212 Plant Micro” program to J.L (LE 2321/1-3) and the Collaborative Research Center program SFB648 to J.L. and D.S. L.E.-L. is supported within the ProNet-T3 program (03ISO2211B). We thank Nicole Bauer, Marina Häußler and Siska Herklotz for excellent technical assistance.
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