A universal stress protein involved in oxidative stress1 is a phosphorylation target for protein kinase CIPK6

A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK61 Emilio Gutiérrez-Beltrán, José María Personat, Fernando de la Torre, and Olga del Pozo Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Sevilla, Spain ORCID IDs: 0000-0001-7978-3164 (E.G.B.); 0000-0003-2273-5000 (J.M.P.); 0000-0002-5143-3709 (F.d.l.T.); 0000-0001-9852-0431 (O.d.P.). Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca2+ signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named “SlRd2”. SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2treatment. Finally, transient expression of SlCipk6 in transgenic N.benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation. Environmental factors, especially those imposing stress, stimulate endogenous cellular cues, which initiate protective responses in plants. Among the concurrent events during stress are changes in the intracellular Ca2+ concentration, which activate an overlapping set of downstream responses. Ca2+ changes are perceived and decoded by an array of Ca2+ sensors including calmodulins or calmodulin-related proteins, Ca2+-dependent protein kinases (CDPKs, CPKs), and calcineurin B-like proteins (CBLs; Dodd et al., 2010). Particularly, the CBL family has been shown to play a crucial role in different Ca2+-dependent processes in plants (Sanyal et al., 2015). CBL proteins present homology to the regulatory B-subunit of calcineurin and the neuronal calcium sensor proteins from animals and yeast (Luan, 2009). The overall structure of CBLs consists of four EF-hands. Spacing of EF-hands is invariable, while the C- and N-terminal extension of CBL proteins varies in length. Posttranslational modifications of CBLs, including protein phosphorylation and lipid modifications affect their subcellular localization and their stability to interact with other proteins (Sanyal et al., 2015; Nagae et al., 2003). Thus, phosphorylation of the conserved Ser residue in the C-terminal PFPF motif of the CBL proteins enhances the interaction with CBL-interacting protein kinases (CIPKs; Du et al., 2011; Hashimoto et al., 2012). Upon Ca2+ binding, CBLs physically interact with CIPKs, Ser/Thr kinases that structurally belong to Suc nonfermenting 1-related kinases, group 3, also called protein kinases related to SOS2 (PKS; Gong et al., 2004; Yu et al., 2014). CIPKs are constituted of a C-terminal or regulatory domain and a conserved kinase catalytic domain at the N terminus. Within the divergent regulatory domain, CIPKs contain an autoinhibitory NAF/ FISL motif and a type 2C protein phosphatase binding site called the “PPI motif”. It is well established that binding of CBLs to the NAF/FISL motif releases the C-terminal (autoinhibitory) domain from the kinase domain, thus leading the kinase into an active state 1 This work was funded in part by the European Regional Development Fund through the Ministerio de Economía y Competitividad (grant nos. BIO2005-02136, BIO2009-08648, and BIO2013-44750R) and by the Junta de Andalucía, Spain (grant no. P07-CVI-03171, to O.d.P.); O.d.P. was also supported in part by the Junta de Andalucía, Spain (Programa de Retorno de Investigadores); F.d.l.T. was supported by Marie Curie Programme through the International Reintegration grants (MIRG-CT-2005-031174) and a Juan de la Cierva contract (Ministerio de Ciencia e Innovación, Spain); E.G.-B. was a recipient of a Formación de Personal Investigador fellowship (Ministerio de Educación, Spain); and J.M.P. was supported by grant no. BIO2013-44750R. * Address correspondence to [email protected]c.es. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Olga del Pozo ([email protected]). E.G.B., O.d.P., and F.d.l.T. designed experiments and analyzed the data; O.d.P. wrote the manuscript and E.G.B. helped with the writing; E.G.B. performed most of experiments; J.M.P. generated Figure 7A. www.plantphysiol.org/cgi/doi/10.1104/pp.16.00949 836 Plant PhysiologyÒ,January 2017, Vol. 173, pp. 836–852, www.plantphysiol.org Ó2017 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. (Guo et al., 2001; Chaves-Sanjuan et al., 2014). In Arabidopsis (Arabidopsis thaliana), there are 10 CBL and 26 CIPK homologs (Yu et al., 2014). By yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays it has been determined that CBLs show a level of specificity in targeting different CIPKs. On the other hand, a specific CIPK can also interact with different CBLs, thus allowing a single CIPK to access different cellular compartments and hence different substrates (Kim et al., 2000, 2007). It is believed that the specificity of the response to a given stimulus is achieved by decoding specificCa 2+ profiles by CBLs followed by the subsequent formation of different CBL/ CIPK complexes in planta, and finally by phosphorylation of CIPK-specific substrates that contribute to the specific output response (Batistic et al., 2010). At the moment, the most numerous and best characterized interactors or substrates for CBL/CIPK complexes are membrane proteins, which include salt overly sensitive 1 (SOS1; Quintero et al., 2002; Katiyar-Agarwal et al., 2006), H+-ATPase 2 (He et al., 2004), nitrate transporter (Ho et al., 2009), K+transporter 1 (AKT1; Xu et al., 2006), high-affinity K+transporter 5 (Ragel et al., 2015), and the respiratory burst oxidase homolog F (Drerup et al., 2013). Additionally, CIPKs havealso been shown to interact with nonmembrane proteins; for example, SOS2- like protein kinase 5 interacts with the chaperone DnaJ (He et al., 2004; Yang et al., 2010); AtCIPK24 interacts with GIGANTEA (Kim et al., 2013), nucleoside diphosphate kinase 2, the catalases CAT2 and CAT3 (Verslues et al., 2007) and with ABA-insensitive 2 (ABI2), a type 2C Ser/Thr phosphatase (Guo et al., 2002; Ohta et al., 2003); and CIPK26 interacts with the RING-type E3 ligase “Keep on Going”and with ABI1, ABI2 (Lyzenga et al., 2013). Although it appears that CBL/CIPK complexes could interact with several proteins, at present only few CIPK phosphorylation targets have been identified. Previously, our group demonstrated, to our knowledge, a novel role for tomato (Solanum lycopersicum)Cipk6 (SlCipk6) in plant innate immunity, thus functionally implicating for the first time the participation of a CBL/ CIPK module in biotic stress signaling in plants (de la Torre et al., 2013). Other studies demonstrated the participation of Cipk6 orthologs from different plant species in diverse abiotic stress responses (Chen et al., 2012, 2013; Tsou et al., 2012; Tripathi et al., 2009). As a first step to investigate SlCipk6 downstream signaling molecular mechanisms, we set to identify SlCipk6-interacting proteins using a Y2H approach. We discovered that tomato Responsive to desiccation 2 (SlRd2), which contains a Universal stress protein (Usp) domain [Pfam (http:// pfam.xfam.org/) accession no. PF00582], interacted with SlCipk6 and by means of a BiFC approach, we found that the complex SlCipk6/SlRd2 is localized in the cytoplasm. In addition, we demonstrated that SlRd2 is a phosphorylation substrate of SlCipk6, thus expanding the previously described substrates for the CIPK family. Interestingly, SlRd2 is an ATP-binding protein that forms homodimers, which is required for its biological role and for interacting with SlCipk6. The universal stress protein A (UspA) superfamily was originally discovered in Escherichia coli, where its expression drastically increased in response to multiple stress conditions and to starvation (Vanbogelen et al., 1990). Importantly, UspA protein accumulation was necessary for bacterial survival at the stationary phase (Nyström and Neidhardt, 1994). It was found later that E.coli has six usp genes (uspA,uspC,uspD,uspF,uspF, and uspG); however, UspA set the nomenclature for the orthologous groups of proteins. UspA family members are classified into two major groups according to their ATP binding capability. The first group is constituted by ATP-binding proteins and is represented by Mj0577 from Methanococcus jannaschii (Zarembinski et al., 1998). Members of the second group have no ATP-binding capability and are represented by Haemophilus influenzae and E.coli UspAs (Sousa and McKay, 2001). Both Mj0577 and HiUspA form homodimers in vivo (Zarembinski et al., 1998). At present, more than 2000 UspA (or Usp containing domain) proteins have been identified from a wide range of organisms such as bacteria, archaea, fungi, protozoa and plants, constituting an ancient and conserved group of proteins (Aravind et al., 2002). In Arabidopsis, at least 44 proteins were found to contain an Usp domain, all of which resemble ATP-binding Mj0577 protein (Kerk et al., 2003) and several plant UspAs seemed to be involved in abiotic stress. In Arabidopsis, two UspA proteins, AtPHOS32 and AtPHOS34, were phosphorylated by AtMPK3 and AtMPK6 in response to bacterial elicitors in cell suspension cultures (Merkouropoulos et al., 2008). Other reports described several UspA members as effectors of low water potential (Merkouropoulos et al., 2008). Several UspA proteins have been characterized in rice (Sauter et al., 2002), tomato (Zegzouti et al., 1999; Loukehaich et al., 2012) legumes (Becker et al., 2001; Hohnjec et al., 2000), Salicornia (Udawat et al., 2016), and cotton (Zahur et al., 2009). Still, the precise structure, regulation, biochemical function, or mechanism of function of UspA proteins in planta, are largely unknown. RESULTS Identification of SlRd2 as a SlCipk6-Interacting Protein To identify SlCipk6-interacting proteins (CIPs), we carried out a Y2H approach in two separate screens, using a tomato cDNA prey library previously developed (Zhou et al., 1995) and SlCipk6 and a mutant derivative, SlCipk6(T172D), as baits (Fig. 1A). SlCipk6(T172D) displayed enhanced kinase and autophosphorylation activity compared to SlCipk6 (de la Torre et al., 2013), and we hypothesized that using either SlCipk6 or SlCipk6(T172D) as a bait, might facilitate the identification of putative regulatory proteins or phosphorylation substrates respectively, because constitutive active kinase versions stabilize the interaction with their substrates (Uno et al., 2009). Approximately 1.1 3103and 4.5 3103yeast transformants were screened for SlCipk6- and SlCipk6(T172D)-interacting proteins Plant Physiol. Vol. 173, 2017 837 SlRd2 Phosphorylation and Role in Stress Defense www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. respectively on selection plates (Supplemental Table S1). The inserts of 11 and 34 candidate prey clones from both screens were sequenced and compared with databases by BLAST in the Arabidopsis database (www. tair.org) for identification. The clone no. 29 (Cip29) was a partial open reading frame (ORF) that encoded a protein with a high similarity to Arabidopsis Response to desiccation 2 (AtRD2; At2g21620), a protein not yet characterized with no assigned function, containing a Universal stress protein domain (Usp). Clones containing different length fragments of Cip29 were identified once and two times in the screens performed with SlCipk6 and with SlCipk6(T172D), respectively, and they were among the strongest interactors. In light of these facts, its characterization was prioritized and the remaining CIPs will be published elsewhere. BLAST performed with Cip29 in the tomato database (www. solgenomics.net) identified unigene SGN-U567775 containing a full length ORF that corresponded to the tomato locus Solyc01g109710 and we will be referred to it as SlRd2 hereafter. We next obtained full-length SlRd2 ORF, cloned it in the prey vector, and confirmed its interaction with SlCipk6 by Y2H (Fig. 1B). Both SlRd2 and SlCipk6 did not show autoactivation activity. To characterize SlCipk6/SlRd2 interaction, different SlCipk6 mutant derivatives were tested (de la Torre et al., 2013). SlRd2 interacted with SlCipk6, SlCipk6(T172D), SlCipk6DNAF (a mutant version lacking the NAF/FISL domain necessary for CBL binding) or SlCipk6DCterm (a deletion mutant version that lacked the C-terminal regulatory domain), but did not interact with SlCipk6DNterm (a deletion mutant version lacking the kinase domain) or very weakly with SlCipk6 (K43M, a reduced kinase activity version where the catalytic Lys-43 has been mutated to Met; Fig. 1, A and B). The interaction was quantitated in a b-galactosidase activity assay (Fig. 1C) and protein expression in yeast was confirmed by immunoblot (Supplemental Fig. S1). SlRd2 did not interact with SlCipk11 (Solyc06g082440) or SlCipk14 (Solyc10g085450; Supplemental Fig. S2A) and SlCipk6 did not interact with SGN-U601569, the closest tomato sequence to SlRd2 (Supplemental Fig. S2B), indicating that SlCipk6 interaction with SlRd2 was specific. Altogether, we confirmed that SlCipk6 interacted with SlRd2 and concluded that SlCipk6 kinase domain and kinase activity seemed to be necessary and important, respectively, for SlCipk6/SlRd2 interaction. SlCipk6 and SlRd2 Interact in Vivo Next, we examined the in vivo interaction between SlCipk6 and SlRd2 by coimmunoprecipitation (co-IP). Although SlCipk6 interacted with SlRd2 in yeast at slightly higher levels, we used SlCipk6(T172D) for co-IP in planta because it has been described that constitutive active kinase versions stabilize the interaction with their substrates (Uno et al., 2009). For that purpose, both SlRd2 (fused to a GFP tag) and SlCipk6(T172D; fused to 2xIgG-BD and 9xMyc tags) were coexpressed via Agrobacterium-mediated transient transformation of leaves (agro-infiltration) in Nicotiana benthamiana. SlRd2 coimmunoprecipitated with SlCipk6(T172D), but did not with the GFP, used as a negative control in this experiment (Fig. 2A). Therefore, SlCipk6(T172D) and Figure 1. SlRd2 and SlCipk6 Interact in a Y2H Assay. A, Schematic structure of SlCipk6 protein representing N-terminal catalytic, junction and C-terminal regulatory domains, PPi, NAF/FISL, and activation loop domains. Residues mutagenized for SlCipk6 characterization Lys-43 to Met (K43M), and Thr-172 to Asp (T172D) are marked. B, Y2H assay performed with SlCipk6 and SlCipk6 mutant derivatives in the bait vector pEG202 and SlRd2 in the prey vector pJG4-5. Wild-type, full-length SlCipk6; K43M and T172D, single amino acid substitution of Lys-43 to Met and Thr-172 to Asp, respectively; DNAF, amino acids 302–321 deletion; DCterm amino acids 302–432 deletion; and DNterm, amino acids 1–302 deletion. Clones were transformed into EGY48 strain, grown in liquid culture, and spotted at OD600 = 0.1 and 0.01 on selective medium. Growth and blue patches indicates interaction. C, Interaction was quantitated by a b-galactosidase assay. Represented is the mean value of three independent experiments, each performed with three independent transformants. Error bars represent the SD of three different experiments. 838 Plant Physiol. Vol. 173, 2017 Gutiérrez-Beltrán et al. www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. SlRd2 interacted in planta. Because kinases and their substrates need a coordinated expression and subcellular localization, we first assessed the likelihood that both SlCipk6 and SlRd2 localized at common cellular compartments. Both proteins were fused to GFP, agroinfiltrated individually into N.benthamiana leaves and the localizations were determined by confocal microscopy (Supplemental Fig. S3). Both SlCipk6-GFP (Supplemental Fig. S3, a and b) and GFP-SlRd2 (Supplemental Fig. S3, c and d) localized in the cytoplasm and in the nucleus, thus showing an overlapping subcellular localization. To gain insight where the complex localized intracellularly, we performed BiFC assays in N.benthamiana leaves (Walter et al., 2004). Fluorescence due to the reconstitution of yellow fluorescent protein (YFP) was observed at the confocal microscope when the combinations SlRd2-YFPCand SlCipk6-YFPNwere agroinfiltrated in N.benthamiana leaves (Fig. 2B, a and d). The YFP signal showed a pattern similar to the one observed for AtFKBP12-CFP, a cytoplasmic marker fused to cyan fluorescent protein [CFP; (Faure et al., 1998)] (Fig. 2B, d–f), but did not overlap with the signal derived from the membrane-bound marker FM4-64 (Fig. 2B, a–c). No fluorescence was detected in the nucleus or when SlRd2- YFPCor SlCipk6-YFPNwere coinfiltrated with empty vectors respectively, and neither when SlRd2-YFPCwas coexpressed with SlCbl10-YFPC, a known interactor of SlCipk6 (Fig. 2B, g–i; de la Torre et al., 2013). These results indicate that SlCipk6 and SlRd2 interact in vivo and form a cytoplasmic complex in N.benthamiana leaves at the conditions assayed. SlRd2 Transcript Accumulates in Response to Abiotic Stress E.coli UspA (EcUspA) protein accumulates in response to a large and diverse number of stresses providing survival cues under adverse bacterial growth conditions (Freestone et al., 1997; Jung et al., 2015). In plants, members of the UspA family have been reported to accumulate in response to different abiotic stress conditions, including AtRD2 (Yamaguchi-Shinozaki et al., 1992; Jung et al., 2015). Both SlCipk6 and SlRd2 displayed an overlapping subcellular localization in N.benthamiana epidermal cells (Supplemental Fig. S3, a–d). Next, we inquired if SlRd2 and SlCipk6 expression were correlated in response to abiotic stress using quantitative (Q) RT-PCR to support synchronous activity in abiotic stress response. SlRd2 mRNA was present in all the tissues examined, however it was substantially more abundant in roots (.25-fold compared to leaves), suggesting that it might play a prominent role in this tissue (Fig. 3A). AtRD2 accumulation increased in response to abiotic stress (Yamaguchi-Shinozaki et al., 1992). Therefore, to determine if SlRd2 and SlCipk6 transcript accumulation were responsive to salt and osmotic stress, we treated hydroponically grown tomato plants with 100 mM NaCl and 300 mMmannitol and analyzed SlRd2 accumulation. NaCl treatment rapidly increased SlRd2 transcript accumulation [approximately 12-fold, after 2 h (h)], which was maintained up to 12 h and decreased 24 h later (Fig. 3B). Osmotic pressure resulted in a similar accumulation of SlRd2 after 2 h and 4 h, but in contrast, SlRd2 accumulation increased up to 30-fold after 8 h to 12 h and decreased to 20-fold at 24 h (Fig. 3C). SlCipk6 orthologs from different plant species Figure 2. SlCipk6 and SlRd2 interact in planta and the complex localizes in the cytosol. A, Protein extracts from N.benthamiana leaves agroinfiltrated with GFP-SlRd2 and SlCipk6(T172D)-Myc, which is fused to two copies of the protein A IgG binding domains (2xIgG-BD), were incubated with IgG agarose beads, eluted and analyzed by immunoblotting using anti-Myc for SlCipk6 and anti-GFP for SlRd2. GFP protein was used as a negative control. No interaction was detected when SlCipk6(T172D) was agro-infiltrated with GFP (right upper panel). B, Fluorescence images of N.benthamiana leaf sections agroinfiltrated with SlRd2-YFPCand SlCipk6-YFPN. Colocalization analyses of SlCipk6/SlRd2 complexes (yellow, a and d) with the fluorescent membrane marker dye FM4-64 (red, b) and the cytoplasmic marker AtFKBP12-CFP (cyan, e; Faure et al., 1998). Merged images are shown in (c) and (f), respectively. White arrows show the cytoplasmic localization observed for the SlCipk6/SlRd2 complexes. Localization analysis of YFPN(g) and YFPC(h) empty vectors and SlCbl10/ SlRd2 (i) were used as an experiment controls. Bars = 10 mm. Plant Physiol. Vol. 173, 2017 839 SlRd2 Phosphorylation and Role in Stress Defense www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. have been described to participate in different abiotic stress responses, and we tested if SlCipk6 transcript accumulated also in response to abiotic stress. For that purpose, we treated tomato plants with NaCl and mannitol, using the same conditions as described above. After NaCl treatment, accumulation of SlCipk6 mRNA increased 6-fold to 8-fold after 2 h and was maintained at the same levels up to 8 h, decreasing gradually thereafter, reaching a 2-fold increase at 24 h (Fig. 3D). Osmotic pressure also resulted in an increased SlCipk6 mRNA accumulation; however, followed a different pattern compared to SlRd2: 2 h after SlCipk6 transcripts increased 6-fold and steadily increased up to 15 fold 24 h after (Fig. 3E). Both SlCipk6 and SlRd2 transcript accumulation pattern showed similar kinetics, thus supporting the possibility of a coordinated role for SlCipk6 and SlRd2 in abiotic stress responses in tomato. SlRd2 Belongs to the Universal Stress Protein A Superfamily The translated sequence of SlRd2 ORF yields a 177 amino acid protein, with a Mrof 19.5 kD and a pI of 5.98. We aligned the Usp domain of SlRd2 with those of N.benthamiana and Arabidopsis orthologs NbRd2 and AtRD2 and other plant proteins containing the Usp domain, including tomato LeER6 (Zegzouti et al., 1999), rice (Oryza sativa) OsUSP1 (Sauter et al., 2002), and Vicia fava Enod18 (Becker et al., 2001) along with bacterial proteins belonging to the UspA family, including E.coli (Nyström and Neidhardt, 1992), H.influenzae (Sousa and McKay, 2001) and M.jannaschii (Zarembinski et al., 1998; Supplemental Fig. S4). Amino acid sequence alignment revealed that SlRd2 contained the residues involved in ATP binding including the Walker motif A or P-loop, (G-2X-G-9X-G(S/T)), also present in Mj0577 and UspA plantrepresentativesbutabsentinE.coli paralogs (Sousa and McKay, 2001). This observation suggests that plant UspA proteins might also be functional ATP-binding proteins (Supplemental Figs. S4 and 5A). Overall, all UspA proteins (plant and bacterial) shared conservation within the dimerization subdomain (Supplemental Fig. S4), thus raising the possibility that plant UspAs could be present as dimers in the cell. In addition to the conserved Usp domain, SlRd2 has an N-terminal domain (amino acids 1 to 38) and a C-terminal extension (amino acids 168 to 177) with unknown function, which is shared with the Arabidopsis and N.benthamiana homologs (Figs. 4A and S5). Subsequently, a phylogenetic analysis was performed using E.coli UspG and the proteins included in the alignment shown in Supplemental Fig. S4. SlRd2 and NbRd2 were located in the same clade as AtRD2, thus supporting their orthology (Fig. 4B). Moreover, SlRd2, and all plant UspAs, are more related to Mj0577 subfamily and E.coli UspG than to the E.coli UspA (Fig. 4B). SlRd2 Is an ATP-binding Protein and Forms Homodimers in Yeast, Bacteria, and Plants In light of the high degree of conservation of SlRd2 amino acids putatively involved in nucleotide binding (see alignments in Figs. 5A and S4), we next inquired if SlRd2 also had the functional competence to bind ATP, as described for bacterial Mj0577. For that purpose, we Figure 3. SlRd2 and SlCipk6 transcripts highly accumulate after nacl and osmotic stress in tomato. A to E, yaxis represents mean values of Q RT-PCR of three experiments, with three biological replicates in each. Error bars represent the SE. The expression levels were normalized to SlActin2. Gene induction (fold increase) in infected or treated plants was compared with the expression level of control or mock inoculated plants at 0 h and is shown as relative expression, excepting in (A). A, SlRd2 transcript accumulates in tomato leaves, petioles, stems, flowers, and roots. Relative expression on yaxis was compared with expression level in leaves. B and C, SlRd2;DandE,SlCipk6 mRNA accumulation in leaves during NaCl (B and D) and osmotic stress (C and E). Hydroponically grown tomato plants were treated with 100 mMNaCl (B and D) or 300 mM mannitol (C and E). OS, osmotic stress. 840 Plant Physiol. Vol. 173, 2017 Gutiérrez-Beltrán et al. www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. purified SlRd2 protein and performed a nucleotide binding assay in vitro by incubating SlRd2 in the presence of [a-32P]ATP. Samples were then analyzed by SDS-PAGE followed by autoradiography (Fig. 5B, upper panel). Indeed, SlRd2 was able to bind [a-32P]ATP, whereas no [a-32P]ATP binding was observed when the GFP protein was incubated in its presence. To further confirm that SlRd2 was also able to bind ATP in vivo, a synthetic analog of ATP, (+)-biotin-hex-acyl-ATP (BHAcATP), consisting of ATP, an acyl-P linker and a biotin tag, was used (Villamor et al., 2013). Incubation of GFP-SlRd2 expressing plant extracts with BHAcATP followed by streptavidin purification revealed that SlRd2 was able to bind BHAcATP in planta (Fig. 5C, upper panel). Moreover, ATP addition suppressed BHAcATP labeling by competing with BHAcATP and saturating the nucleotide-binding site of SlRd2 (Fig. 5C, upper panel). These findings indicate that SlRd2 binds ATP and therefore functionally belongs to the ATP- binding subgroup of the UspA family represented by Mj0577. The dimerization motif, present in UspA family proteins, is also conserved in SlRd2 and is localized close to the C terminus (Figs. 5A and S4). Mj0577 and HiUspA were shown to exist as homodimers in vivo (Sousa and McKay, 2001; Zarembinski et al., 1998). Mj0577 crystallizes as a homodimer, and each monomer binds the other through antiparallel hydrogen bonds in the fifth beta sheet within each subunit. To test whether SlRd2 was also able to form homodimers, we performed a Y2H analysis in which the yeast strain expressed SlRd2 both in the prey (pJG4-5) and in the bait (pEG202) plasmids. Indeed, SlRd2 can form homodimers because growth was observed in restrictive media and blue color developed in the presence of X-gal (Fig. 6A). No interaction was detected when Y2H analysis was performed using a SlRd2 mutant version, SlRd2Δdim (amino acids 163 to 166 deletion), in which the putative dimerization domain VIIV was deleted (Fig. 6A). SlRd2 and SlRd2Δdim, were expressed in yeast (Fig. 6B). Therefore, SlRd2 forms dimers and the conserved VIIV domain is necessary for dimerization in vivo. It has been described that E.coli UspC is able to form tetramers in vivo (Nachin et al., 2008). To check if SlRd2 also formed homotetramers in vivo, an E.coli culture overexpressing SlRd2 tagged at the N terminus with the epitope His (His-SlRd2) was treated with a cross-linking agent, disuccinimidyl glutarate (DSG), for 30 min. Thereafter, protein extracts were obtained, analyzed by SDS-PAGE and His-SlRd2 detected by immunoblot. Two bands of approximately 42 and 24 kD were observed in the extracts treated with the crosslinker, which corresponded likely with SlRd2 dimer and monomer, whereas only the lower Mrband was observed in control conditions (Fig. 6C). Thus, we concluded that SlRd2 is able to form homodimers but not homotetramers in vivo. To determine that SlRd2 formed homodimers in planta and their putative subcellular localization, BiFC assays were performed. SlRd2 cDNA was cloned in the BiFC vectors, transformed into Agrobacterium and agroinfiltrated in N.benthamiana leaves. Reconstitution of YFP fluorescence was observed 2 d after under the confocal microscope when SlRd2-YFPCand SlRd2-YFPN Figure 4. Tomato SlRd2 protein structure and phylogenetic analysis. A, Schematic structure of SlRd2 containing N-terminal and C-terminal domains and a conserved Usp domain, which encompasses a dimerization motif. Special symbols indicate the conserved residues described to be involved in ATP-binding. B, Phylogenetic relationship of proteins containing Usp domains from plants and bacteria including A.thaliana,AtRD2;tomato,SlRd2 and LeER6; N.benthamiana, NbRd2; O. sativa, OsUSP1; V. faba, VfENOD18; E. coli, EcUspG and EcUspA; H. influenzae,HiUspA;andM. jannaschii, Mj0577. SlCbl10 was used as the outgroup. The numbers on the tree represent bootstrap scores. Plant Physiol. Vol. 173, 2017 841 SlRd2 Phosphorylation and Role in Stress Defense www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. were coexpressed. The signal colocalized with that obtained by the expression of the cytoplasmic marker AtFKBP12-CFP (Fig. 6D, d–f)andwithDAPInuclear marker (Fig. 6D, g–i) but not with the cell membrane marker FM4-64 (Fig. 6D, a–c). These results demonstrate that SlRd2 can form homodimers in planta in the cytoplasm and in the nucleus. SlRd2 Is a Phosphorylation Substrate of SlCipk6 Because SlCipk6 kinase domain and activity was necessary and important respectively for SlCipk6/SlRd2 interaction (Fig. 1), we inquired if SlRd2 was a phosphorylation target of SlCipk6. For that purpose, we set up an in vitro kinase assay in the presence of [g-32P]ATP using purified SlCipk6 (fused to the GST at the C terminus) and as substrates, SlRd2 (fused to the His epitope at the N terminus) and a mutant version, SlRd2(UspA; fused to GST at the N terminus), in which the specific putative regulatory N-terminal domain (amino acids 1 to 38) and C-terminal extension (amino acids 168 to 177) were deleted to leave only a core UspA domain. Myelin Basic Protein (MBP), a universal kinase substrate was included as a positive control for SlCipk6 kinase activity. SlCipk6 was expressed and purified from yeast, whereas SlRd2 and SlRd2(UspA) were purified from bacteria. Figure 7A shows that SlRd2 is phosphorylated by SlCipk6 but does not phosphorylate SlRd2(UspA), thus indicating that SlCipk6 phosphorylates SlRd2 specific regulatory N- or C-terminal regions. SlCipk6 also phosphorylates MBP. A faint signal can be still detected in the absence of SlCipk6, indicating that SlRd2 binds ATP (Fig. 7A, left). Protein loading levels were monitored by Coomassie staining (Fig. 7A). Quantification of SlRd2 [g-32P]ATP incorporation is presented in Figure 7B. Therefore, we conclude that SlRd2 is a phosphorylation substrate of SlCipk6 and represents, to our knowledge, a novel phosphorylation target of the CIPK family. Figure 5. SlRd2 is an ATP-binding protein. A, Alignment of Usp domain of SlRd2 and AtRD2 and the full amino acid sequence of Mj0577. Conserved residues are marked in black boxes while similar residues are marked in gray boxes. Black bars underline residues putatively involved in ATP-binding. Walker motif or P loop motif G-2x-G-9x-G-(S/T) for ATP binding is marked with a blue line. Amino acid residues responsible for protein dimerization are marked as (D). B, For ATP-binding in vitro assay, 1 mgof purified His-SlRd2 and His-GFP proteins from E.coli were incubated with [a-32P]ATP and corresponding buffer during 1 h at 30°C. Autoradiograph indicates ATP-binding (top panel); immunoblot analysis indicates protein level in the ATP binding assay (low panel). C, For ATP-binding in vivo assay, N.benthamiana leaf extracts from wild type and GFP-Rd2 overexpressing line 6 plants were incubated with 20 mg BHAcATP. Biotinylated proteins were purified using streptavidin beads, separated on protein gel, and detected using anti-GFP (top panel). Samples labeled with BHAcATP in presence of 10 mMof ATP were used as a control. Ponceau S staining was used as a loading control (bottom panel). Adenine, A; phosphate, P; ribose, R. 842 Plant Physiol. Vol. 173, 2017 Gutiérrez-Beltrán et al. www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. SlRd2 Overexpression in Yeast Confers Resistance to LiCl In light of the strong transcriptional response of SlRd2 to NaCl and osmotic stress, we decided to functionally test SlRd2 participation in mediating different stress responses using yeasts as a model system. Yeast has been successfully used in functional studies to characterize the role of plant proteins in stress responses (Matsumoto et al., 2001; Quintero et al., 2002; Shitan et al., 2013; Bernard et al., 2012). SlRd2 was overexpressed in the yeast strain Saccharomyces cerevisiae BY4741 under control of the constitutive expression vector p426GPD (p426GPD-SlRd2). As a control, BY4741 strain transformed with empty vector (EV) p426GPD was used. Both yeasts lines were subjected to the following stress-causing treatments: hygromycin B, generates changes in membrane potential (Wang et al., 2009); MnCl2, causes programmed cell death at high concentrations (Liang and Zhou, 2007); CaCl2and KCl, cause osmotic stress (Liang and Zhou, 2007); DTT and H2O2, generate oxidative stress (Babiychuk et al., 1995); bleomycin, causes DNA damage (Aouida et al., 2004); and finally, NaCl and LiCl, cause changes in ionic homeostasis (Ye et al., 2006). SlRd2 overexpressing line grew at a similar rate than EV strain in control conditions and in all the different treatments, except in 300 mMLiCl, where it grew significantly better than EV line (Fig. 8A). Next, we tested if SlRd2 dimerization was crucial for its biological role. For that purpose, we cloned SlRd2Δdim into p426GPD vector, transformed it into S.cerevisiae BY4741 strain and performed a LiCl resistance experiment along with p426GPD-SlRd2 and EV. Figure 8B shows that a functional SlRd2 dimer was required for conferring LiCl resistance, because SlRd2Δdim line grew at the same rate as EV line and loss its protection against LiCl. Both SlRd2 and SlRd2Δdim expression in yeast was confirmed by immunoblotting (Fig. 8C). We concluded that SlRd2 dimerization was necessary for its molecular function in conferring resistance to LiCl. Next, we asked if SlRd2 dimerization was also necessary for its interaction with SlCipk6. For that purpose, we performed a Y2H assay with SlCipk6 and SlCipk6(T172D) as baits and SlRd2Δdim as a prey (Fig. 9A). Neither SlCipk6 nor SlCipk6(T172D) interacted with SlRd2Δdim by Y2H (Fig. 9A). To test their interaction in planta, we performed a BiFC experiment, and agro-infiltration SlRd2Δdim-YFPCand SlCipk6-YFPNin N.benthamiana leaves did not reconstitute YFP fluorescence, indicating that SlCipk6 was not able to interact with SlRd2Δdim (Fig. 9B). Hence, SlRd2 homodimerization is required for interaction with SlCipk6. SlRd2 Protects Bacteria from Oxidative Stress and Negatively Regulates Reactive Oxygen Species in Plants E.coli mutant strain TN3151 (a knock-out of the uspA gene) was susceptible to H2O2treatments, which did not affect the wild-type strain, W3101 (Nyström and Neidhardt, 1994). Recent results demonstrated that a Figure 6. SlRd2 homodimerizes in the cytosol and in the nucleus. A, Y2H assay with both SlRd2 and SlRd2Ddim cloned in the bait pEG202 (with a LexA fusion tag at the C terminus) and prey pJG45 (with a HA fusion tag at the C terminus) vectors in EGY48 strain, grown in liquid culture and spotted at OD600 = 0.1 and 0.01 on selective medium. Growth and blue patches indicates interaction. B, Expression of both SlRd2 and SlRd2Ddim proteins in yeast were confirmed by immunoblotting using anti-LexA (pEG202) and anti-HA (pJG45) antibodies, respectively. C, The cross-linking agent DSG was added to a His-SlRd2 overexpressing E.coli culture for 30 min. Polymerization of His-SlRd2 protein was calculated according to its molecular weight: 13, monomer; and 23, dimer. D, Colocalization analyses of SlRd2/SlRd2 complexes (yellow, a, d, and g) with the membrane marker dye FM4-64 (red, b), cytoplasmic marker AtFKBP12 (cyan, e), or nuclear marker DAPI (blue, h). Merged images are shown in (c), (f), and (i). Arrow denotes localization in the nucleus. Bars = 10 mm. Plant Physiol. Vol. 173, 2017 843 SlRd2 Phosphorylation and Role in Stress Defense www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. UspA protein from the pathogenic bacteria Mycobacterium tuberculosis provided protection for the parasite against host reactive oxygen species (ROS) generated by mammalian macrophages defense (Drumm et al., 2009). SlCipk6 was demonstrated to participate in ROS generation during plant responses to bacterial pathogen attack, which was dependent on the NADPH oxidase, RbohB (de la Torre et al., 2013). Given the striking conserved structural and functional features between SlRd2 and bacterial UspA proteins, we decided to test if SlRd2 could functionally complement TN3151 in protecting bacteria in response to H2O2treatment. E.coli wild-type W3101 (wild type), uspa mutant TN3151 and TN3151 complemented with either SlRd2 (TN3151- SlRd2) or with the native UspA gene (TN3151-UspA) were grown in minimal media (morpholine propane sulphonic acid, MOPS) until OD600 reached 0.7. Then, H2O2(5 mM) was added and survival was measured at different time points after (Fig. 10A). A strong growth inhibition was observed for TN3151 strain after H2O2 treatment, as previously described (Nyström and Neidhardt, 1994); 40 min after H2O2addition, only 45% of TN3151 survived whereas 100% TN3151-SlRd2 survived, showing a similar survival rate as either TN3151- EcUspA or wild-type W3101. SlRd2 and EcUspA expression in TN3151 was confirmed by immunoblotting (Fig. 10B) and lack of UspA gene expression in TN3151 was confirmed by PCR (Fig. 10C). In light of these results, we concluded that tomato SlRd2 functionally complements TN3151 mutant strain in protecting bacteria against oxidative stress damage and thus SlRd2 and E.coli UspA are functionally conserved in their ROS protection role in bacteria. We have previously shown that kinase activity of SlCipk6 is associated with ROS production in N. benthamiana (de la Torre et al., 2013), so we next inquired if SlRd2 is required for SlCipk6-mediated ROS generation. To this end, c-Myc tagged versions of SlCipk6, SlCipk6(T172D) and SlCipk6(K43M; cloned into pTAPa-pYL436 vector) or EV were agro-infiltrated in N.benthamiana wild-type and overexpressing GFP- SlRd2 (OE-6, OE-7) leaves and production of ROS was quantified by a chemiluminescence assay in a luminometer (Fig. 10D). As expected, SlCipk6 and SlCipk6(T172D) expression in wild-type leaf discs resulted in ROS generation, which was significantly reduced in SlCipk6(K43M) expressing leaf discs (de la Torre et al., 2013). However, SlCipk6 and SlCipk6(T172D) agro-infiltration in OE-6 and OE-7 plants resulted in a significant reduction of ROS (Fig. 10D) whereas SlCipk6(K43M)-induced ROS levels remained as in wild-type discs. SlCipk6, SlCipk6(T172D), SlCipk6(K43M), and GFP-SlRd2 expression were confirmed by immunoblotting (Supplemental Fig. S6). This result clearly indicated that overexpression of SlRd2 negatively regulates SlCipk6-mediated ROS generation and that a functional link exists between both proteins. DISCUSSION SlRd2, a Member of the Universal Stress Protein Family, Interacts with SlCipk6 The identification and characterization of SlCipk6 targets and regulatory components is an important step for understanding how downstream SlCipk6 signaling specificity is achieved and to identify the pathways it regulates. As a firststep,wesettoidentify Figure 7. SlCipk6 Phosphorylates SlRd2. A, SlCipk6, SlRd2, SlRd2(UspA) and MBP alone and with SlCipk6 were incubated in the presence of [g-32 P] ATPand kinase buffer, electrophoresed on SDS polyacrylamide gel and autoradiographed. They were expressed and purified as fusion proteins as follows: SlCipk6-GST, His-SlRd2, GST-SlRd2(UspA). Autoradiographs indicate SlCipk6 autophosphorylation (upper panels) and SlRd2 phosphorylation (lower panels); Coomassie protein staining indicates SlRd2, SlRd2(UspA), MBP, and SlCipk6 protein loads. Protein amounts loaded: 0.3 mg for SlCipk6, 2.5 mg for SlRd2 and SlRd2(UspA), and 0.5 mgfor MBP. B, Phosphorylation was quantified using Cyclone Phosphorimager Optiquant software (Packard Bioscience, Perkin Elmer). Signal from MBP phosphorylation was not normalized to protein content to better quantify SlRd2 phosphorylation and ATP binding. Similar results were obtained in two additional experiments. a.u., arbitrary units. 844 Plant Physiol. Vol. 173, 2017 Gutiérrez-Beltrán et al. www.plantphysiol.orgon November 4, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. Loukehaich R, Wang T, Ouyang B, Ziaf K, Li H, Zhang J, Lu Y, Ye Z (2012) SpUSP, an annexin-interacting universal stress protein, enhances drought tolerance in tomato. 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