a tomato universal stress protein involved in oxidative ... · 5 130 introduction 131 132...

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Running head 1 SlRd2 phosphorylation and role in stress defense 2 3 4 Corresponding Author 5 Olga del Pozo 6 Instituto de Bioquiacutemica Vegetal y Fotosiacutentesis Universidad de SevillaConsejo Superior de 7 Investigaciones Cientiacuteficas Avda Ameacuterico Vespucio 49 41092 Sevilla Spain 8 9 Email olga_delpozoibvfcsices 10 11 Phone +34 954489518 12 Fax +34 954460065 13 14 Research Area 15 Signaling and Response 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Plant Physiology Preview Published on November 29 2016 as DOI101104pp1600949

Copyright 2016 by the American Society of Plant Biologists

wwwplantphysiolorg on December 1 2016 - Published by wwwplantphysiolorgDownloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved

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33 Title 34 A Universal Stress Protein Involved in Oxidative Stress is a Phosphorylation Target for protein kinase 35 CIPK6 36 Authors 37 Emilio Gutieacuterrez-Beltraacuten Joseacute Mariacutea Personat Fernando de la Torre and Olga del Pozo 38 39 Institution Address 40 Instituto de Bioquiacutemica Vegetal y Fotosiacutentesis Universidad de SevillaConsejo Superior de 41 Investigaciones Cientiacuteficas Avda Ameacuterico Vespucio 49 41092 Sevilla Spain 42 43 44 One-Sentence Summary 45 Universal stress protein SlRd2 is a Cipk6 target and regulates Cipk6-mediated ROS 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Footnotes 64


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This work was funded in part by the European Regional Development Fund through the Ministerio 65 de Economiacutea y Competitividad (grant nos BIO2005-02136 BIO2009-08648 and BIO2013-66 44750R) and by the Junta de Andaluciacutea Spain (grant no P07-CVI-03171) (OdP) OdP was 67 supported in part by the Junta de Andaluciacutea Spain (Programa de Retorno de Investigadores) 68 FdlT by Marie Curie Programme through the International Reintegration grants (MIRG-CT-69 2005-031174) and a Juan de la Cierva contract (Ministerio de Ciencia e Innovacioacuten Spain) EG-B 70 was a recipient of a Formacioacuten de Personal Investigador fellowship (Ministerio de Educacioacuten 71 Spain) and JM Personat was supported by grant BIO2013-44750R 72 73 74 Corresponding Author 75 Olga del Pozo 76 Email olga_delpozoibvfcsices 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98


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ABSTRACT 99 Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction 100 with the calcium sensors calcineurin B like proteins (CBLs) into phosphorylation events that result 101 into adaptation to environmental stresses Few phosphorylation targets of CIPKs are known and 102 therefore the molecular mechanisms underlying their downstream output responses are not fully 103 understood Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible 104 Programmed cell death (PCD) in immunity transforming Ca2+ signals into Reactive Oxygen 105 Species (ROS) signaling To investigate SlCipk6-induced molecular mechanisms and identify 106 putative substrates a yeast two hybrid (Y2H) approach was carried on and a protein was identified 107 that contained a Universal stress protein (Usp) domain present in bacteria protozoa and plants 108 which we named SlRd2 SlRd2 was an ATP-binding protein that formed homodimers in planta 109 SlCipk6 and SlRd2 interacted using co-immunoprecipitation and bimolecular fluorescence 110 complementation (BiFC) assays in Nicotiana benthamiana (N benthamiana) leaves and the 111 complex localized in the cytosol SlCipk6 phosphorylated SlRd2 in vitro thus defining a novel 112 target for CIPKs Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic 113 LiCl whereas SlRd2 expression in Escherichia coli (E coli) UspA mutant restored bacterial 114 viability in response to H2O2 treatment Finally transient expression of SlCipk6 in transgenic N 115 benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild 116 type (WT) plants Taken together our results establish that SlRd2 a tomato UspA is a novel 117 interactor and phosphorylation target of a member of the CIPK family SlCipk6 and functionally 118 regulates SlCipk6-mediated ROS generation 119 120 121 122 123 124 125 126 127 128 129


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INTRODUCTION 130 131 Environmental factors especially those imposing stress stimulate endogenous cellular 132 cues which initiate protective responses in plants Among the concurrent events during stress are 133 changes in the intracellular Ca2+ concentration which activate an overlapping set of downstream 134 responses Ca2+ changes are perceived and decoded by an array of Ca2+ sensors including 135 calmodulins (CaMs) or CaM-related proteins Ca2+ dependent protein kinases (CDPKs CPKs) and 136 calcineurin B-like proteins (CBLs) (Dodd et al 2010) Particularly the CBL family has been 137 shown to play a crucial role in different Ca2+ -dependent processes in plants (Sanyal et al 2015) 138 CBL proteins present homology to the regulatory B-subunit of calcineurin (CNB) and the neuronal 139 calcium sensor (NCS) proteins from animals and yeast (Luan 2009) The overall structure of CBLs 140 consists of four EF hands Spacing of EF hands is invariable while the C- and N -terminal 141 extension of CBL proteins vary in length Post-translational modifications of CBLs including 142 protein phosphorylation and lipid modifications affect their subcellular localization and their 143 stability to interact with another proteins (Sanyal et al 2015 Nagae et al 2003) Thus 144 phosphorylation of the conserved Ser residue in the C-terminal PFPF motif of the CBL proteins 145 enhances the interaction with CIPKs (Du et al 2011 Hashimoto et al 2012) 146 Upon Ca2+ binding CBLs physically interact with CBL-Interacting Protein Kinases 147 (CIPKs) SerThr kinases that structurally belong to sucrose non-fermenting 1-related kinases 148 group 3 (SnRK3s) also called PKS (protein kinases related to SOS2) (Gong et al 2004 Yu et al 149 2014) CIPKs are constituted of a C-terminal or regulatory domain and a conserved kinase catalytic 150 domain at the N-terminus Within the divergent regulatory domain CIPKs contain an 151 autoinhibitory NAFFISL motif and a type 2C protein phosphatase (PP2C) binding site called ldquoPPI 152 motifrdquo It is well established that binding of CBLs to the NAFFISL motif releases the C-terminal 153 (auto-inhibitory) domain from the kinase domain thus leading the kinase into an active state (Guo 154 et al 2001 Chaves-Sanjuan et al 2014) In Arabidopsis thaliana (Arabidopsis) there are 10 CBL 155 and 26 CIPK homologues (Yu et al 2014) By yeast two hybrid (Y2H) and bimolecular 156 fluorescence complementation (BiFC) assays it has been determined that CBLs show a level of 157 specificity in targeting different CIPKs On the other hand a specific CIPK can also interact with 158 different CBLs thus allowing a single CIPK to access different cellular compartments and hence 159 different substrates (Kim et al 2000 Kim et al 2007) It is believed that the specificity of the 160 response to a given stimulus is achieved by decoding specific Ca2+ profiles by CBLs followed by 161 the subsequent formation of different CBLCIPK complexes in planta and finally by 162 phosphorylation of CIPK specific substrates that contribute to the specific output response (Batistic 163 et al 2010) 164 At the moment the most numerous and best characterized interactors or substrates for 165 CBLCIPK complexes are membrane proteins which include salt overly sensitive 1 (SOS1) 166


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(Quintero et al 2002 Katiyar-Agarwal et al 2006) H+-ATPase 2 (AHA2) (He et al 2004) 167 nitrate transporter (CHL1NRT11) (Ho et al 2009) K+ transporter 1 (AKT1) (Xu et al 2006) 168 high-affinity K+ transporter 5 (HAK5) (Ragel et al 2015) and the respiratory burst oxidase 169 homolog F (RBOHF) (Drerup et al 2013) Additionally CIPKs have also been shown to interact 170 with non-membrane proteins for example AtPKS5 (SOS2-like protein kinase 5 AtCIPK11) 171 interacts with the chaperone DnaJ (He et al 2004 Yang et al 2010) AtCIPK24 interacts with 172 GIGANTEA (Kim et al 2013) NADPK2 (nucleoside diphosphate kinase 2) the catalases CAT2 173 and CAT3 (Verslues et al 2007) and with ABI2 (ABA-insensitive 2) a type 2C SerThr 174 phosphatase (Guo et al 2002 Ohta et al 2003) and CIPK26 interacts with the RING-type E3 175 ligase ldquoKeep on Goingrdquo (KEG) and with ABI1 ABI2 (Lyzenga et al 2013) Although it appears 176 that CBLCIPK complexes could interact with several proteins at present only few CIPK 177 phosphorylation targets have been identified 178 Previously our group demonstrated a novel role for tomato (Solanum lycopersicum) Cipk6 179 (SlCipk6) in plant innate immunity thus functionally implicating for the first time the participation 180 of a CBLCIPK module in biotic stress signaling in plants (de la Torre et al 2013) Other studies 181 demonstrated the participation of Cipk6 orthologs from different plant species in diverse abiotic 182 stress responses (Chen et al 2013 Tsou et al 2012 Chen et al 2012 Tripathi et al 2009) As a 183 first step to investigate SlCipk6 downstream signaling molecular mechanisms we set to identify 184 SlCipk6-interacting proteins using a Y2H approach We discovered that tomato Responsive to 185 desiccation 2 (SlRd2) which contains a universal stress protein (Usp) domain (Pfam accession 186 number PF0582) interacted with SlCipk6 and by means of a BiFC approach we found that the 187 complex SlCipk6SlRd2 is localized in the cytoplasm In addition we demonstrated that SlRd2 is a 188 phosphorylation substrate of SlCipk6 thus expanding the previously described substrates for the 189 CIPK family Interestingly SlRd2 is an ATP-binding protein that forms homodimers which is 190 required for its biological role and for interacting with SlCipk6 191 The universal stress protein A (UspA) superfamily was originally discovered in E coli 192 where its expression drastically increased in response to multiple stress conditions and to starvation 193 (VanBogelen et al 1990) Importantly UspA protein accumulation was necessary for bacterial 194 survival at the stationary fase (Nystrom and Neidhardt 1994) It was found later that E coli has 6 195 usp genes (uspA uspC uspD uspF uspF uspG) however UspA set the nomenclature for the 196 orthologous groups of proteins UspA family members are classified into two major groups 197 according to their ATP binding capability The first group is constituted by ATP-binding proteins 198 and is represented by Mj0577 from Methanococcus jannaschii (Zarembinski et al 1998) 199 Members of the second group have no ATP-binding capability and are represented by Haemophilus 200 influenzae and E coli UspAs (Sousa and McKay 2001) Both Mj0577 and HiUspA form 201 homodimers in vivo (Zarembinski et al 1998) At present more than 2000 UspA (or Usp 202 containing domain) proteins have been identified from a wide range of organisms such as bacteria 203


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archaea fungi protozoa and plants constituting an ancient and conserved group of proteins 204 (Aravind et al 2002) In Arabidopsis at least 44 proteins were found to contain an Usp domain all 205 of which resemble ATP-binding Mj0577 protein (Kerk et al 2003) and several plant UspAs 206 seemed to be involved in abiotic stress In Arabidopsis two UspA proteins AtPHOS32 and 207 AtPHOS34 were phosphorylated by AtMPK3 and AtMPK6 in response to bacterial elicitors in cell 208 suspension cultures (Merkouropoulos et al 2008) Other reports described several UspA members 209 as effectors of low water potential (Merkouropoulos et al 2008) Several UspA proteins have been 210 characterized in rice (Sauter et al 2002) tomato (Zegzouti et al 1999 Loukehaich et al 2012) 211 legumes (Becker et al 2001 Hohnjec et al 2000) Salicornia (Udawat et al 2016) and cotton 212 (Zahur et al 2009) Still the precise structure regulation biochemical function or mechanism of 213 function of UspA proteins in planta are largely unknown 214 215 216


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RESULTS 217 218 Identification of SlRd2 as a SlCipk6-Interacting Protein 219 In order to identify SlCipk6-interacting proteins (CIPs) we carried out a Y2H approach in 220 two separate screens using a tomato cDNA prey library previously developed (Zhou et al 1995) 221 and SlCipk6 and a mutant derivative SlCipk6(T172D) as baits (Figure 1A) SlCipk6(T172D) 222 displayed enhanced kinase and autophosphorylation activity compared to SlCipk6 (de la Torre et 223 al 2013) and we hypothesized that using either SlCipk6 or SlCipk6(T172D) as a bait might 224 facilitate the identification of putative regulatory proteins or phosphorylation substrates 225 respectively since constitutive active kinase versions stabilize the interaction with their substrates 226 (Uno et al 2009) Approximately 11 x 103 and 45 x 103 yeast transformants were screened for 227 SlCipk6- and SlCipk6(T172D)-interacting proteins respectively on selection plates (Supplemental 228 Table 1) The inserts of 11 and 34 candidate prey clones from both screens were sequenced and 229 compared with databases by BLAST in the Arabidopsis database (wwwtairorg) for identification 230 The clone number 29 (Cip29) was a partial open reading frame (ORF) that encoded a protein with a 231 high similarity to Arabidopsis Response to desiccation 2 (AtRD2 At2g21620) a protein not yet 232 characterized with no assigned function containing a Universal stress protein domain (Usp) 233 Clones containing different length fragments of Cip29 were identified once and 2 times in the 234 screens performed with SlCipk6 and with SlCipk6(T172D) respectively and they were among the 235 strongest interactors In light of these facts its characterization was prioritized and the remaining 236 CIPs will be published elsewhere BLAST performed with Cip29 in the tomato database 237 (wwwsolgenomicsnet) identified unigene SGN-U567775 containing a full length ORF that 238 corresponded to the tomato locus Solyc01g109710 and we will be referred to it as SlRd2 hereafter 239 We next obtained full length SlRd2 ORF cloned it in the prey vector and confirmed its 240 interaction with SlCipk6 by Y2H (Figure 1B) Both SlRd2 and SlCipk6 did not show 241 autoactivation activity To characterize SlCipk6SlRd2 interaction different SlCipk6 mutant 242 derivatives were tested (de la Torre et al 2013) SlRd2 interacted with SlCipk6 SlCipk6(T172D) 243 SlCipk6ΔNAF (a mutant version lacking the NAFFISL domain necessary for CBL binding) or 244 SlCipk6ΔCterm (a deletion mutant version that lacked the C-terminal regulatory domain) but did 245 not interact with SlCipk6ΔNterm (a deletion mutant version lacking the kinase domain) or very 246 weakly with SlCipk6(K43M) (a reduced kinase activity version where the catalytic lysine 43 has 247 been mutated to methionine) (Figure 1A Figure 1B) The interaction was quantitated in a β-248 galactosidase activity assay (Figure 1C) and protein expression in yeast was confirmed by 249 immunoblot (Supplemental Figure 1) SlRd2 did not interact with SlCipk11 (Solyc06g082440) or 250 SlCipk14 (Solyc10g085450) (Supplemental Figure 2A) and SlCipk6 did not interact with SGN-251 U601569 the closest tomato sequence to SlRd2 (Supplemental Figure 2B) indicating that SlCipk6 252


9

interaction with SlRd2 was specific Altogether we confirmed that SlCipk6 interacted with SlRd2 253 and concluded that SlCipk6 kinase domain and kinase activity seemed to be necessary and 254 important respectively for SlCipk6SlRd2 interaction 255 256 SlCipk6 and SlRd2 Interact In vivo 257


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Next we examined the in vivo interaction between SlCipk6 and SlRd2 by co-258 immunoprecipitation (co-IP) Although SlCipk6 interacted with SlRd2 in yeast at slightly higher 259 levels we used SlCipk6(T172D) for co-IP in planta since it has been described that constitutive 260 active kinase versions stabilize the interaction with their substrates (Uno et al 2009) For that 261 purpose both SlRd2 (fused to a GFP tag) and SlCipk6(T172D) (fused to 2xIgG-BD and 9xMyc 262 tags) were co-expressed via Agrobacterium-mediated transient transformation of leaves (agro-263 infiltration) in Nicotiana benthamiana (N benthamiana) SlRd2 co-immunoprecipitated with 264 SlCipk6(T172D) but did not with the GFP used as a negative control in this experiment (Figure 265 2A) Therefore SlCipk6(T172D) and SlRd2 interacted in planta Since kinases and their substrates 266 need a coordinated expression and subcellular localization we first assessed the likelihood that 267 both SlCipk6 and SlRd2 localized at common cellular compartments Both proteins were fused to 268 GFP agro-infiltrated individually into N benthamiana leaves and the localization was determined 269 by confocal microscopy (Supplemental Figure 3) Both SlCipk6-GFP (Supplemental Figure 3ab) 270 and GFP-SlRd2 (Supplemental Figure 3cd) localized in the cytoplasm and in the nucleus thus 271 showing an overlapping subcellular localization 272 To gain insight where the complex localized intracellularly we performed BiFC assays in 273 N benthamiana leaves (Walter et al 2004) Fluorescence due to the reconstitution of yellow 274 fluorescent protein (YFP) was observed at the confocal microscope when the combinations SlRd2-275 YFPC and SlCipk6-YFPN were agro-infiltrated in N benthamiana leaves (Figure 2Bad) The YFP 276 signal showed a pattern similar to the one observed for AtFKBP12-CFP a cytoplasmic marker 277 fused to cyan fluorescent protein [CFP (Faure et al 1998)] (Figure 2Bd-f) but did not overlap 278 with the signal derived from the membrane-bound marker FM4-64 (Figure 2Ba-c) No 279 fluorescence was detected in the nucleus or when SlRd2-YFPC or SlCipk6-YFPN were co-280 infiltrated with empty vectors respectively and neither when SlRd2-YFPC was co-expressed with 281 SlCbl10-YFPC a known interactor of SlCipk6 (Figure 2Bg-i) (de la Torre et al 2013) These 282 results indicate that SlCipk6 and SlRd2 interact in vivo and form a cytoplasmic complex in N 283 benthamiana leaves at the conditions assayed 284 285 SlRd2 Transcript Accumulates in Response to Abiotic Stress 286 E coli UspA (EcUspA) protein accumulates in response to a large and diverse number of 287 stresses providing survival cues under adverse bacterial growth conditions (Freestone et al 1997 288 Jung et al 2015) In plants members of the UspA family have been reported to accumulate in 289 response to different abiotic stress conditions including AtRD2 (Yamaguchi-Shinozaki et al 1992 290 Jung et al 2015) Both SlCipk6 and SlRd2 displayed an overlapping subcellular localization in N 291 benthamiana epidermal cells (Supplemental Figure 3a-d) Next we enquired if SlRd2 and SlCipk6 292 expression were correlated in response to abiotic stress using quantitative (Q) RT-PCR in order to 293 support synchronous activity in abiotic stress response 294


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SlRd2 mRNA was present in all the tissues examined however it was substantially more 295 abundant in roots (gt25 fold compared to leaves) suggesting that it might play a prominent role in 296 this tissue (Figure 3A) AtRD2 accumulation increased in response to abiotic stress (Yamaguchi-297 Shinozaki et al 1992) Therefore to determine if SlRd2 and SlCipk6 transcript accumulation were 298 responsive to salt and osmotic stress we treated hydroponically grown tomato plants with 100 mM 299 NaCl and 300 mM mannitol and analyzed SlRd2 accumulation NaCl treatment rapidly increased 300 SlRd2 transcript accumulation [aprox 12 fold after 2 hours (h)] which was maintained up to 12 h 301


12

and decreased 24 h later (Figure 3B) Osmotic pressure resulted in a similar accumulation of SlRd2 302 after 2 and 4 h but in contrast SlRd2 accumulation increased up to 30 fold after 8-12 h and 303 decreased to 20 fold at 24 h (Figure 3C) SlCipk6 orthologs from different plant species have been 304 described to participate in different abiotic stress responses and we tested if SlCipk6 transcript 305 accumulated also in response to abiotic stress For that purpose we treated tomato plants with NaCl 306 and mannitol using the same conditions as described above After NaCl treatment accumulation of 307 SlCipk6 mRNA increased 6-8 fold after 2 h and was maintained at the same levels up to 8 hours 308


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decreasing gradually thereafter reaching a 2 fold increase at 24 h (Figure 3D) Osmotic pressure 309 also resulted in an increased SlCipk6 mRNA accumulation however followed a different pattern 310 compared to SlRd2 2 h after SlCipk6 transcripts increased 6 fold and steadily increased up to 15 311 fold 24 h after (Figure 3E) Both SlCipk6 and SlRd2 transcript accumulation pattern showed similar 312 kinetics thus supporting the possibility of a coordinated role for SlCipk6 and SlRd2 in abiotic 313 stress responses in tomato 314 315 SlRd2 Belongs to the Universal Stress Protein A Superfamily 316 The translated sequence of SlRd2 ORF yields a 177 amino acid protein with a molecular 317 weight of 195 kD and an isoelectric point of 598 We aligned the Usp domain of SlRd2 with those 318 of N benthamiana and Arabidopsis orthologs NbRd2 and AtRD2 and other plant proteins 319 containing the Usp domain including tomato LeER6 (Zegzouti et al 1999) rice OsUSP1 (Sauter 320 et al 2002) Vicia fava Enod18 (Becker et al 2001) along with bacterial proteins belonging to the 321 UspA family including E coli (Nystrom and Neidhardt 1992) H influenzae (Sousa and McKay 322 2001) and M jannaschii (Zarembinski et al 1998) (Supplemental Figure 4) Amino acid sequence 323 alignment revealed that SlRd2 contained the residues involved in ATP binding including the 324 Walker motif A or P-loop (G-2X-G-9X-G(ST)) also present in Mj0577 and UspA plant 325 representatives but absent in E coli paralogs (Sousa and McKay 2001) This observation suggests 326 that plant UspA proteins might also be functional ATP-binding proteins (Supplemental Figure 4 327 Figure 5A) Overall all UspA proteins (plant and bacterial) shared conservation within the 328 dimerization subdomain (Supplemental Figure 4) thus raising the possibility that plant UspAs 329 could be present as dimers in the cell In addition to the conserved Usp domain SlRd2 has an N-330 terminal domain (aminoacids 1 to 38) and a C-terminal extension (aminoacids 168 to 177) with 331 unknown function which is shared with the Arabidopsis and N benthamiana homologs (Figure 4A 332 and Supplemental Figure 5) Subsequently a phylogenetic analysis was performed using Ecoli 333 UspG and the proteins included in the alignment shown in Supplemental Figure 4 SlRd2 and 334 NbRd2 were located in the same clade as AtRD2 thus supporting their orthology (Figure 4B) 335 Moreover SlRd2 and all plant UspAs are more related to Mj0577 subfamily and E coli UspG 336 than to the E coli UspA (Figure 4B) 337 338 SlRd2 is an ATP-binding Protein and Forms Homodimers in Yeast Bacteria and Plants 339 In light of the high degree of conservation of SlRd2 amino acids putatively involved in 340 nucleotide binding (see alignments in Figure 5A and Supplemental Figure 4) we next enquired if 341 SlRd2 also had the functional competence to bind ATP as described for bacterial Mj0577 For that 342 purpose we purified SlRd2 protein and performed a nucleotide binding assay in vitro by incubating 343 SlRd2 in the presence of [α-32P]ATP Samples were then analyzed by SDS-PAGE followed by 344 autoradiography (Figure 5B upper panel) Indeed SlRd2 was able to bind [α-32P]ATP whereas no 345


14

[α-32P]ATP binding was observed when the GFP protein was incubated in its presence To further 346 confirm that SlRd2 was also able to bind ATP in vivo a synthetic analogue of ATP (+)-biotin-hex-347 acyl-ATP (BHAcATP) consisting of ATP an acyl-phosphate linker and a biotin tag was used 348 (Villamor et al 2013) Incubation of GFP-SlRd2 expressing plant extracts with BHAcATP 349 followed by streptavidin purification revealed that SlRd2 was able to bind BHAcATP in planta 350 (Figure 5C upper panel) Moreover ATP addition suppressed BHAcATP labeling by competing 351 with BHAcATP and saturating the nucleotide-binding site of SlRd2 (Figure 5C upper panel) 352 These findings indicate that SlRd2 binds ATP and therefore functionally belongs to the ATP-353 binding subgroup of the UspA family represented by Mj0577 354 The dimerization motif present in UspA family proteins is also conserved in SlRd2 and is 355 localized close to the C-terminus (Figure 5A and Supplemental Figure 4) Mj0577 and HiUspA 356 were shown to exist as homodimers in vivo (Sousa and Mackay 2001 Zarembinski et al 1998) 357 Mj0577 crystallizes as a homodimer and each monomer binds the other through antiparallel 358 hydrogen bonds in the fifth beta sheet within each subunit To test whether SlRd2 was also able to 359 form homodimers we performed a Y2H analysis in which the yeast strain expressed SlRd2 both in 360 the prey (pJG4-5) and in the bait (pEG202) plasmids Indeed SlRd2 can form homodimers since 361 growth was observed in restrictive media and blue color developed in the presence of X-gal (Figure 362


15

6A) No interaction was detected when Y2H analysis was performed using a SlRd2 mutant version 363 SlRd2∆dim (amino acids 163 to 166 deletion) in which the putative dimerization domain VIIV 364 was deleted (Figure 6A) SlRd2 and SlRd2∆dim were expressed in yeast (Figure 6B) Therefore 365 SlRd2 forms dimers and the conserved VIIV domain is necessary for dimerization in vivo 366 It has been described that E coli UspC is able to form tetramers in vivo (Nachin et al 367 2008) To check if SlRd2 also formed homotetramers in vivo an E coli culture overexpressing 368 SlRd2 tagged at the N-terminus with the epitope histidine (His-SlRd2) was treated with a cross-369 linking agent disuccinimidyl glutarate (DSG) for 30 min Thereafter protein extracts were 370 obtained analyzed by SDS-PAGE and His-SlRd2 detected by immunoblot Two bands of 371 approximately 42 and 24 kD were observed in the extracts treated with the crosslinker which 372


16

corresponded likely with SlRd2 dimer and monomer whereas only the lower molecular weight 373 band was observed in control conditions (Figure 6C) Thus we concluded that SlRd2 is able to 374 form homodimers but not homotetramers in vivo 375 To determine that SlRd2 formed homodimers in planta and their putative subcellular 376 localization BiFC assays were performed SlRd2 cDNA was cloned in the BiFC vectors 377


17

transformed into Agrobacterium and agro-infiltrated in N benthamiana leaves Reconstitution of 378 YFP fluorescence was observed two days after under the confocal microscope when SlRd2-YFPC 379 and SlRd2-YFPN were co-expressed The signal co-localized with that obtained by the expression 380 of the cytoplasmic marker AtFKBP12-CFP (Figure 6Dd-f) and with DAPI nuclear marker (Figure 381 6Dg-i) but not with the cell membrane marker FM4-64 (Figure 6Da-c) These results demonstrate 382 that SlRd2 can form homodimers in planta in the cytoplasm and in the nucleus 383 384 SlRd2 is a Phosphorylation Substrate of SlCipk6 385 Since SlCipk6 kinase domain and activity was necessary and important respectively for 386 SlCipk6SlRd2 interaction (Figure 1) we enquired if SlRd2 was a phosphorylation target of 387 SlCipk6 For that purpose we set up an in vitro kinase assay in the presence of [γ-32P]ATP using 388 purified SlCipk6 (fused to the GST at the C-terminus) and as substrates SlRd2 (fused to the His 389 epitope at the N-terminus) and a mutant version SlRd2(UspA) (fused to GST at the N-terminus) in 390 which the specific putative regulatory N-terminal domain (amino acids 1 to 38) and C-terminal 391 extension (amino acids 168 to 177) were deleted to leave only a core UspA domain Myelin Basic 392 Protein (MBP) a universal kinase substrate was included as a positive control for SlCipk6 kinase 393 activity SlCipk6 was expressed and purified from yeast whereas SlRd2 and SlRd2(UspA) were 394 purified from bacteria Figure 7A shows that SlRd2 is phosphorylated by SlCipk6 but does not 395 phosphorylate SlRd2(UspA) thus indicating that SlCipk6 phosphorylates SlRd2 specific regulatory 396 N- or C-terminal regions SlCipk6 also phosphorylates MBP A faint signal can be still detected in 397 the absence of SlCipk6 indicating that SlRd2 binds ATP (panel A left) Protein loading levels 398 were monitored by Coomasie staining (Figure 7A) Quantification of SlRd2 [γ-32P]ATP 399 incorporation is presented in Figure 7B Therefore we conclude that SlRd2 is a phosphorylation 400 substrate of SlCipk6 and represents a novel phosphorylation target of the CIPK family 401 402 SlRd2 Overexpression in Yeast Confers Resistance to LiCl 403 In light of the strong transcriptional response of SlRd2 to NaCl and osmotic stress we 404 decided to functionally test SlRd2 participation in mediating different stress responses using yeasts 405 as a model system Yeast has been successfully used in functional studies to characterize the role of 406 plant proteins in stress responses (Matsumoto et al 2001 Quintero et al 2002 Shitan et al 2013 407 Bernard et al 2012) SlRd2 was overexpressed in the yeast strain Saccharomyces cerevisiae (S 408 cerevisiae) BY4741 under control of the constitutive expression vector p426GPD (p426GPD-409 SlRd2) As a control BY4741 strain transformed with empty vector p426GPD (EV) was used 410 Both yeasts lines were subjected to the following stress-causing treatments hygromycin B 411 generates changes in membrane potential (Wang et al 2009) MnCl2 causes PCD at high 412 concentrations (Liang and Zhou 2007) CaCl2 and KCl cause osmotic stress (Liang and Zhou 413 2007) DTT and H2O2 generate oxidative stress (Babiychuk et al 1995) bleomycin causes DNA 414


18

damage (Aouida et al 2004) and finally NaCl and LiCl cause changes in ionic homeostasis (Ye 415 et al 2006) SlRd2 overexpressing line grew at a similar rate than EV strain in control conditions 416 and in all the different treatments except in 300 mM LiCl where it grew significantly better than 417 EV line (Figure 8A) Next we tested if SlRd2 dimerization was crucial for its biological role For 418 that purpose we cloned SlRd2∆dim into p426GPD vector transformed it into S cerevisiae 419 BY4741 strain and performed a LiCl resistance experiment along with p426GPD-SlRd2 and EV 420


19

Figure 8B shows that a functional SlRd2 dimer was required for conferring LiCl resistance since 421 SlRd2∆dim line grew at the same rate as EV line and loss its protection against LiCl Both SlRd2 422 and SlRd2∆dim expression in yeast was confirmed by immunoblotting (Figure 8C) We concluded 423 that SlRd2 dimerization was necessary for its molecular function in conferring resistance to LiCl 424 Next we asked if SlRd2 dimerization was also necessary for its interaction with SlCipk6 425 For that purpose we performed a Y2H assay with SlCipk6 and SlCipk6(T172D) as baits and 426 SlRd2∆dim as a prey (Figure 9A) Neither SlCipk6 nor SlCipk6(T172D) interacted with 427


20

SlRd2∆dim by Y2H (Figure 9A) To test their interaction in planta we performed a BiFC 428 experiment and agro-infiltration SlRd2∆dim-YFPC and SlCipk6-YFPN in N benthamiana leaves 429 did not reconstitute YFP fluorescence indicating that SlCipk6 was not able to interact with 430 SlRd2∆dim (Figure 9B) Hence SlRd2 homodimerization is required for interaction with SlCipk6 431 432 SlRd2 Protects Bacteria from Oxidative Stress and Negatively Regulates ROS in Plants 433 E coli mutant strain TN3151 (a knock out [KO] of the uspA gene) was susceptible to H2O2 434 treatments which did not affect the WT strain W3101 (Nystrom and Neidhardt 1994) Recent 435 results demonstrated that a UspA protein from the pathogenic bacteria Mycobacterium tuberculosis 436 provided protection for the parasite against host reactive oxygen species (ROS) generated by 437 mammalian macrophages defense (Drumm et al 2009) SlCipk6 was demonstrated to participate 438 in ROS generation during plant responses to bacterial pathogen attack which was dependent on the 439


21

NADPH oxidase RbohB (de la Torre et al 2013) Given the striking conserved structural and 440 functional features between SlRd2 and bacterial UspA proteins we decided to test if SlRd2 could 441 functionally complement TN3151 in protecting bacteria in response to H2O2 treatment E coli wild 442 type W3101 (WT) uspa mutant TN3151 and TN3151 complemented with either SlRd2 (TN3151-443 SlRd2) or with the native UspA gene (TN3151-UspA) were grown in minimal media (MOPS) until 444 OD600 reached 07 Then H2O2 (5 mM) was added and survival was measured at different time 445 points after (Figure 10A) A strong growth inhibition was observed for TN3151 strain after H2O2 446


22

treatment as previously described (Nystrom and Neidhardt 1994) 40 min after H2O2 addition 447 only 45 of TN3151 survived whereas 100 TN3151-SlRd2 survived showing a similar survival 448 rate as either TN3151-EcUspA or WT W3101 SlRd2 and EcUspA expression in TN3151 was 449 confirmed by immunoblotting (Figure 10B) and lack of UspA gene expression in TN3151 was 450 confirmed by PCR (Figure 10C) In light of these results we concluded that tomato SlRd2 451 functionally complements TN3151 mutant strain in protecting bacteria against oxidative stress 452 damage and thus SlRd2 and E coli UspA are functionally conserved in their ROS protection role in 453 bacteria 454 We have previously shown that kinase activity of SlCipk6 is associated with ROS production in N 455 benthamiana (de la Torre et al 2013) so we next enquired if SlRd2 is required for SlCipk6-456 mediated ROS generation To this end c-Myc tagged versions of SlCipk6 SlCipk6(T172D) and 457 SlCipk6(K43M) (cloned into pTAPa-pYL436 vector) or empty vector (EV) were agro-infiltrated in 458 N benthamiana WT and over-expressing GFP-SlRd2 (OE-6 OE-7) leaves and production of ROS 459 was quantified by a chemiluminiscence assay in a luminometer (Figure 10D) As expected 460 SlCipk6 and SlCipk6(T172D) expression in WT leaf discs resulted in ROS generation which was 461 significantly reduced in SlCipk6(K43M) expressing leaf discs (de la Torre et al 2013) However 462 SlCipk6 SlCipk6(T172D) agro-infiltration in OE-6 and OE-7 plants resulted in a significant 463 reduction of ROS (Figure 10D) whereas SlCipk6(K43M)-induced ROS levels remained as in WT 464 discs SlCipk6 SlCipk6(T172D) SlCipk6(K43M) and GFP-SlRd2 expression were confirmed by 465 immunoblotting (Supplemental Figure 6) This result clearly indicated that overexpression of 466 SlRd2 negatively regulates SlCipk6-mediated ROS generation and that a functional link exists 467 among both proteins 468 469


23

DISCUSSION 470 SlRd2 a Member of the Universal Stress Protein Family Interacts with SlCipk6 471 The identification and characterization of SlCipk6 targets and regulatory components is an 472 important step for understanding how downstream SlCipk6 signaling specificity is achieved and to 473 identify the pathways it regulates As a first step we set to identify SlCipk6-interacting proteins 474 using a Y2H approach with either SlCipk6 or its enhanced kinase activity version SlCipk6(T172D) 475 as baits and a tomato library as a prey (Zhou et al 1995) We expected to identify putative 476 phosphorylation substrates or regulatory proteins SlRd2 was identified in both screens and 477 displayed one of the strongest interactions SlCipk6SlRd2 interaction was later confirmed and 478 appeared to have some level of specificity In addition no assigned function was available for this 479 protein Hence we prioritized its characterization For SlCipk6SlRd2 interaction SlCipk6 kinase 480 domain and kinase activity was required thus suggesting that SlRd2 could be a phosphorylation 481 substrate of SlCipk6 SlCipk6SlRd2 interaction was further confirmed in planta using co-482 immunoprecipitation and BiFC approaches Thus we compiled data using different approaches 483 demonstrating that SlCipk6SlRd2 interacted in N benthamiana 484 BiFC experiments indicated that SlCipk6SlRd2 complex localized in the cytosol Both 485 GFP tagged SlCipk6 or SlRd2 localized mainly in the nucleus and in the cytoplasm of N 486 benthamiana epidermal cells thus showing a coordinated subcellular localization pattern 487 However SlCipk6SlRd2 complex was only detected in the cytosol We cannot rule out that the 488 complex could localize to the nucleus or to different cellular compartments when complexed with 489 CBLs or under stress conditions In fact it has been demonstrated that CIPKs can be targeted to 490 different intracellular compartments via their interacting CBLs (Batistic et al 2010) Accordingly 491 tomato Cipk6 was found to interact with SlCbl10 and with NbRbohB a membrane based NADPH 492 oxidase at the plasma membrane in N benthamiana epidermal cells (de la Torre et al 2013) 493 whereas Brassica napus CIPK6 (also plasma membrane- and nucleus-localized) was found at the 494 plasma membrane in complex with BnCBL1 (Chen et al 2012) Here we describe the localization 495 of a CIPK in complex with its substrate which poses additional unsolved questions whether CBLs 496 translocate CIPKs to meet their substrate at the final destination or whether they could also 497 transport the attached substrates as cargoes CBLs and CIPKs interact with some but not with all 498 CIPK and CBL partners respectively thus providing an enormous combinatorial signaling 499 flexibility (Luan 2009 Yu et al 2014) At this point we do not know which CBL(s) relay Ca2+ 500 signals to SlCipk6 and SlRd2 501 502 SlCipk6 Phosphorylates SlRd2 a Universal Stress Protein and a Novel Target of CIPK 503 Family 504


24

We have demonstrated that SlCipk6 phosphorylates SlRd2 in vitro In plants Ca2+ 505 intracellular increases occur after several intra- and extracellular stimuli including biotic abiotic 506 and developmental changes (Hashimoto and Kudla 2011) It is noteworthy that tomato exposure to 507 abiotic stress (osmotic stress and salinity) results in a rapid and strong accumulation of SlRd2 508 transcripts which resembles SlCipk6 accumulation pattern Similarly in silico data indicates that 509 under osmotic stress (mannitol 300 mM) in Arabidopsis AtCIPK6 and AtRD2 are transcriptionally 510 upregulated (Arabidopsis eFP Browser) Based on our results we propose that SlRd2 is a 511 phosphorylation target of SlCipk6 and therefore a Ca2+ signaling downstream effector or more 512 likely a downstream Ca2+ signaling component Our results also indicate that CIPKs in addition to 513 membrane proteins phosphorylate additional substrates thus expanding their role in regulating 514 different physiological processes Previous work by others have shown that different UspA proteins 515 from bacteria and plants are phosphorylated in response to stress indicating that this post-516 translational modification may be important for their role in the cell (Merkouropoulos et al 2008 517 Lenman et al 2008 Freestone et al 1997) In addition it was also found that the phosphosites 518 were localized outside the Usp domain as likely occurs in SlRd2 (Merkouropoulos et al 2008) 519 Likely the ancient and conserved function of the Usp domain adapts to different stress signals in 520 different organisms acquiring specific signaling domains to modulate specific output responses In 521 the future we will identify the exact SlRd2 residues phosphorylated by SlCipk6 Once identified 522 functional analysis in plants will be performed using transgenic plants with altered expression 523 levels of SlRd2 or SlRd2 mutant versions in which the residues phosphorylated by SlCipk6 will be 524 mutated to A (not a substrate) or to D (phosphomimics) These studies will help clarify SlRd2 role 525 and SlCipk6 phosphorylation contribution in ROS generation and regulation in plants 526

Using in vitro and in vivo assays we demonstrate that SlRd2 is also an ATP-binding 527 protein like E coli UspFG subfamily members and group representative Mj0577 Mj0577 and 528 plant UspAs share the common motif present in Usps experimentally proven to bind ATP (Kvint et 529 al 2003) Obtaining SlRd2 nucleotide binding impaired mutant versions will help to understand 530 the physiological relevance of this feature in plants under stress responses The ATP binding 531 capability in some members of the UspA family has led to the speculation that nucleotide binding 532 Usps could function as molecular switches by sensing ATP levels during stress signaling detecting 533 cellular energy or metabolic status (OToole et al 2003 Persson et al 2007 Drumm et al 2009) 534 In fact autoadenylation is observed in bacteria in late stationary phase (Weber and Jung 2006) and 535 it has been shown to be a key factor in microbial survival under O2 depletion during growth arrest 536 and in virulence In this line it has been described that the ability of UspA protein Rv2623 from 537 Mycobacterium tuberculosis to regulate its growth and latency in the host is dependent on its ATP-538 binding activity (Drumm et al 2009) Understanding the molecular mechanisms by which Usp-539 proteins act has broader implications in human health since they contribute to human pathogenacutes 540 virulence and survival in the host (Seifart Gomes et al 2011 Liu et al 2007) 541


25

Although several reports described that UspA proteins contain a dimerization domain in its 542 sequence little is known about its functional implication It has been described that EcUsp proteins 543 have the capability to form homodimers andor heterodimers in vivo leading to a higher adaptation 544 to stress (Nachin et al 2008 Heermann et al 2009) In the present work we have found that 545 SlRd2 forms homodimers in vivo at the cytoplasm and nucleus However we cannot discard that 546 SlRd2 might form heterodimers with other UspA proteins in the plant cell Interestingly we found 547 that the dimerization of SlRd2 is required for both its biological stress-protection role in yeast and 548 its interaction with SlCipk6 Similarly Weber et al described that the dimerization of EcUspG was 549 necessary for its cellular function (Weber and Jung 2006) Unlike plants the importance of UspA 550 dimerization is well documented in E coli Thus the Usp domain of KdpD (K+ transport system) 551 functions as a binding surface for EcUspC and it is essential for its signaling role (Heermann et al 552 2009) Recently it has been found that Arabidopsis AtUSP (Universal Stress Protein) is able to 553 switch from low molecular weight species to high molecular weight complexes suggesting a 554 chaperone function in stress tolerance to heat shock and oxidative stress (Jung et al 2015) 555 Notably dimerization of Hypoxia Responsive Universal Stress Protein 1 (HRU1) is also important 556 for ROS regulation and apparently for subcellular localization (Gonzali et al 2015) An important 557 question is whether SlRd2 dimerization is also required for ATP binding which could in turn 558 regulate the interaction with SlCipk6 These aspects deserve further analyses in the future 559 560 SlRd2 protects bacteria against ROS and regulates SlCipk6-mediated ROS in plants 561 Despite UspA proteins are widely represented in plants [with 48 members in Arabidopsis 562 (Kerk et al 2003 Isokpehi et al 2011)] very little is known about their function regulation 563 molecular mechanisms or their participation in physiological responses Previously two 564 Arabidopsis UspA proteins (At5g54430 At4g27320) were identified as differential 565 phosphorylation substrates in response to pathogen derived elicitors (Lenman et al 2008 566 Merkouropoulos et al 2008) In vitro kinase assays identified AtPHOS32 (At5g54430) as a 567 mitogen-activated protein kinases (MAPKs) AtMPK3 and AtMPK6 substrate (Merkouropoulos et 568 al 2008) This observation along with SlRd2 being phosphorylated by SlCipk6 (a Ca2+-regulated 569 kinase) indicates that plant UspA members might be regulated distinctly in response to a plethora 570 of stimuli thus receiving and integrating signals from different pathways 571 An interesting observation is that overexpression of SlRd2 in S cerevisiae results in an 572 increased tolerance to LiCl In yeast increased LiCl tolerance is promoted by a rise of activity of 573 the K+ transporter Trk12 which is in turn controlled by phosphorylation and dephosphorylation 574 modifications (Zaidi et al 2012 Yenush et al 2005) In plants CIPKCBL complexes regulate the 575 cellular K+ flux by interaction with the transporter AKT1 (Li et al 2014) According to the yeast 576 data it is tempting to speculate that SlCipk6 and SlRd2 might act together in plants to regulate the 577 activity of the plant K+ transporter AKT1 In fact in Arabidopsis AtCIPK6 interacts with different 578


26

CBLs to regulate AKT1 and AKT2 (Lee et al 2007 Lan et al 2011 Held et al 2011) However 579 further studies should be carried out in order to figure out this functional implication Significantly 580 we have found that SlRd2 complements an E coli Uspa mutant line restoring bacterial viability to 581 WT levels to otherwise lethal doses of H2O2 for the mutant Because EcUspA does not contain 582 ATP-binding regions whereas SlRd2 can bind ATP both proteins might play a common and 583 conserved molecular role that does not imply ATP binding at least in bacteria (Weber and Jung 584 2006) The functional complementation of E coli Uspa mutant by the expression of tomato Rd2 585 protein indicates that the molecular function of SlRd2 a plant UspA protein in protecting cells 586 against the toxic effects of oxidative stress in bacteria have been conserved in evolution 587 588 A rapid ROS burst have been implicated in different physiological responses Recently we 589 reported that SlCipk6 contributed to ROS generation during biotic stress response in N 590 benthamiana which largely depends on RbohB (de la Torre et al 2013) Also SlCipk6 591 overexpression resulted in ROS generation which was dependent on SlCipk6 kinase activity but 592 did not occur in RbohB silenced N benthamiana plants (de la Torre et al 2013) The fact that 593 overexpression of SlRd2 results in reduced SlCipk6-mediated ROS in planta clearly indicates a 594 functional relationship among both proteins in which SlRd2 negatively regulates SlCipk6-mediated 595 ROS output Similarly overexpression of Salicornia brachiata Universal Stress Protein (SbUSP) 596 in tobacco plants resulted in reduced accumulation of ROS during stresses (Udawat et al 2016) 597 Since RBOHs require post-translational modifications for their activation (Kobayashi et al 2012) 598 we propose that SlCipk6 phosphorylates the N-terminal regulatory domain of RbohB and SlRd2 599 directly or indirectly modulates this event thus affecting ROS output Since SlRd2 cancelled 600 SlCipk6-dependent ROS it might work in a negative feedback loop tempering the SlCipk6-RbohB 601 signaling If this was true it will be important to determine whether SlRd2 inhibited SlCipk6 in a 602 phosphorylation assay using MBP or RbohB as substrates On the other hand similar to UspA 603 family members the small GTP-binding proteins (RacRop) have been postulated to act as 604 molecular switches regulating a wide variety of important physiological functions in cells (Nibau et 605 al 2006 Xu et al 2010) In this context OsRac1 was required to activate OsRbohB in N 606 benthamiana cells (Wong et al 2007) Interestingly HRU1 has been found to interact with the 607 GTPase ROP2 and RbohD participating in the modulation of ROS levels under anoxia (Gonzali et 608 al 2015) Similarly SlRd2 might act as a regulatory element of RbohB by affecting SlCipk6 609 activity In the future the molecular mechanism underlying SlRd2 regulation of SlCipk6-mediated 610 ROS will be studied in higher detail 611 612 MATERIALS AND METHODS 613 614 Bacteria Yeast and Plant Materials 615


27

Agrobacterium tumefaciens (Agrobacterium) strain C58C1 was grown at 30ordmC in Luria-Bertani 616 (LB) medium with appropriate antibiotics Yeast strains EGY48 (Matα trp1 his3 ura3 617 leu26LexAop-LEU2) BY4741 (Matα met15Δ0 his3Δ1 ura3Δ1 leu2Δ0) and GRF-167 (MAT618 α his3Δ200 ura3-167) were grown at 30ordmC in synthetic dropout medium (SD) with glucose as a 619 carbon source E coli strain W3101 (F- galT22 λ- IN (rrnD-rrnE)1 rph-1) and TN3151 (F- λ- 620 IN (rrnD-rrnE)1 uspA1kan rph-1) were grown at 37ordmC in liquid MOPS (morpholine propane 621 sulphonic acid) supplemented with glucose 04 (wv) N benthamiana) was grown in the 622 greenhouse with 16 h of light and at 24ordmC (day) and 22ordmC (night) Solanum lycopersicum line Rio 623 Grande-PtoR (RG-PtoR PtoPto PrfPrf) was grown in hydroponic culture (Hoagland medium) in 624 a growth chamber at the same growth conditions as N benthamiana The ORF of SlRd2 was cloned 625 into the binary vector pGWB6 under the control of a cauliflower mosaic virus 35S (CaMV35S) 626 promoter Transgenic N benthamiana plants were obtained according to the procedures of Rajput 627 et al (Rajput et al 2014 Park et al 2013) and the homozygous transgenic lines 6 and 7 from T3 628 progeny were used (OE-6 and OE-7) Salt and osmotic shock treatments were performed adding 629 NaCl or mannitol to final 100 mM or 300 mM concentration respectively to the media on 4-week 630 old tomato plants For ROS detection and measurement N benthamiana leaf disks (028 cm2) were 631 floated on 100 μL of distilled water in a 96-well white-bottom plate over night at room 632 temperature Water was later replaced with 50 μL of distilled water and then incubated for 8 to 12 h 633 at room temperature For ROS detection 50 μL of a 2x solution containing 100 μM luminol 634 (Sigma-Aldrich) and 1 μg of horseradish peroxidase was quickly added to each well and ROS were 635 measured in vivo as luminescence using a Varioskan Flash Multimode Reader 636 637 SlRd2 Open Reading Frame cDNA Cloning and Deletion Mutant Generation 638 SlRd2 open reading frame was amplified from a tomato cDNA library prepared from leaf tissue by 639 RT-PCR using primers OPS291 (5acute-ATGGAAACGGTTATGGA-3acute) OPS292 (5acute-640 TTAAATCACAGAGACTT-3acute) and cloned into Gateway entry vector pDONR207 (Invitrogen) 641 PCR-based site mutagenesis was performed to generate deletion of dimerization domain in SlRd2 642 using the QuickChange kit (Stratagene) using OPS405 (5acute-643 CACAACTGTAAGATAGCACCGCCTGGAAAAGAAGCTGGGG-3acute) and OPS406 (5acute-644 CCCCAGCTTCTTTTCCAGGCGGTGCTATCTTACAGTTGTG-3acute) primers 645 646 Yeast Two-Hybrid Assay 647 Yeast strain EGY48 (containing pS18-34 vector) was used for Y2H assays SlCipk6 cDNA and its 648 mutant derivatives (de la Torre et al 2013) were cloned into the bait vector pEG202 and SlRd2 649 cDNA was cloned into the prey vector pJG4-5 To generate ΔNterm mutant derivate in SlCipk6 a 650 PCR reaction was performed using OPS501 (5acute-ATGTTGAATGCTTTTCATATCATTTC-3acute) and 651 OPS165 (5acute-CGGAATTCATGGGGACAGAAGAAAAATGTGC-3acute) primers Yeast 652


28

transformation was performed by LiAcPEG method as described in Yeast Protocols Handbook 653 (Clontech) Growth and blue colonies on SD (X-GalGal-Ura-His-Trp-Leu) plates indicated 654 positive interaction Finally β-galactosidase assay was performed as described in Yeast Protocols 655 Handbook Expression of bait and prey fusion proteins was verified by immunoblotting using anti-656 LexA mouse monoclonal antibody (Santa Cruz Biotechnology) or anti-HA rat monoclonal 657 antibody (Roche) 658 659 Quantitative Real-Time PCR (Q RT-PCR) 660 Total RNA was isolated from tomato leaves using TRIZOL reagent (Invitrogen) and subjected to 661 DNAse treatment using TURBO DNA-free (Ambion) 2 μg of total RNA were used to synthesize 662 cDNA using random primers and Superscript II reverse transcriptase (Invitrogen) following the 663 manufactureracutes protocol Quantitative real-time PCR was performed with SsoFasttrade EvaGreenreg 664 Supermix (BIORAD) and a BIORAD real-time PCR system The thermal cycle used was 95ordmC for 665 10 min 40 cycles of 95ordmC for 30 s 60ordmC for 30 s and 72ordmC for 30s Q RT-PCR reactions were 666 carried out with the following oligonucleotides OPS389 (5acute-667 AGCAAACACGCTTTTGATTGGGC-3acute)OPS390 (5acute-CACTGTCTTCACCATAGCAACC-3acute) 668 for SlRd2 OPS305 (5acute-ATCCATGCACTTAATATCTTCC-3acute) OPS306 (5acute-669 GCAATGATGGGTATCTGATAGCG-3acute) for SlCipk6 and OPS281 (5acute-670 AGCCACACAGTTCCCATCTAC-3acute) OPS282 (5acute-AACTTCTCCTTCACTCCCTA-3acute) for 671 SlActin2 as an internal standard Relative expression levels were determined as described 672 previously (de la Torre et al 2013) 673 674 Co-Immunoprecipitation 675 For Co-IP experiment SlRd2 and SlCipk6(T172D) coding sequences were cloned into pMDC43 676 (with a N-terminal GFP epitope) and pTAPa-pYL436 (with C-terminal 2xIgG-BD and 9xMyc 677 tags) vectors respectively by gateway technology (Invitrogen) (Rubio et al 2005) Agrobacterium 678 strains C58C1 carrying GFP-SlRd2 and SlCipk6(T172D)-Myc respectively were co-infiltrated into 679 N benthamiana leaves Co-infiltration Agrobacterium C58C1 cultures carrying GFP and 680 SlCipk6(T172D) were used as a negative control Two days after leaves were collected frozen and 681 grounded in liquid nitrogen and resuspended in three volumes of extraction buffer [50 mM Tris-682 HCl pH8 01 NP-40 1x Complete protease inhibitors (Roche)] and centrifuged at 14000 g for 20 683 min at 4ordmC Supernatant was filtered through two layers of Miracloth (Calbiochem) 2 mL was 684 incubated with 50 μL IgG beads (Amersham Biosciences) for 2 h at 4ordmC with gentle rotation 685 Beads were washed five times with 2 mL of washing buffer (50 mM Tris-HCl pH 8 01 NP-40) 686 Elution from the IgG beads was performed by boiling the samples with 1x Laemmli buffer 687 688 ATP-binding Assay 689


29

For in vitro ATP binding assay 1 μg of SlRd2 and GFP proteins purified from E coli were 690 incubated in binding buffer (20 mM Tris-HCl pH 75 15 mM MgCl2 1 mM DTT) 50 μM ATP 691 and 10 μCi of [α-32P]ATP [Amersham 3000 Cimmol (1 Ci = 37 GBq)] during 60 min at 30ordmC 692 Then the reactions were stopped by adding 10 μL of 4x Laemmli loading buffer The samples were 693 denatured by boiling 5 min and later were run on 12 SDS-PAGE gel ATP binding was 694 visualized by autoradiography Protein levels were verified by immunoblot using anti-His mouse 695 monoclonal antibody (Roche) The in vivo ATP-binding assay was performed as described 696 previously (Villamor et al 2013) N benthamiana leaf samples (1 g fresh weight) expressing GFP-697 SlRd2 were ground in liquid nitrogen and thawed in 2 volumes of extraction buffer (50 mM Tris 698 pH 75) The lysate was later cleared via centrifugation and subjected to gel filtration using DG10 699 columns (Bio-Rad) Labeling was performed adding 10 mm MgCl2 and 20 μM of BHAcATP 700 (Thermo Scientific) to each sample and then incubated at room temperature for 1 h For inhibition 701 experiments the lysate was incubated with 10 mM ATP for 30 min before labeling with 702 BHAcATP Biotinylated proteins were affinity purified by incubating the samples with 703 Streptavidin beads (Thermo Scientific) for 1 h at room temperature The beads were washed three 704 times with 6 M urea Finally the purified proteins were boiled in 4X Laemmli buffer and analyzed 705 by SDS-PAGE proteins gel and immunoblotted using anti-GFP mouse monoclonal antibody 706 707 Cross-linking Assay 708 The cross-linking assay was performed as previously described (Nachin et al 2008) Fresh LB 709 containing 50 μgmL ampicillin was inoculated to a final OD600 of 03 with an overnight culture of 710 E coli harbouring pDEST17-SlRd2 construct and grown at 37 ordmC After 1 h protein expression was 711 induced by adding 1 mM Isopropyl -D-1-thiogalactopyranoside (IPTG) After 4 h at 37 ordmC cells 712 were harvested by centrifugation and resuspended in phosphate buffered saline 1x (PBS) to a final 713 OD600 of 07 Subsequently 05 mM of cross-linking agent disuccinimidyl glutarate (DSG) was 714 added to 200 μL of E coli culture during 30 min at room temperature The reaction was stopped by 715 adding 40 μL of TS (200 mM Tris-HCl pH 88 5 mM EDTA 1 M sucrose and 005 (wv) 716 bromophenol blue) TD (18 SDS and 03 M DTT) (ratio 21) Finally the samples were 717 denatured by boiling 5 min and then were run on 12 SDS-PAGE gel Protein molecular weight 718 was determined by immunoblot using anti-His mouse monoclonal antibody (Roche) 719 720 In Vitro Phosphorylation Assays 721 For protein kinase assays SlCipk6 full length was amplified by PCR using the primers OPS606 722 (5rsquo-TCTAGACATGGGGACAGAAGAAAAATGT-3rsquo) and OPS607 (5rsquo-723 GTCGACCTCAAGCAATTGTTGGATTCTC-3rsquo) and cloned as SalIXbaI fragment in the yeast 724 expression vector pEG(KT) (Mitchell et al 1993) and then purified from yeast (strain GRF-167) 725 using Glutathione Sepharose 4B affinity resin (GE Healthcare) SlRd2 cDNA was cloned into 726


30

pET28a and purified from E coli using a Histidine column kit (GE Healthcare) SlRd2(UspA) was 727 amplified by PCR using primers OPS675 (5rsquo- 728 AAAAAGCAGGCTCTATGGGCCGTGATATAGTGATC-3rsquo) and OPS676 (5rsquo- 729 AGAAAGCTGGGTTTAAGGAACTATGATGACCGG-3rsquo) cloned into pDEST15 vector 730 (Invitrogen) and purified using Glutathione Sepharose 4B affinity beads For kinase assays 03 microg 731 of SlCipk6 proteins and 25 microg of SlRd2 25 microg of SlRd2(UspA) or 05 microg of MBP proteins were 732 incubated in a final volume of 30 microL in kinase buffer (50 mM Tris-HCl pH 75 2 mM MnCl2 2 733 mM DTT) 10 microM ATP and 10 μCi of [γ-32P]ATP [Amersham 3000 Cimmol (1 Ci = 37 GBq)] 734 during 60 min at 30ordmC The reactions were stopped by adding 10 μL of 4x Laemmli loading buffer 735 The samples were denatured by boiling 5 min and then were run on 12 SDS-PAGE gel Kinase 736 activity was visualized by autoradiography Protein levels were verified by colloidal Coomassie 737 Brilliant Blue G-250 staining 738 739 BiFC Assay 740 For BiFC assays SlRd2 and SlCipk6 coding sequences were cloned into pYFPC (C-terminal YFP 741 fragment) and pYFPN (N-terminal YFP fragment) respectively by gateway technology (Invitrogen) 742 Agrobacterium strains C58C1 carrying SlRd2-YFPC and SlCipk6-YFPN were co-infiltrated into N 743 benthamiana leaves Agrobacterium cultures carrying YFPC YFPN empty vectors and the mix 744 SlRd2-YFPCSlCbl10-YFPN were used as negative controls Staining of N benthamiana cells with 745 FM4-64 was performed as previously described (Bolte et al 2004) Fluorescence images were 746 obtained 48 h after infiltration using a Leica TCS Sp2DMRE confocal microscope with excitation 747 wavelengths of 514 nm (YFP) 543 nm (FM4-64) and 440 nm (CFP) Transient expression of 748 proteins in N benthamiana leaves via agro-infiltration was performed as previously described (He 749 et al 2004) 750 751 Yeast Stress Tolerance Assays 752 For stress tolerance assays yeast strain BY4741 harbouring p426GPD empty vector (EV) and 753 p426GPD-SlRd2 constructs were grown in liquid SD medium lacking Uracile (SD -Ura) 754 containing 1 glucose (wv) during 24 h at 30 ordmC Subsequently they were diluted to the same 755 concentrations (OD600 = 10-1 10-2 10-3 and 10-4) and 10 μL of each dilution was spotted onto solid 756 YPD medium supplemented with the different stress agents Finally yeast were grown at 30ordmC 757 during 3 days and photographed 758 Preparation and Purification of SlRd2 Antibody 759 SlRd2 open reading frame was cloned into pET-28a in frame with an N-terminal His-tag) The 760 pET-28a-SlRd2 construct was transformed in BL21 (DE3) RIL (Stratagene) E coli cells 761 Preparation of recombinant protein was performed as described previously (San-Miguel et al 762


31

2013) Briefly protein expression was induced at OD600 = 05 by adding of 1 mM IPTG to LB 763 medium supplemented with 50 μg mL ampicillin Cells were harvested by centrifugation at 3000 g 764 for 10 min at room temperature and frozen overnight at minus80degC Preparation of His-tagged 765 recombinant protein from pET-28a-SlRd2 was performed according to the manufacturerrsquos 766 instructions (Quiagen) The antiserum was raised in rabbits using the full length SlRd2 as the 767 antigen 768 769 Oxidative Stress Complementation Assay in E coli 770 The complementation assay was performed as described by Nystrom and Neidhardt (1994) Fresh 771 MOPS medium containing 50 μgmL ampicillin was inoculated with overnight cultures of W3101 772 (F- galT22 λ- IN (rrnD-rrnE)1 rph-1) and TN3151 (F- λ- IN (rrnD-rrnE)1 uspA1kan rph-1) 773 strains harbouring pDEST17-SlRd2 and pDEST17-UspA constructs at an OD600 of 01 and grown at 774 37 ordmC until they reached an OD600 = 07 A final concentration of 5 mM H2O2 was then added 1 775 mL of each culture was harvested to perform the growth curve Viability is expressed as the 776 number of colony forming unit (CFU) at time divided by the number of CFU before the imposition 777 of stress Expression of SlRd2 and UspA proteins was determined by immunoblotting using anti-778 His mouse monoclonal antibody (Roche) mRNA UspA and 16S rRNA abundance were verified by 779 RT-PCR using gene specific primers OPS446 (5acute-ATGGCTTATAAACACATTCTC-3acute) and 780 OPS448 (5acute-TTATTCTTCTTCGTCGCGCAGC -3acute) for UspA and OPS600 (5acute-781 CTCCTACGGGAGGCAGCAG-3acute) and OPS601 (5acute-ATTACCGCGGCKGCTG-3acute) for 16S 782 rRNA 783 784 Accession Numbers 785 Sequence data from this article can be found in the GenBankEMBL data libraries under the 786 following accession numbers SlRd2 (KP843662) SlCipk6 (JF831200) SlCipk11 (JF831201) and 787 SlCipk14 (JF831202) 788 789 Author Contributions 790 EGB OdP and FdlT designed experiments and analyzed the data OdP wrote the manuscript 791 and EGB helped with the writing EGB performed most of experiments JM Personat generated 792 Figure 7A 793 794 Acknowledgements 795 We thank Jose M Pardo (Instituto de Bioquiacutemica Vegetal y Fotosiacutentesis Consejo Superior de 796 Investigaciones Cientiacuteficas Sevilla Spain) for critically reading the manuscript 797 798 FIGURE LEGENDS 799


32

800 Figure 1 SlRd2 and SlCipk6 Interact in a Yeast Two Hybrid Assay (A) Schematic structure of 801 SlCipk6 protein representing N-terminal catalytic junction and C-terminal regulatory domains 802 PPi NAFFISL and activation loop domains Residues mutagenized for SlCipk6 characterization 803 Lys43 to Met (K43M) and Thr172 to Asp (T172D) are marked (B) Yeast two hybrid assay 804 performed with SlCipk6 and SlCipk6 mutant derivatives in the bait vector pEG202 and SlRd2 in 805 the prey vector pJG4-5 WT full length SlCipk6 K43M and T172D single amino acid substitution 806 of Lys43 to Met and Thr172 to Asp respectively ΔNAF amino acids 302-321 deletion ΔCterm 807 amino acids 302-432 deletion ΔNterm amino acids 1-302 deletion Clones were transformed into 808 EGY48 strain grown in liquid culture and spotted at OD600= 01 and 001 on selective medium 809 Growth and blue patches indicates interaction (C) Interaction was quantitated by a β -810 galactosidase assay Represented are the mean value of three independent experiments each 811 performed with three independent transformants Error bars represent the standard deviation of 812 three different experiments 813 814 Figure 2 SlCipk6 and SlRd2 Interact in Planta and the Complex Localizes in the Cytosol (A) 815 Protein extracts from N benthamiana leaves agro-infiltrated with GFP-SlRd2 and 816 SlCipk6(T172D)-Myc which is fused to two copies of the protein A IgG binding domains (2xIgG-817 BD) were incubated with IgG agarose beads eluted and analyzed by immunoblotting using anti-818 Myc for SlCipk6 and anti-GFP for SlRd2 GFP protein was used as a negative control No 819 interaction was detected when SlCipk6(T172D) was agro-infiltrated with GFP (right upper panel) 820 (B) Fluorescence images of N benthamiana leaf sections agro-infiltrated with SlRd2-YFPC and 821 SlCipk6-YFPN Co-localization analyses of SlCipk6SlRd2 complexes (yellow a and d) with the 822 fluorescent membrane marker dye FM4-64 (red b) and the cytoplasmic marker AtFKBP12-CFP 823 (cyan e Faure et al 1998) Merged images are shown in (c) and (f) respectively White arrows 824 show the cytoplasmic localization observed for the SlCipk6SlRd2 complexes Localization 825 analysis of YFPN (g) and YFPC (h) empty vectors and SlCbl10SlRd2 (i) were used as an 826 experiment controls Bars = 10 μm 827 828 Figure 3 SlRd2 and SlCipk6 Transcripts Highly Accumulate after NaCl and Osmotic Stress 829 in Tomato (A-E) Y axis represents mean values of Quantitative Real-Time PCR (Q RT-PCR) of 3 830 experiments with three biological replicates in each Error bars represent the standard error The 831 expression levels were normalized to SlActin2 Gene induction (fold increase) in infected or treated 832 plants was compared with the expression level of control or mock inoculated plants at 0 h and is 833 shown as relative expression excepting in (A) (A) SlRd2 transcript accumulates in tomato leaves 834 petioles stems flowers and roots Relative expression on Y axis was compared with expression 835


33

level in leaves (B C) SlRd2 and (D E) SlCipk6 mRNA accumulation in leaves during NaCl (B D) 836 and osmotic stress (OS) (C E) Hydroponically grown tomato plants were treated with 100 mM 837 NaCl (B D) or 300 mM mannitol (C E) 838 839 Figure 4 Tomato SlRd2 Protein Structure and Phylogenetic Analysis (A) Schematic structure 840 of SlRd2 containing N-terminal and C-terminal domains and a conserved universal stress protein 841 domain which encompasses a dimerization motif Marks ( ) indicate the conserved residues 842 described to be involved in ATP-binding (B) Phylogenetic relationship of proteins containing Usp 843 domains from plants and bacteria including Arabidopsis thaliana AtRD2 tomato SlRd2 and 844 LeER6 Nicotiana benthamiana NbRd2 Oryza sativa OsUSP1 Vicia faba VfENOD18 845 Escherichia coli EcUspG and EcUspA Haemophilus influenzae HiUspA and Methanococcus 846 jannaschii Mj0577 SlCbl10 was used as the outgroup The numbers on the tree represent 847 bootstrap scores 848 849 Figure 5 SlRd2 is an ATP-Binding Protein (A) Alignment of Usp domain of SlRd2 and AtRD2 850 and the full amino acid sequence of Mj0577 Conserved residues are marked in black boxes while 851 similar residues are marked in grey boxes Black bars underline residues putatively involved in 852 ATP-binding adenine (A) phosphate (P) or ribose (R) Walker motif or P loop motif G-2x-G-9x-853 G-(ST) for ATP binding is marked with a blue line Amino acid residues responsible for protein 854 dimerization are marked as (D) (B) For ATP-binding in vitro assay 1 μg of purified His-SlRd2 855 and His-GFP proteins from E coli were incubated with [α-32P]ATP and corresponding buffer 856 during 1 h at 30 ordmC Autoradiograph indicates ATP-binding (top panel) immunoblot analysis 857 indicates protein level in the ATP binding assay (low panel) (C) For ATP-binding in vivo assay N 858 benthamiana leaf extracts from WT and GFP-Rd2 overexpressing line 6 plants were incubated with 859 20 μg BHAcATP Biotinylated proteins were purified using streptavidin beads separated on 860 protein gel and detected using anti-GFP (top panel) Samples labelled with BHAcATP in presence 861 of 10 mM of ATP were used as a control Ponceau S staining was used as a loading control (bottom 862 panel) 863 864 Figure 6 SlRd2 Homodimerizes in the Cytosol and in the Nucleus (A) Yeast two hybrid assay 865 with both SlRd2 and SlRd2Δdim cloned in the bait pEG202 (with a LexA fusion tag at the C 866 terminus) and prey pJG45 (with a HA fusion tag at the C-terminus) vectors in EGY48 strain grown 867 in liquid culture and spotted at OD600= 01 and 001 on selective medium Growth and blue patches 868 indicates interaction (B) Expression of both SlRd2 and SlRd2Δdim proteins in yeast were 869 confirmed by immunoblotting using anti-LexA (pEG202) and anti-HA (pJG45) antibodies 870 respectively (C) The cross-linking agent disuccinimidyl glutarate (DSG) was added to a His-SlRd2 871 over-expressing E coli culture for 30 min Polymerization of His-SlRd2 protein was calculated 872


34

according to its molecular weight 1X monomer and 2X dimer (D) Co-localization analyses of 873 SlRd2SlRd2 complexes (yellow a d and g) with the membrane marker dye FM4-64 (red b) 874 cytoplasmic marker AtFKBP12 (cyan e) or nuclear marker DAPI (blue h) Merged images are 875 shown in (c f and i) Arrow denote localization in the nucleus Bars = 10 μm 876 877 Figure 7 SlCipk6 Phosphorylates SlRd2 (A) SlCipk6 SlRd2 SlRd2(UspA) and MBP alone and 878 with SlCipk6 were incubated in the presence of [γ-32P] ATP and kinase buffer electrophoresed on 879 SDS polyacrylamide gel and autoradiographed They were expressed and purified as fusion 880 proteins as follows SlCipk6-GST His-SlRd2 GST-SlRd2(UspA) Autoradiographs indicate 881 SlCipk6 auto-phosphorylation (upper panels) and SlRd2 phosphorylation (lower panels) Coomasie 882 protein staining indicates SlRd2 SlRd2(UspA) MBP and SlCipk6 protein loads Protein amounts 883 loaded 03 microg for SlCipk6 25 microg for SlRd2 and SlRd2(UspA) and 05 microg for MBP (B) 884 Phosphorylation was quantified using Cyclone Phosphorimager Optiquant software (Packard 885 Bioscience Perkin Elmer) au arbitrary units Signal from MBP phosphorylation was not 886 normalized to protein content to better quantify SlRd2 phosphorylation and ATP binding Similar 887 results were obtained in two additional experiments 888 889 Figure 8 SlRd2 Expression Confers LiCl Resistance in Yeast and its Dimerization is 890 Required for its Biological Function (A) BY4741 yeast strain expressing SlRd2 (cloned into 891 p426GPD) was subjected to different stress conditions As a control BY4741 was transformed 892 with the empty vector (EV) Both SlRd2 and EV lines were grown in liquid culture and spotted on 893 specific medium at different dilutions Over-expression of SlRd2 showed a higher growth than EV 894 in response to LiCl (300 mM) (B) Over-expression of SlRd2Δdim showed the same growth rate 895 than EV line in response to LiCl The experiment was performed as described in A (C) Expression 896 of SlRd2 and SlRd2Δdim proteins in yeast were confirmed by immunoblotting using anti-SlRd2 897 antibodies 898 899 Figure 9 SlRd2 Dimerization is Required for its Interaction with SlCipk6 (A) Yeast two 900 hybrid assay performed with SlCipk6 and SlCipk6(T1172D) in the bait vector pEG202 and 901 SlRd2Δdim in the prey vector pJG4-5 Clones were transformed into EGY48 strain grown in 902 liquid culture and spotted at OD600= 01 and 001 on selective medium Growth and blue patches 903 indicates interaction A Y2H experiment performed with SlRd2 and SlCipk6 was used as a control 904 (B) Fluorescence images of N benthamiana epidermal cells expressing SlRd2Δdim-YFPC and 905 SlCipk6-YFPN YFP signal was not reconstituted in the BiFC experiment (a) confirming that 906 SlRd2 dimerization is required for the interaction with SlCipk6 in planta A BiFC experiment 907 performed with SlRd2 and SlCipk6 was used as a control (b) Bars = 10 μm 908 909


35

Figure 10 Tomato SlRd2 Restores E coli TN3151 (Δuspa) Survival and Negatively Regulates 910 SlCipk6-mediated ROS (A) WT [W3101 (F- IN(rrnD-rrnE)1 rph-1)] Uspa mutant TN3151 [F- 911 IN(rrnD-rrnE)1 UspAkan rph-1)] and the overexpressing TN3151 SlRd2 (OE-SlRd2) and 912 TN3151 UspA (OE-UspA) E coli lines were grown in MOPS medium and treated with H2O2 (5 913 mM) when the cultures reached an OD600 of 07 (B) Immunoblot experiment confirmed SlRd2 914 (upper panel) and UspA (lower panel) expression in TN3151 E coli lines (C) Transcript 915 abundance of UspA cDNA in TN3151 and WT strains was determined by RT-PCR 16S rRNA 916 transcript abundance was used as an internal standard control (D) Measurement of ROS 917 production in N benthamiana leaf disks of GFP-SlRd2 overexpressing (OE-6 and OE-7) and WT 918 plants after agro-infiltration of SlCipk6-Myc (WT) SlCipk6(T172D)-Myc (T172D) 919 SlCipk6(K43M)-Myc (K43M) or empty vector (EV) ROS accumulation was quantified as relative 920 light units (RLU) 8 to 12 h after Data are presented as means plusmn standard errors of four independent 921 experiments Means with different letters are significantly different at P lt 005 Studentrsquos t test 922 Anti-GFP and anti-Myc were used as primary antibodies 923 924 SUPPLEMENTAL FIGURES 925 Supplemental Figure 1 SlRd2 SlCipk6 and SlCipk6 Mutant Derivative Proteins are Expressed in 926 Yeast 927 Supplemental Figure 2 Specificity of SlCipk6 SlRd2 Interaction 928 Supplemental Figure 3 Both SlCipk6 and SlRd2 are Localized in the Cytosol and in the Nucleus 929 Supplemental Figure 4 SlRd2 Protein Sequence Alignment 930 Supplemental Figure 5 SlRd2 Protein Sequence Alignment 931 Supplemental Figure 6 SlCipk6-Myc (WT) SlCipk6(T172D)-Myc (T172D) and SlCipk6(K43M)-932 Myc (K43M) are expressed in N benthamiana GFP-SlRd2 overexpressing (OE-6 and OE-7) and 933 WT plants 934 Supplemental Table 1 Summary of the Yeast Two Hybrid Screen 935 936 Supplemental Figure 1 SlRd2 SlCipk6 and SlCipk6 Mutant Derivative Proteins are 937 Expressed in Yeast SlCipk6 and SlCipk6 mutant derivatives were cloned in the bait vector 938 pEG202 (with a LexA fusion tag at the C-terminus) and SlRd2 was cloned in the prey vector pJG4-939 5 (with a HA fusion tag at the C-terminus) Yeast transformants were grown in SD medium which 940 lacks histidine tryptophan and uracil for 24 h at 30ordmC Yeast cells from 1 mL culture were 941 collected protein extracted and SlCipk6 and SlRd2 protein expression was detected by 942 immunoblot using anti-LexA (upper panel) and anti-HA (lower panel) antibodies respectively 943 944 Supplemental Figure 2 Specificity of SlCipk6 SlRd2 Interaction (A) SlRd2 did not interact 945 with SlCipk11 or SlCipk14 (closest tomato CIPK to SlCipk6 expressed in leaves) SlCipk11 and 946


36

SlCipk14 were used as baits and both were cloned into pEG202 (with a LexA fusion tag at the C-947 terminus) SlRd2 was used as prey and it was cloned into pJG4-5 (with a HA fusion tag at the C-948 terminus) (B) SlCipk6 (in pEG202) did not interact with SGN-U601569 (in pJG4-5) the closest 949 tomato protein to SlRd2 For (A) and (B) the yeast two-hybrid assay was performed as described in 950 Figure 1 Blue colour (panel 1) or growth (panel 2) indicates interaction Expression of SlCipk11 951 SlCipk14 or SlCipk6 and SlRd2 or SGN-U601569 proteins in yeast were confirmed by 952 immunoblotting using anti-LexA and anti-HA antibodies respectively 953 954 Supplemental Figure 3 Both SlCipk6 and SlRd2 are Localized in the Cytosol and in the 955 Nucleus Fluorescence images of N benthamiana epidermal cells expressing SlCipk6-GFP and 956 GFP-SlRd2 (a) and (b) Co-localization analyses of GFP-SlCipk6 (green) with the fluorescent 957 membrane marker dye FM4-64 (red) (c) Co-localization analyses of GFP-SlRd2 (green) with the 958 cytoplasmic marker AtFKBP12-CFP (cyan) and fluorescent membrane marker dye FM4-64 (red) 959 Arrows denote localization in the nucleus Bars = 10 μm 960 961 Supplemental Figure 4 SlRd2 Protein Sequence Alignment Multiple protein sequence 962 alignment of Usp domains of Usp proteins from bacteria Escherichia coli (EcUspA) Haemophilus 963 influenzae (HiUspA) Methanococcus jannaschii (Mj0577) and plants Oryza sativa (OsUsp) 964 Vicia faba (VfENOD18) Solanum lycopersicum (LeER6) Arabidopsis thaliana (AtRD2) 965 Nicotiana benthamiana (NbRd2) Residues conserved in five or more sequences are marked in 966 black boxes while similar residues are marked in grey boxes Black bars underline residues as 967 adenine (A) phosphate (P) or ribose (R) are putatively involved in ATP-binding Walker motif or P 968 loop motif G-2x-G-9x-G-(ST) is marked with a blue line Amino acid residues responsible for 969 protein dimerization are marked as (D) 970 971 Supplemental Figure 5 SlRd2 Protein Sequence Alignment Rd2 protein sequence alignment 972 was performed with amino acid sequences from tomato SlRd2 Nicotiana benthamiana NbRd2 973 and Arabidopsis thaliana AtRD2 Residues conserved in the three sequences are marked in black 974 boxes while similar residues are marked in grey boxes Usp domain is boxed in solid red line the 975 N-terminal region is boxed in solid blue line and the C-terminal extension is boxed in solid green 976 line 977 Supplemental Figure 6 SlCipk6-Myc (WT) SlCipk6(T172D)-Myc (T172D) and 978 SlCipk6(K43M)-Myc (K43M) are expressed in N benthamiana GFP-SlRd2 overexpressing 979 (OE-6 and OE-7) and WT plants Anti-GFP and anti-Myc were used as primary antibodies 980 981 Supplemental Table 1 Summary of the Yeast Two Hybrid Screen 21 x 105 and 13 x 105 982 transformants were obtained for SlCipk6 and SlCipk6(T172D) respectively and 11 x 103 and 45 x 983


37

103 colonies were analyzed by selection medium 11 and 34 CIPs were identified using either 984 SlCipk6 or SlCipk6(T172D) as baits 985 986 REFERENCES 987 988 Aouida M Tounekti O Leduc A Belhadj O Mir L Ramotar D (2004) Isolation and 989 characterization of Saccharomyces cerevisiae mutants with enhanced resistance to 990 the anticancer drug bleomycin Curr Genet 45 265-72 991 Aravind L Anantharaman V Koonin EV (2002) Monophyly of class I aminoacyl 992 tRNA synthetase USPA ETFP photolyase and PP-ATPase nucleotide-binding 993 domains implications for protein evolution in the RNA Proteins 48 1-14 994 Babiychuk E Kushnir S Belles-Boix E Van Montagu M Inze D (1995) Arabidopsis 995 thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the 996 thiol-oxidizing drug diamide J Biol Chem 270 26224-31 997 Batistic O Waadt R Steinhorst L Held K Kudla J (2010) CBL-mediated targeting 998 of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular 999 stores Plant J 61 211-22 1000 Becker JD Moreira LM Kapp D Frosch SC Puhler A Perlic AM (2001) The 1001 nodulin vfENOD18 is an ATP-binding protein in infected cells of Vicia faba L nodules 1002 Plant Mol Biol 47 749-59 1003 Bernard A Domergue F Pascal S et al (2012) Reconstitution of plant alkane 1004 biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and 1005 ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex 1006 Plant Cell 24 3106-18 1007 Bolte S Talbot C Boutte Y Catrice O Read ND Satiat-Jeunemaitre B (2004) FM-1008 dyes as experimental probes for dissecting vesicle trafficking in living plant cells J 1009 Microsc 214 159-73 1010 Chaves-Sanjuan A Sanchez-Barrena MJ Gonzalez-Rubio JM et al (2014) 1011 Structural basis of the regulatory mechanism of the plant CIPK family of protein 1012 kinases controlling ion homeostasis and abiotic stress Proc Natl Acad Sci U S A 111 1013 E4532-41 1014 Chen L Ren F Zhou L Wang QQ Zhong H Li XB (2012) The Brassica napus 1015 calcineurin B-Like 1CBL-interacting protein kinase 6 (CBL1CIPK6) component is 1016 involved in the plant response to abiotic stress and ABA signalling J Exp Bot 63 1017 6211-22 1018 Chen L Wang QQ Zhou L Ren F Li DD Li XB (2013) Arabidopsis CBL-interacting 1019 protein kinase (CIPK6) is involved in plant response to saltosmotic stress and ABA 1020 Mol Biol Rep 40 4759-67 1021 de la Torre F Gutierrez-Beltran E Pareja-Jaime Y Chakravarthy S Martin GB 1022 del Pozo O (2013) The tomato calcium sensor Cbl10 and its interacting protein 1023 kinase Cipk6 define a signaling pathway in plant immunity Plant Cell 25 2748-64 1024 Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev 1025 Plant Biol 61 593-620 1026 Drerup MM Schlucking K Hashimoto K et al (2013) The Calcineurin B-like 1027 calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26 1028 regulate the Arabidopsis NADPH oxidase RBOHF Mol Plant 6 559-69 1029


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Katiyar-Agarwal S Zhu J Kim K et al (2006) The plasma membrane Na+H+ 1078 antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in 1079 Arabidopsis Proc Natl Acad Sci U S A 103 18816-21 1080 Kerk D Bulgrien J Smith DW Gribskov M (2003) Arabidopsis proteins containing 1081 similarity to the universal stress protein domain of bacteria Plant Physiol 131 1209-1082 19 1083 Kim BG Waadt R Cheong YH et al (2007) The calcium sensor CBL10 mediates salt 1084 tolerance by regulating ion homeostasis in Arabidopsis Plant J 52 473-84 1085 Kim KN Cheong YH Gupta R Luan S (2000) Interaction specificity of Arabidopsis 1086 calcineurin B-like calcium sensors and their target kinases Plant Physiol 124 1844-1087 53 1088 Kim WY Ali Z Park HJ et al (2013) Release of SOS2 kinase from sequestration with 1089 GIGANTEA determines salt tolerance in Arabidopsis Nat Commun 4 1352 1090 Kobayashi M Yoshioka M Asai S et al (2012) StCDPK5 confers resistance to late 1091 blight pathogen but increases susceptibility to early blight pathogen in potato via 1092 reactive oxygen species burst New Phytol 196 223-37 1093 Kvint K Nachin L Diez A Nystrom T (2003) The bacterial universal stress protein 1094 function and regulation Curr Opin Microbiol 6 140-5 1095 Lan WZ Lee SC Che YF Jiang YQ Luan S (2011) Mechanistic analysis of AKT1 1096 regulation by the CBL-CIPK-PP2CA interactions Mol Plant 4 527-36 1097 Lee SC Lan WZ Kim BG et al (2007) A protein 1098 phosphorylationdephosphorylation network regulates a plant potassium channel 1099 Proc Natl Acad Sci U S A 104 15959-64 1100 Lenman M Sorensson C Andreasson E (2008) Enrichment of phosphoproteins and 1101 phosphopeptide derivatization identify universal stress proteins in elicitor-treated 1102 Arabidopsis Mol Plant Microbe Interact 21 1275-84 1103 Li J Long Y Qi GN et al (2014) The Os-AKT1 channel is critical for K+ uptake in rice 1104 roots and is modulated by the rice CBL1-CIPK23 complex Plant Cell 26 3387-402 1105 Liang Q Zhou B (2007) Copper and manganese induce yeast apoptosis via different 1106 pathways Mol Biol Cell 18 4741-9 1107 Liu WT Karavolos MH Bulmer DM et al (2007) Role of the universal stress 1108 protein UspA of Salmonella in growth arrest stress and virulence Microb Pathog 42 1109 2-10 1110 Loukehaich R Wang T Ouyang B et al (2012) SpUSP an annexin-interacting 1111 universal stress protein enhances drought tolerance in tomato J Exp Bot 63 5593-1112 606 1113 Luan S (2009) The CBL-CIPK network in plant calcium signaling Trends Plant Sci 14 1114 37-42 1115 Lyzenga WJ Liu H Schofield A Muise-Hennessey A Stone SL (2013) Arabidopsis 1116 CIPK26 interacts with KEG components of the ABA signalling network and is 1117 degraded by the ubiquitin-proteasome system J Exp Bot 64 2779-91 1118 Matsumoto TK Pardo JM Takeda S Bressan RA Hasegawa PM (2001) Tobacco 1119 and Arabidiopsis SLT1 mediate salt tolerance of yeast Plant Mol Biol 45 489-500 1120 Merkouropoulos G Andreasson E Hess D Boller T Peck SC (2008) An 1121 Arabidopsis protein phosphorylated in response to microbial elicitation AtPHOS32 1122 is a substrate of MAP kinases 3 and 6 J Biol Chem 283 10493-9 1123 Mitchell DA Marshall TK Deschenes RJ (1993) Vectors for the inducible 1124 overexpression of glutathione S-transferase fusion proteins in yeast Yeast 9 715-22 1125


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Nachin L Brive L Persson KC Svensson P Nystrom T (2008) Heterodimer 1126 formation within universal stress protein classes revealed by an in silico and 1127 experimental approach J Mol Biol 380 340-50 1128 Nagae M Nozawa A Koizumi N et al (2003) The crystal structure of the novel 1129 calcium-binding protein AtCBL2 from Arabidopsis thaliana J Biol Chem 278 42240-1130 6 1131 Nibau C Wu HM Cheung AY (2006) RACROP GTPases hubs for signal integration 1132 and diversification in plants Trends Plant Sci 11 309-15 1133 Nystrom T Neidhardt FC (1992) Cloning mapping and nucleotide sequencing of a 1134 gene encoding a universal stress protein in Escherichia coli Mol Microbiol 6 3187-1135 98 1136 Nystrom T Neidhardt FC (1994) Expression and role of the universal stress protein 1137 UspA of Escherichia coli during growth arrest Mol Microbiol 11 537-44 1138 OToole R Smeulders MJ Blokpoel MC Kay EJ Lougheed K Williams HD (2003) 1139 A two-component regulator of universal stress protein expression and adaptation to 1140 oxygen starvation in Mycobacterium smegmatis J Bacteriol 185 1543-54 1141 Ohta M Guo Y Halfter U Zhu JK (2003) A novel domain in the protein kinase SOS2 1142 mediates interaction with the protein phosphatase 2C ABI2 Proc Natl Acad Sci U S A 1143 100 11771-6 1144 Park HJ Kim WY Yun DJ (2013) A role for GIGANTEA keeping the balance between 1145 flowering and salinity stress tolerance Plant Signal Behav 8 e24820 1146 Persson O Valadi A Nystrom T Farewell A (2007) Metabolic control of the 1147 Escherichia coli universal stress protein response through fructose-6-phosphate Mol 1148 Microbiol 65 968-78 1149 Quintero FJ Ohta M Shi H Zhu JK Pardo JM (2002) Reconstitution in yeast of the 1150 Arabidopsis SOS signaling pathway for Na+ homeostasis Proc Natl Acad Sci U S A 99 1151 9061-6 1152 Ragel P Rodenas R Garcia-Martin E et al (2015) The CBL-Interacting Protein 1153 Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis 1154 Roots Plant Physiol 169 2863-73 1155 Rajput NA Zhang M Ru Y et al (2014) Phytophthora sojae effector PsCRN70 1156 suppresses plant defenses in Nicotiana benthamiana PLoS One 9 e98114 1157 Rubio V Shen Y Saijo Y et al (2005) An alternative tandem affinity purification 1158 strategy applied to Arabidopsis protein complex isolation Plant J 41 767-78 1159 San-Miguel T Perez-Bermudez P Gavidia I (2013) Production of soluble 1160 eukaryotic recombinant proteins in is favoured in early log-phase cultures induced at 1161 low temperature Springerplus 2 89 1162 Sanyal SK Pandey A Pandey GK (2015) The CBL-CIPK signaling module in plants a 1163 mechanistic perspective Physiol Plant 1164 Sauter M Rzewuski G Marwedel T Lorbiecke R (2002) The novel ethylene-1165 regulated gene OsUsp1 from rice encodes a member of a plant protein family related 1166 to prokaryotic universal stress proteins J Exp Bot 53 2325-31 1167 Seifart Gomes C Izar B Pazan F et al (2011) Universal stress proteins are 1168 important for oxidative and acid stress resistance and growth of Listeria 1169 monocytogenes EGD-e in vitro and in vivo PLoS One 6 e24965 1170 Shitan N Dalmas F Dan K et al (2013) Characterization of Coptis japonica 1171 CjABCB2 an ATP-binding cassette protein involved in alkaloid transport 1172 Phytochemistry 91 109-16 1173


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Sousa MC McKay DB (2001) Structure of the universal stress protein of 1174 Haemophilus influenzae Structure 9 1135-41 1175 Tripathi V Parasuraman B Laxmi A Chattopadhyay D (2009) CIPK6 a CBL-1176 interacting protein kinase is required for development and salt tolerance in plants 1177 Plant J 58 778-90 1178 Tsou PL Lee SY Allen NS Winter-Sederoff H Robertson D (2012) An ER-targeted 1179 calcium-binding peptide confers salt and drought tolerance mediated by CIPK6 in 1180 Arabidopsis Planta 235 539-52 1181 Udawat P Jha RK Sinha D Mishra A Jha B (2016) Overexpression of a Cytosolic 1182 Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and 1183 Osmotic Stress in Transgenic Tobacco Plants Front Plant Sci 7 518 1184 Uno Y Rodriguez Milla MA Maher E Cushman JC (2009) Identification of proteins 1185 that interact with catalytically active calcium-dependent protein kinases from 1186 Arabidopsis Mol Genet Genomics 281 375-90 1187 VanBogelen RA Hutton ME Neidhardt FC (1990) Gene-protein database of 1188 Escherichia coli K-12 edition 3 Electrophoresis 11 1131-66 1189 Verslues PE Batelli G Grillo S et al (2007) Interaction of SOS2 with nucleoside 1190 diphosphate kinase 2 and catalases reveals a point of connection between salt stress 1191 and H2O2 signaling in Arabidopsis thaliana Mol Cell Biol 27 7771-80 1192 Villamor JG Kaschani F Colby T et al (2013) Profiling protein kinases and other 1193 ATP binding proteins in Arabidopsis using Acyl-ATP probes Mol Cell Proteomics 12 1194 2481-96 1195 Walter M Chaban C Schutze K et al (2004) Visualization of protein interactions in 1196 living plant cells using bimolecular fluorescence complementation Plant J 40 428-38 1197 Wang L Tsuda K Sato M Cohen JD Katagiri F Glazebrook J (2009) Arabidopsis 1198 CaM binding protein CBP60g contributes to MAMP-induced SA accumulation and is 1199 involved in disease resistance against Pseudomonas syringae PLoS Pathog 5 1200 e1000301 1201 Weber A Jung K (2006) Biochemical properties of UspG a universal stress protein of 1202 Escherichia coli Biochemistry 45 1620-8 1203 Wong HL Pinontoan R Hayashi K et al (2007) Regulation of rice NADPH oxidase 1204 by binding of Rac GTPase to its N-terminal extension Plant Cell 19 4022-34 1205 Xu J Li HD Chen LQ et al (2006) A protein kinase interacting with two calcineurin 1206 B-like proteins regulates K+ transporter AKT1 in Arabidopsis Cell 125 1347-60 1207 Xu T Wen M Nagawa S et al (2010) Cell surface- and rho GTPase-based auxin 1208 signaling controls cellular interdigitation in Arabidopsis Cell 143 99-110 1209 Yamaguchi-Shinozaki K Koizumi M Urao S Shinozaki K (1992) Molecular cloning 1210 and characterization of 9 cDNAs for genes that are responsive to desiccation in 1211 Arabidopsis thaliana sequence analysis of one cDNA clone that encode a putative 1212 transmembrane channel protein Plant Cell Physiol 33 217-24 1213 Yang Y Qin Y Xie C et al (2010) The Arabidopsis chaperone J3 regulates the 1214 plasma membrane H+-ATPase through interaction with the PKS5 kinase Plant Cell 1215 22 1313-32 1216 Ye T Garcia-Salcedo R Ramos J Hohmann S (2006) Gis4 a new component of the 1217 ion homeostasis system in the yeast Saccharomyces cerevisiae Eukaryot Cell 5 1611-1218 21 1219 Yenush L Merchan S Holmes J Serrano R (2005) pH-Responsive posttranslational 1220 regulation of the Trk1 potassium transporter by the type 1-related Ppz1 phosphatase 1221 Mol Cell Biol 25 8683-92 1222


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Yu Q An L Li W (2014) The CBL-CIPK network mediates different signaling 1223 pathways in plants Plant Cell Rep 33 203-14 1224 Zahur M Maqbool A Ifran M et al (2009) [Isolation and functional analysis of 1225 cotton universal stress protein promoter in response to phytohormones and abiotic 1226 stresses] Mol Biol (Mosk) 43 628-35 1227 Zaidi I Gonzalez A Touzri M et al (2012) The wheat MAP kinase phosphatase 1 1228 confers higher lithium tolerance in yeast FEMS Yeast Res 12 774-84 1229 Zarembinski TI Hung LW Mueller-Dieckmann HJ et al (1998) Structure-based 1230 assignment of the biochemical function of a hypothetical protein a test case of 1231 structural genomics Proc Natl Acad Sci U S A 95 15189-93 1232 Zegzouti H Jones B Frasse P et al (1999) Ethylene-regulated gene expression in 1233 tomato fruit characterization of novel ethylene-responsive and ripening-related 1234 genes isolated by differential display Plant J 18 589-600 1235 Zhou J Loh YT Bressan RA Martin GB (1995) The tomato gene Pti1 encodes a 1236 serinethreonine kinase that is phosphorylated by Pto and is involved in the 1237 hypersensitive response Cell 83 925-35 1238 1239


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Yenush L Merchan S Holmes J Serrano R (2005) pH-Responsive posttranslational regulation of the Trk1 potassium transporterby the type 1-related Ppz1 phosphatase Mol Cell Biol 25 8683-92


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33 Title 34 A Universal Stress Protein Involved in Oxidative Stress is a Phosphorylation Target for protein kinase 35 CIPK6 36 Authors 37 Emilio Gutieacuterrez-Beltraacuten Joseacute Mariacutea Personat Fernando de la Torre and Olga del Pozo 38 39 Institution Address 40 Instituto de Bioquiacutemica Vegetal y Fotosiacutentesis Universidad de SevillaConsejo Superior de 41 Investigaciones Cientiacuteficas Avda Ameacuterico Vespucio 49 41092 Sevilla Spain 42 43 44 One-Sentence Summary 45 Universal stress protein SlRd2 is a Cipk6 target and regulates Cipk6-mediated ROS 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Footnotes 64


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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations

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Parsed Citations

Reviewer PDF

Parsed Citations




















































































































































































Parsed Citations

Reviewer PDF

Parsed Citations

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