Phosphorylation of the CAMTA3 transcription factor triggers its destabilization and nuclear export

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3’s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes. One sentence summary Treatment with flg22 activates two independent kinase pathways that effect CAMTA3 phosphorylation and degradation.

The Arabidopsis calmodulin-binding transcription activator 3 (CAMTA3, AT2G22300, also 109 abbreviated as SR1) belongs to a six-membered gene family (Finkler et al., 2007). Key conserved 110 domains include an N-terminal CG-1 domain that binds a conserved 6-bp motif (with the consensus 111 sequence (A/C/G)CGCG(G/T/C)) in the promoter of target genes (Yang and Poovaiah, 2002) and two 112 IQ motifs and a CaMBD near the C-terminus, which are thought to mediate Ca 2+ -independent and -113 dependent calmodulin binding, respectively (Finkler et al., 2007;. Transcriptomics 114 analysis of two T-DNA insertion knockout mutants (camta3-1 and camta3-2) revealed up-regulation of 115 many defense genes (e.g. PRs, NDR1, PAD4, ZAT10 and various WRKYs) (Galon et al., 2008). Under CAMTA3 is alleviated through its degradation. This proteasome-dependent degradation of CAMTA3 127 is promoted via interaction with SR1IP1 (SR1 Interaction Protein 1), a substrate-adaptor for cullin3-128 based E3 ubiquitin ligase ). Contrary to above-described understanding of CAMTA3 129 function as a negative regulator, the enhanced defense phenotype of camta3 is recently reported to be a 130 form of ETI-like autoimmunity triggered by the activation of two nucleotide-binding domain leucine-131 rich repeat-containing (NLR) proteins (Lolle et al., 2017). A recent transcriptome analysis further 132 pinpoints CAMTA3 as an early convergence point between PTI and ETI (Jacob et al., 2018). Thus, 133 despite ambiguity about its negative regulator function, CAMTA3 has been independently isolated in a 134 number of disease resistance genetic screens and is clearly important for plant immunity 135 Jing et al., 2011;Nie et al., 2012). 136 In contrast to its role as a repressor, CAMTA3 was initially reported to act as transcriptional activator 137 in cold stress response. For instance, expression of the cold-responsive gene CBF2 (C-repeat-Binding 138 factor 2) is positively regulated by CAMTA3 in response to cold stress (Doherty et al., 2009). It was 139 also found to be a transcriptional activator in general stress response and regulation of glucosinolate 140 metabolism (Laluk et al., 2012;Benn et al., 2014). Therefore, CAMTA3 appears to function as a 141 transcriptional repressor for immunity-related genes but acts as a transcriptional activator when bound 142 to promoters of genes involved in cold acclimation or other general stresses. How CAMTA3 switches 143 between activator and repressor functions remains unclear. One possibility is through molecular 144 interactions, either with other proteins or intra-molecularly. A recent study showed that in unstressed 145 plants, CAMTA3 represses transcription of SA pathway genes through an N-terminal repression module 146 (NRM) of CAMTA3; cold stress promotes CaM binding to IQ and/or CaMB domains, resulting in 147 conformation change that interferes with the repressor activity of the NRM (Kim et al., 2017). 148 Additionally, post-translational modifications such as phosphorylation may also be involved (Zhang et 149 al., 2014). Proteomics analysis identified CAMTA3 as a putative MPK3/6 substrate (Hoehenwarter et 150 al., 2012). In addition, in the PhosPhAt database (an Arabidopsis database for experimentally 151 determined phosphorylation sites) (Heazlewood et al., 2008), five phosphopeptides are annotated, which 152 contain typical MAPK phosphorylation sites. 153 In this work, we investigated CAMTA3 phosphorylation and how this affects its function. We show that 154 CAMTA3 is indeed phosphorylated by flg22-responsive MAPKs, which promotes its destabilization. 155 We could not confirm physical interaction between CAMTA3 and SR1IP1 but show that they have 156 distinct cellular localization and therefore cannot physically interact prior to PTI activation. Finally, we 157 also uncovered that there are additional flg22-responsive kinases that phosphorylate CAMTA3, which 158 may be a CDPK or another kinase downstream of CDPK. 159 160

CAMTA3 negatively regulates defense-related gene expression 162
Datamining of expression databases (Hruz et al., 2008) indicated that besides salicylic acid, treatment 163 with PAMPs (e.g. flg22, elf26 or chitin) induces the accumulation of CAMTA3 transcripts. By RT-164 qPCR, we confirmed the increase in CAMTA3 expression in Arabidopsis thaliana Col-0 seedlings 165 stimulated with flg22 (Fig. 1A). CAMTA3 mRNA levels increased rapidly within 30-60 min of flg22 166 treatment and subsided to near basal levels after ~4 h. Since CAMTA3 is a transcription factor, this 167 increased expression may contribute to the flg22-induced transcriptional response. CAMTA3 is known 168 to regulate expression positively, such as for the cold-responsive gene, CBF2 (Doherty et al., 2009), or 169 negatively, as was shown for several defense genes, e.g. EDS1 , NDR1 and EIN3 (Nie 170 et al., 2012). Notably, promoters of all of these genes contain CGCG box as cis-elements. To investigate 171 the effect of CAMTA3 on PAMP-regulated gene expression, CAMTA3 overexpression (OE) lines were 172 generated. The enhanced basal expression (i.e. in the mock treated tissues) of CAMTA3 in two 173 independent OE lines was confirmed by RT-qPCR. Flg22 treatment further raised CAMTA3 expression 174 in the OE lines (Fig. 1B), which is presumably the sum of expression arising from the transgene and the 175 inducible expression of the endogenous gene. Next, we analyzed the effect of CAMTA3 on expression 176 of EDS1. As expected, camta3 mutants show high basal EDS1 transcript levels and CAMTA3 177 overexpression reduced EDS1 transcript accumulation. Flg22-induced EDS1 expression was enhanced 178 in the camta3 mutants and reciprocally, it was reduced in the two independent OE lines (Fig. 1C). A 179 similar effect was also shown on NHL10 (Fig. 1D), which has not been reported as a direct CAMTA3 180 target but includes a potential CAMTA3-binding site in its promoter. These observations are consistent 181 with CAMTA3 functioning as a negative regulator (repressor) during plant immunity response. The Phosphorylation has been speculated to be involved in the destabilization of CAMTA3 after bacterial 202 infection . We therefore investigated phosphorylation status upon flg22 elicitation. 203 Here, we employed Manganese (II)-Phos-Tag TM -based western blot analysis, where phosphorylated 204 forms show retarded mobility in the gels compared to the non-phosphorylated proteins (Kinoshita et al., 205 2009). Unlike the well-defined single band in standard PAGE, CAMTA3 from unstressed or H2O treated 206 protoplasts already appears as broad smeary bands on Phos-Tag-based western blot, thus suggesting that 207 it exists as several partially phosphorylated forms. By contrast, a mobility shift was seen for CAMTA3 208 from flg22-treated protoplasts within 10 min of treatment (Fig. 2B, upper panel). λ-phosphatase 209 treatment of the protoplast extracts (Fig. 2B, lower panel) abrogated this mobility shift, suggesting that the smeary bands or reduced mobility bands are due to phosphorylation. Taken together, the Phos-Tag 211 and the phosphatase analyses indicate an in vivo flg22-induced CAMTA3 phosphorylation. 212 By contrast, cold stress (transfer to ice-cold media and placed into a 4°C cold room) did not induce any 213 CAMTA3 degradation within the tested time period and in fact, there appeared to be enhanced protein 214 stability (or less protein degradation) despite termination of protein translation by CHX treatment (Fig.  215 2C). This is in line with CAMTA3 functioning as a transcriptional activator for cold stress (Doherty et 216 al., 2009). Cold treatment also did not lead to phospho-mobility shift of CAMTA3 in the Phos-Tag 217 analysis (Fig. 2D, upper panel), where MAPKs were correspondingly not activated within the tested 218 time points (Fig. 2D, lower panel). Taken together, unlike PAMP (flg22) elicitation, cold stress does not 219 trigger CAMTA3 phosphorylation and degradation, suggesting differential mechanism exists to 220 determine positive or negative functions of CAMTA3 in these distinct stress context. 221

CAMTA3 interacts with and is phosphorylated by PAMP-responsive MAPKs 223
MAPKs are rapidly activated after flg22 treatment and are thus candidate kinases responsible for the 224 autophosphorylation of the kinases were seen, CAMTA3 was phosphorylated by MPK3 and MPK6, but 232 barely by MPK4 ( Fig 3B). on the protein levels were then monitored by Phos-Tag or standard western blot, respectively. CAMTA3 238 was phospho-shifted when co-expressed with CA-MPK6 and accumulated to lower levels. For CA-239 MPK3 co-expression, some phosphorylation (but weaker compared to MPK6) was observed but a strong 240 CAMTA3 destabilization was seen. Consistent with in vitro kinase assay, CA-MPK4 co-expression did 241 not induce CAMTA3 phospho-shift (Fig. 3C). Thus, MPK4 is probably not involved in inducing in vivo 242 phosphorylation and destabilization of CAMTA3. However, some caveats to the above interpretations 243 include: (1) the CA-MPK4 displayed lower autophosphorylation activity than the other two kinases; (2) 244 it cannot be excluded that substrate specificities or activities of these mutated MAPKs are comparable 245 to the MAPKs natively activated through upstream MAPK kinases. 246 To validate our results, we transiently expressed in protoplasts a constitutively active MKK5 (MKK5 DD ) 247 to specifically activate endogenous MPK3 and MPK6 but not MPK4 (Lee et al., 2004;Lassowskat et 248 al., 2014). In this case, MPK3/6 are activated "naturally" through phosphorylation of its kinase 249 activation loop and not through mutation. Similar to observations above for CA-MPK3 and CA-MPK6, 250 CAMTA3 protein level was reduced through MKK5 DD co-expression. The phosphorylated CAMTA3 251 proteins are difficult to visualize in the Phos-Tag gels since the strongly reduced protein levels are 252 further separated into smeary, barely detectable, bands. However, the slower mobility of these fuzzy 253 bands is in agreement with MPK3/6-mediated phosphorylation. Control transfection without any MKKs 254 or co-transfection with a kinase-inactive MKK5 (MKK5 KR ) did not show these effects (Fig. 3D). Hence, 255 the results above suggest that two out of three known flg22-responsive MAPKs phosphorylate 256 CAMTA3, and in vivo flg22-induced CAMTA3 destabilization may be triggered by phosphorylation 257 via MPK3 and MPK6. 258 259 CAMTA3 is phosphorylated by PAMP-responsive MPK3/6 at multiple sites, which contribute to 260

CAMTA3 protein destabilization 261
After confirming in vitro and in vivo CAMTA3 phosphorylation by MPK3 and MPK6, we proceeded to 262 determine the phospho-sites in CAMTA3. CAMTA3 contains 11 potential MAPKs phosphorylation 263 sites, which are serine or threonine followed by a proline (S/TP) ( To increase MS sequence coverage of CAMTA3 we repeated the experiment but digested the protein 279 with both trypsin and endoproteinase Glu-C prior to MS analysis. This resulted in more than 60% 280 sequence coverage that encompass most of the SP/TP motifs, leading to the identification of 281 phosphorylation at T243, S587 and S780 (Fig. S1). Notably, T243 (Fig. 4A, marked in green) is a novel 282 phospho-site, never detected in any previous reports. When the phospho-mutant CAMTA3-mutP2+ 283 (containing phospho-site mutations at S8, T243, S272, S454, S587 and S780) was tested, in vitro MPK3-284 or MPK6-mediated phosphorylation was strongly reduced compared to CAMTA3-WT (Fig. 4B), so that 285 these six sites are likely the major MPK3/6-targeted sites. We also generated CAMTA3-mutP3 (as a 286 phospho-null for all 11 potential phospho-sites) and the corresponding phospho-mimetic mutant 287 (CAMTA3-mimic), in which all 11 potential sites (S/T) were substituted by aspartic acids (D) (Fig. 4A). 288 Phosphorylation of these variants was completely lost (Fig. 4B), which also proved that MPK3/6 did 289 not unspecifically phosphorylate other residues in CAMTA3. 290 To investigate the role of phosphorylation in protein destabilization, we compare the stability of the 291 different CAMTA3 phosphosite variants. Co-expression of MKK5 DD (or the kinase-inactive MKK5 KR 292 variant as a control) was employed to investigate the actions of MPK3/6. A quantitative western blot 293 system (Li-COR Odyssey® CLx multiplex imaging system) was used to determine the protein levels in 294 five replicates (consisting of independent protoplast samples). differences in protein stability may be partially masked. However, note that basal expression of all the 300 phospho-site mutants are higher levels than the CAMTA3-WT while the opposite is true for the 301 CAMTA3-mimic ( Fig 4D). Thus, the MAPK-targeted phospho-sites may be crucial for maintaining 302 protein stability. 303 As seen in the representative western blot (Fig. 4C), the mutP2+ and mutP3 phospho-mutants appear 304 slightly more stable than the CAMTA3-WT protein after MPK3/6 activation, which is substantiated by 305 statistical analysis for the mutP3 variant. Although interpretation for mutP2+ lacked statistical 306 robustness, this may be a type II (false negative) statistical error because we do see this qualitative 307 difference in most of the independent replicates. As seen in the recalculation of the percent decrease 308 The potential mechanism behind CAMTA3 destabilization through phosphorylation is still unknown. A 313 previous study proposed that the CAMTA3-interacting SR1IP1 recruits CAMTA3 for ubiquitination and degradation by the 26S proteasome . To test the involvement of SR1IP1, we 315 aimed to check if phosphorylation affected SR1IP1-CAMTA3 interaction. We performed several assays 316 including yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and split-assays in 317 transfected Arabidopsis protoplasts (Fig. S2), but none of the assays supported interaction between 318 SR1IP1 and CAMTA3. 319 320

MAPK-induced phosphorylation triggers CAMTA3 subcellular relocalization 321
To clarify the discrepancy to the literature, we looked at the cellular distribution of the two proteins. 322 CAMTA3 is a transcription factor and mainly localized in the nucleus (Yang and Poovaiah, 2002), 323 which we could also confirm in unstressed protoplasts using a CAMTA3-YFP fusion construct ( Thus, the recruitment of the reporter to the plasma membrane may permit interaction. Nevertheless, the 330 distinct localization of CAMTA3 and SR1IP1 suggests that they cannot physically interact unless there 331 is a re-localization of either proteins from the nucleus or the plasma membrane, respectively. 332 Interestingly, the CAMTA3-YFP signal relocated from nucleus to the cytoplasm after flg22 elicitation, 333 and multiple cytoplasmic aggregates were often observed ( Fig. 5B; see additionally the time course 334 experiment in Fig S3A). The aggregates are not cleaved YFP fragments since western blot analysis 335 revealed intact CAMTA3-YFP protein bands (Fig. S3B). By contrast, SR1IP1 localization was not 336 visibly affected by flg22 treatment. Although the CAMTA3 relocalization into the cytoplasm may 337 potentially allow interaction with SR1IP1 and indeed, there seems to be partial co-localization between 338 CAMTA3-YFP and SR1IP1-CFP after flg22 treatment (Fig. 5B), we never obtain any BiFC or split-339 LUC signals between CAMTA3 and SR1IP1 even upon flg22 elicitation (Fig. S2). Nonetheless, we 340 cannot exclude that CAMTA3-SR1IP1 does interact upon flg22 elicitation but is degraded too rapidly 341 for detection of the complex. 342 Notably, activation of MPK3/6 (through MKK5 DD co-expression) also induced the CAMTA3 343 relocalization to the cytoplasm (Fig. 5B, left lower panels), thus phenocopying the effects of flg22 344 elicitation. The non-phosphorylatable CAMTA3-mutP3 remained nuclear-localized with the MKK5 DD 345 co-expression (Fig. 5B, right lower panels), demonstrating that the mutated phospho-sites are crucial for 346 the relocalization and that CAMTA3 nuclear export can be triggered by its MPK3/6-mediated 347 phosphorylation. On the other hand, these phospho-sites also appear to be irrelevant for the flg22 348 response since the CAMTA3-mutP3 mutant still re-localized to the cytoplasm upon flg22 treatment ( were transiently co-expressed with CAMTA3 in the protoplasts, most of them induce a phospho-shift 370 of CAMTA3 and, in part, a change in CAMTA3 protein levels (Fig. 6B). According to the quantitative 371 western blot and statistical analysis, CAMTA3 protein stability was significantly reduced by co-372 expression of CPK1, CPK2, or CPK5, while the tested members from subfamily III and IV did not 373 reduce but increased CAMTA3 levels significantly (Fig. 6B, lower panel). Note that although through 374 overexpression, many of the constitutively-active CDPKs can biochemically modulate CAMTA3  Thus, we tested the role of CPK5 by transiently expressing CAMTA3-WT (or mutP3) together with 381 CPK5-VK or a kinase-deficient version (CPK5m-VK) into protoplasts. CPK5-VK expression led to a 382 strong destabilization of CAMTA3. Extended exposure of the western blots was necessary to visualize 383 the low levels of CAMTA3-WT or mutP3. Importantly, the strong phospho-shift of CAMTA3 induced by CPK5-VK is comparable to the shift induced by flg22 stimulation (Fig. 6C) shown to interact with CAMTA3 as well (Fig. 7A, upper panel), which indicates that the interaction 394 between CAMTA3 and CPK5 is not dependent on its kinase activity. BiFC signals co-localized with a 395 presented here support the latter, i.e. CAMTA3 is a negative regulator of defence genes, where 413 CAMTA3 (overexpression) suppresses both the basal and flg22-induced expression of defence genes 414 (Fig 1C,D). Furthermore, upon flg22 perception, activated MPK3 and MPK6 can phosphorylate 415 CAMTA3 directly, which promotes CAMTA3 degradation via a proteasome-dependent pathway and 416 also the relocalization of CAMTA3 from the nucleus to cytoplasm. These two processes can thus lead 417 thus represent a negative feedback mechanism to replace the degraded CAMTA3 proteins and shut down 419 defence gene expression. 420 Besides MAPKs, additional flg22-responsive kinase(s) can induce CAMTA3 phosphorylation that 421 strongly promotes its degradation. This is most likely an unknown kinase downstream of CPK5; 422 although we cannot exclude that CPK5 can directly phosphorylate CAMTA3 under optimal conditions 423 (such as the presence of additional co-factors). Taken together, our findings suggest that CAMTA3 may  In agreement to pathogen-induced CAMTA3 degradation, we show that flg22-induced CAMTA3 452 phosphorylation leads to its in vivo destabilization and propose this may de-repress expression of 453 downstream target genes. Equally possible would be a direct phospho-dependent reduction in DNA 454 binding properties of CAMTA3. If phosphorylation is involved, only allosteric effects can be expected 455 since the key MAPK-targeted phosphosites are not within the DNA-binding domain (see Fig. 4A). 456 Unfortunately, we were unable to obtain convincing EMSA (electrophoretic mobility shift assay) data 457 proving specific CAMTA3 binding to "CGCG"-core containing DNA probes (Fig S4) In conclusion, the isolation of CAMTA3 mutants in numerous independent genetic screens for 512 pathogen/stress responses coupled with its post-translational control through (direct/indirect) 513 phosphorylation by two major stress-responsive kinase pathways, namely MAPKs and CDPKs, 514 highlights it as a node in abiotic and biotic stress signalling network. The convergence between 515 environmental (cold) and pathogen (or other biotic stresses) signalling on CAMTA3 suggests it may 516 control the interplay and trade-offs between growth and stress response. There are still many gaps in the 517 understanding of how CAMTA3 navigates these different roles but multiple phosphorylation and 518 nucleo-cytoplasmic trafficking may be involved.

Plant growth conditions, protoplast assays and immunoblot analysis 541
Arabidopsis thaliana plants were grown on soil in climate chambers for 5-6 weeks (22° C; 8 h of light, 542 16 h of darkness; 140 µE). Protoplast isolation and transfection were performed as described (Yoo et 543 al., 2007). Proteins were extracted by directly adding SDS-loading buffer to the pelleted protoplasts and 544 processed for immunoblotting as described (Lee et al., 2004).  Table S1 and S4. Site-551 directed mutagenesis was performed using the primers listed in Table S2 as previously described (Palm-552 554

Real-time PCR analysis 555
Total RNA was extracted from plant tissues using TRIzol reagent (Roth). 2 µg of RNA was used for 556 cDNA synthesis (Thermo Fisher Scientific). Diluted cDNA (1:10) was used for real-time PCR following 557 the manufacturer's protocol from 5×QPCR Mix EvaGreen® (ROX) (Bio & Sell). Primers and probes 558 are listed in Table S3. The PCR was performed in MX3005P cyclers (Agilent), and the program 559 After wash steps, target proteins were imaged (800 nm channel) as described above. To compensate for 594 loading differences, the target protein signals (800 nm) were normalized against the total protein 595 quantification (700 nm) according to the Odyssey® CLx Application Protocol Manual (Li-COR).

Protein immunoprecipitation from protoplasts 607
To obtain CPK5 or constitutively-active MPKs for kinase assays, protoplasts were transfected with the 608 corresponding constructs and proteins were immunoprecipitated after an overnight expression period. 609 The following plasmids were used: pEXSG encoding CPK5-FL-strepII or CPK5m-FL-strepII (Dubiella peptides were separated using C18 reverse phase chemistry employing a pre-column (EASY column 638 SC001, length 2 cm, ID 100 μm, particle size 5 μm) in line with an EASY column SC200 with a length 639 of 10 cm, an inner diameter (ID) of 75 μm and a particle size of 3 μm on an EASY-nLC II (all from 640 Thermo Fisher Scientific). Peptides were eluted into a Nanospray Flex ion source (Thermo Fisher 641 Scientific) with a 60 or 120 min gradient increasing from 5% to 40% acetonitrile in ddH2O with a flow 642 rate of 300 nl/min and electrosprayed into an Orbitrap Velos Pro mass spectrometer (Thermo Fisher 643 Scientific). The source voltage was set to 1.9 kV, the S Lens RF level to 50%. The delta multipole offset 644 was -7.00. The AGC target value was set to 1e06 and the maximum injection time (max IT) to 500 ms specificity was set to trypsin and two missed cleavages were tolerated. Carbamidomethylation of 654 cysteine was set as a fixed modification and oxidation of methionine and phosphorylation of serine and threonine as variable modifications. The precursor tolerance was set to 7 ppm and the product ion mass 656 tolerance was set to 0.8 Da. A decoy database search was performed to determine the peptide spectral 657 match (PSM) and peptide identification false discovery rates (FDR). Phosphorylated peptides with a 658 score surpassing the false discovery rate threshold of 0.01 (q-value<0.01) were considered positive 659 identifications. The phosphoRS module was used to specifically map phosphorylation to amino acid 660 residues within the primary structure of phosphopeptides.  Table S4  Supplemental Information 689 Figure S1 MS2 spectra of peptides with phosphorylated site found in CAMTA3. 690 Figure S2 Yeast-or plant-based interaction assays between CAMTA3 and SR1IP1. 691 Figure S3 Flg22 treatment induces subcellular re-localization of CAMTA3. 692 Figure S4 EMSA for in vitro binding of full-length CAMTA3 to a "CGCG-containing" promoter 693 fragment of EDS1. 694 Figure S5 CAMTA3 is not phosphorylated by CPK5 in vitro. 695 Figure S6 Compilation of western blots validating the expression of intact fusion proteins for the BiFC 696 or co-localization experiments. 697 698 699 Table S1: Primers for cloning into entry vector pENTR/D 700 Table S2: Primers used for site-directed mutagenesis of CAMTA3 in entry vectors 701 Table S3: Primers and probes for RT-qPCR 702

869
Cold treatment was performed as described in C and the proteins analyzed for phospho-mobility shift of CAMTA3 after     Table summarizes 903 the different phosphosite mutations generated in this study. (Note substitution to alanine was used to generate non-904 phosphorytable residues in all cases except at S8, S198, S272, T243, S469 and T736, where glycine was used due to the