bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Short title: SUMO conjugation activity in nuclear bodies

2 Corresponding Author: Dr. Harrold A. van den Burg 3 Molecular Plant Pathology 4 SILS, University of Amsterdam 5 Science Park 904 6 1098XH, Amsterdam 7 the Netherlands 8 Tel. +31 (0)20 525 7797 9 Title: 10 The SUMO conjugation complex self-assembles into

11 nuclear bodies independent of SIZ1 and COP1

12 13 Authors: 14 Magdalena J. Mazur1,3¶, Mark Kwaaitaal1¶, Manuel Arroyo Mateos1, Francesca 15 Maio1, Ramachandra K. Kini1,4, Marcel Prins1,2 and Harrold A. van den Burg1,* 16 17 Author affiliations: 18 ¶Authors contributed equally to this work. 19 1Molecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), 20 University of Amsterdam, 21 2Keygene N.V., Wageningen, The Netherlands 22 23 One sentence Summary (max 200 characters): 24 SUMO conjugation activity causes formation of SUMO nuclear bodies, which 25 strongly overlap with COP1 bodies thanks to a substrate-binding (VP) motif in the 26 E3 ligase SIZ1 that acts as bridge protein. 27

1 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

28 Footnotes: 29 Author contributions: 30 HB conceptualized the project. MM, MK, FM and HB designed experiments. MM, 31 MK, MAM, FM and RK performed experiments. MM, MK, MA, FM and HB 32 analysed the data. MM, MK and HB wrote the MS. MM, MK, FM, MAM, MP and 33 HB reviewed and edited the MS. HB and MP acquired funding and supervised the 34 project. MM and MK contributed equally to this work. 35 36 Funding information: 37 The Netherlands Scientific Organisation (ALW-VIDI grant 864.10.004 to HvdB) and 38 the Topsector T&U program Better Plants for Demands (grant 1409-036 to HvdB), 39 including the partnering breeding companies, supported this work; FM is financially 40 supported by Keygene N.V. (The Netherlands). 41 42 Current address: 43 3Current address: Laboratory of Virology, Wageningen University, Wageningen, 44 The Netherlands 45 4Current address: Department of Studies in Biotechnology, University of Mysore, 46 Manasagangotri, Mysore- 570 006, India 47 48 Corresponding author email: [email protected] 49

2 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

50 Abstract

51 Attachment of the small ubiquitin-like modifier SUMO to substrate proteins 52 modulates their turnover, activity or interaction partners. An unresolved question is 53 how this SUMO conjugation activity concentrates the enzymes involved and the 54 substrates into uncharacterized nuclear bodies (NBs). We here define the 55 requirements for the formation of SUMO NBs and for their subsequent co- 56 localisation with the master regulator of growth, the E3 ubiquitin ligase COP1. COP1 57 activity results in degradation of transcription factors, which primes the 58 transcriptional response that underlies elongation growth induced by night-time and 59 high ambient temperatures (skoto- and thermomorphogenesis, respectively). 60 SUMO conjugation activity itself is sufficient to target the SUMO machinery into NBs. 61 Co-localization of these bodies with COP1 requires besides SUMO conjugation 62 activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SIZ1. We find that 63 SIZ1 docks in the substrate-binding pocket of COP1 via two VP motifs - a known 64 peptide motif of COP1 substrates. The data reveal that SIZ1 physically connects 65 COP1 and SUMO conjugation activity in the same NBs that can also contain the 66 blue-light receptors CRY1 and CRY2. Our findings thus suggest that sumoylation 67 apparently coordinates COP1 activity inside these NBs; a mechanism that 68 potentially explains how SIZ1 and SUMO both control the timing and amplitude of 69 the high-temperature growth response. The strong co-localization of COP1 and 70 SUMO in these NBs might also explain why many COP1 substrates are sumoylated. 71 72 Word count Abstract: 229 73 74 Word count body text: 7174

75

3 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

76 Introduction

77 The post-translational modification of proteins by SUMO1 and -2 (SMALL 78 UBIQUITIN-LIKE MODIFIER 1 and 2) is an essential process in Arabidopsis 79 (Arabidopsis thaliana) affecting hundreds of proteins (Saracco et al., 2007; Miller et 80 al., 2010; Rytz et al., 2018). These two closely related proteins are the nearest 81 homologs of the ancestral SUMO protein (Hammoudi et al., 2016). Their attachment 82 to substrates is catalysed in two steps by the SUMO E1 ACTIVATING ENZYME 83 (SAE1/2) dimer and the SUMO E2 CONJUGATING ENZYME (SCE1) (Colby et al., 84 2006; Saracco et al., 2007). SCE1 directly recognizes and conjugates a SUMO 85 moiety to the acceptor lysine embedded in a short consensus motif ΨKxE (where Ψ 86 denotes a bulky hydrophobic residue, K the acceptor lysine, x any residue, and E is 87 Glutamate) (Bernier-Villamor et al., 2002; Yunus and Lima, 2006). SUMO 88 conjugation (sumoylation) can also be promoted by SUMO E3 ligases, but the 89 conditions by which E3s are recruited to SCE1 remain unclear (Gareau and Lima, 90 2010). SUMO proteome analyses with human cells suggested that ~25-50% of the 91 SUMO targets are modified at non-consensus sites (Hendriks et al., 2014; 92 Lamoliatte et al., 2017). Structural studies exposed that the E3 ligases orientate the 93 E2 enzyme such that they favour SUMO donor transfer to non-consensus lysines 94 (Yunus and Lima, 2009; Streich and Lima, 2016). Alternatively, substrate selection 95 can involve non-covalent interactions between SUMO and a short hydrophobic 96 peptide in substrates, called the SUMO-interaction motif (SIM) (Zhu et al., 2008; 97 Flotho and Melchior, 2013). SIM peptides bind to SUMO by forming an additional 98 alien β-strand in the β-sheet of SUMO (Song et al., 2005; Hecker et al., 2006; 99 Sekiyama et al., 2008). 100 SUMO often acts as a monomeric adduct, but it can also form SUMO chains 101 (Colby et al., 2006). In yeast and mammals SUMO chains are important signals in 102 meiosis, genome maintenance and (proteotoxic) stress (Vertegaal, 2007; Ulrich, 103 2008; Bruderer et al., 2011; Tatham et al., 2011). These SUMO chains are 104 recognized by SUMO-targeted Ubiquitin E3 ligases (StUbls) that mark the SUMO- 105 modified proteins for degradation (Perry et al., 2008; Guo et al., 2014). Distant 106 homologues of these StUbls were identified in the Arabidopsis genome, but their 107 biological function remains to be determined (Elrouby et al., 2013). In Arabidopsis,

4 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

108 two SP-RING domain-containing proteins, PROTEIN INHIBITOR OF ACTIVATED 109 STAT LIKE1 (PIAL1) and PIAL2, were shown to promote SUMO chain formation 110 (Tomanov et al., 2014). There is also a role for the SUMO E2 enzyme in SUMO 111 chain formation (Tomanov et al., 2018). The SUMO E2 interacts via two sites with 112 SUMO: (i) a thioester bond (~) between the catalytic cysteine in the E2 and the C- 113 terminal glycine of SUMO (covalent SUMO loading in the catalytic pocket, 114 E2~SUMO) and (ii) a strong nearly constitutive, non-covalent ‘SIM-like’ interaction 115 (non-covalent SUMO binding, SUMO∙E2) (Bencsath et al., 2002; Knipscheer et al., 116 2007; Streich and Lima, 2016). This second non-covalent interaction between 117 SUMO and SCE1 is apparently needed for chain formation, as mutations in the 118 human SUMO E2 enzyme (Ubc9) that disrupt this interaction also strongly reduce 119 SUMO chain formation in vitro (Knipscheer et al., 2007). 120 Many studies on the SUMO (de)conjugation machinery revealed that the 121 SUMO enzymes and their targets often reside in uncharacterized punctate nuclear 122 structures of various shapes and sizes (Murtas et al., 2003; Conti et al., 2008; 123 Cheong et al., 2009; Castaño-Miquel et al., 2013; Xiong and Wang, 2013). The E3 124 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which is a 125 SUMO substrate, also localizes to nuclear bodies (NBs) (Kim et al., 2016; Lin et al., 126 2016). COP1 is essential for the dark and high-temperature induced growth 127 responses (skoto- and thermomorphogenesis, respectively) (Lau and Deng, 2012; 128 Park et al., 2017; Hammoudi et al., 2018). Both conditions cause COP1 to 129 translocate from the cytosol to the nucleus where it localizes to a subclass of NBs 130 (Stacey et al., 1999; Stacey and von Arnim, 1999; Park et al., 2017), which are 131 proposed sites for degradation of COP1 substrates (Van Buskirk et al., 2012). COP1 132 activity is stimulated by the SUMO E3 ligase SIZ1 and correspondingly both skoto- 133 and thermomorphogenesis are strongly compromised in the SIZ1 null mutant siz1- 134 2 and the knockdown mutant sumo1-1;amiR-SUMO2 (Delker et al., 2014; Lin et al., 135 2016; Park et al., 2017; Hammoudi et al., 2018). Correspondingly, we previously 136 showed that the high temperature induced transcriptional response of many PIF4 137 and BZR1 genomic targets was both delayed in timing and reduced in amplitude in 138 the siz1-2 and sumo1-1;amiR-SUMO2 backgrounds (Hammoudi et al., 2018). In 139 particular, PIF4 is the master regulator of thermomorphogenesis, whose gene 140 expression is tightly controlled by COP1 activity (Koini et al., 2009; Quint et al.,

5 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

141 2016). Notably, COP1 contains a single SUMO acceptor site (Lys193) that is 142 embedded in a non-consensus site and whose sumoylation strictly depends on SIZ1 143 (Lin et al., 2016). 144 As the formation and role of these SUMO and COP1 NBs is poorly 145 understood, we examined how the SUMO conjugation complex and COP1 146 physically interact in NBs. We find that the formation of SUMO∙SCE1 NBs is 147 dynamic and depends on catalytic activity of the SUMO E1 and E2 enzymes. 148 Likewise, only the conjugation-competent SUMOGG form of SUMO1 can stimulate 149 formation of the SUMO∙SCE1 (SUMO∙E2) and SUMO∙SIZ1 (SUMO∙E3) NBs, while 150 the non-covalent SUMO∙SCE1 interaction via the SIM has apparently a dual role in 151 their formation. Co-localization of these SUMO∙SCE1/SIZ1 NBs with COP1 152 depends on the SUMO acceptor site in COP1. Conversely, we expose that SIZ1 is 153 a COP1-dependent ubiquitination substrate due to two VP motifs that can directly 154 bind to the COP1 substrate binding pocket. Our data thus provide a mechanistic link 155 between the subcellular localization of the SUMO conjugation complex and COP1 156 in one and the same NBs, but recruitment to these bodies depends on the intrinsic 157 properties of the proteins involved, i.e. conjugation activity of SCE1 and substrate 158 selection of COP1.

159 Results

160 Arabidopsis SUMO1 interacts with SCE1 and SIZ1 via its SIM-binding site

161 To this point it remains undefined whether Arabidopsis SUMO1 and -2 interact with 162 partnering proteins via SIMs. Based on sequence homology between Arabidopsis 163 and the human SUMOs, we mutated two conserved hydrophobic residues (Phe32, 164 Ile34) in the β2-strand of Arabidopsis SUMO1 to test in the Y2H assay if – in analogy 165 to the yeast and mammalian systems – these two residues determine binding of 166 SIM-containing proteins (Supplemental Fig. S1A). Wild type SUMO1 and the 167 F32A+I34A mutant (Supplemental Table S1, SUMO1SIM) were expressed as bait 168 fusions with the GAL4 binding domain (BD) and four human proteins with a known 169 SIM were used as preys (as GAL4 AD fusions) (Hecker et al., 2006). To only test 170 for non-covalent interactions between SUMO1 and these SIM-containing proteins, 171 we expressed a conjugation-deficient SUMO1 variant; this variant lacks the C-

6 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

172 terminal diGly motif needed for SUMO attachment to acceptor lysines (SUMO1ΔGG). 173 As expected, SUMO1ΔGG interacted with the three of the four tested SIM-containing 174 proteins and these interactions were suppressed by the F32A+I34A mutations for 175 all except PIAS1 (Supplemental Fig. S1A; SUMO1ΔGG+SIM). Deletion of the diGly 176 motif (‘ΔGG’) and/or introduction of the F32A+I34A double mutation (‘SIM’) did not 177 reduce the protein accumulation of SUMO1 in yeast (Supplemental Fig. S1G). Thus, 178 the β2-strand of Arabidopsis SUMO1 apparently facilitates binding of SIM peptides, 179 similar to its human and yeast counterparts. 180 To assess if Arabidopsis SUMO1 interacts via this SIM interface with SCE1 181 or SIZ1, the BD-SUMO1 fusions were expressed together with SCE1 or SIZ1 fused 182 to the GAL4 AD in the Y2H. Both SCE1 and SIZ1 interacted with SUMO1GG and 183 SUMO1ΔGG. The SUMO1-SCE1 interaction was impaired when the SIM-binding 184 pocket was mutated (Supplemental Fig. S1, B and C, GG+SIM and ΔGG+SIM). The 185 interaction between SUMO1 and SIZ1, was reduced for all SUMO1 mutant versions 186 (Supplemental Fig. S1, B and C, ΔGG, GG+SIM and ΔGG+SIM). As the different 187 SUMO1 variants all accumulated at least to same protein level as wild type SUMO1 188 in yeast (Supplemental Fig. S1G), we conclude that SIM(-like) interactions also play 189 a role in the SUMO-SCE1 interaction. Additional support comes from our recent 190 work where we demonstrated that a conserved SIM-like motif in the N-terminal tail 191 of SCE1 is essential for SUMO1 binding (Mazur et al., 2017). Our findings here 192 suggest the same for SUMO-SIZ1 protein complexes in Arabidopsis, as previously 193 reported for the homologous proteins from yeast and mammals (Cheong et al., 194 2010; Mascle et al., 2013; Kaur et al., 2017); however, functional mutants of the SIM 195 domain in SIZ1 still need to be tested.

196 SUMO1 conjugation activity causes SCE1 and SIZ1 to re-localize to nuclear 197 bodies

198 We then examined if these SIM(-like) interactions affected the subcellular 199 localization of SCE1 and SIZ1 in planta. We first analysed the localization of the 200 individual proteins expressing them as GFP fusions in N. benthamiana. YFP- 201 SUMO1, GFP-SCE1, and GFP-SIZ1 localized to the nucleus, cytosolic pockets near 202 the FM4-64-marked plasma membrane and in cytoplasmic strands (Supplemental 203 Fig. S2, A to C). GFP-SUMO1 accumulated in the cytoplasm and nucleus

7 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

204 independent of its diGly motif and SIM-binding site (Supplemental Fig. S1D). 205 Likewise, SCE1 resided both in the cytoplasm and nucleus (Supplemental Fig. S2, 206 B and D), while SIZ1 accumulated primarily in the nucleus, but a residual signal 207 remained visible in the cytoplasm (Supplemental Fig. S2C). 208 To determine if the interaction with SUMO changes the subcellular 209 localization of SCE1 or the E3 ligase SIZ1, the proteins were expressed as BiFC 210 pairs. To this end SUMO1 was fused via its N-terminus to super-CFPN (fragment 211 SCFP1-173), while SCE1 was fused at its N-terminus to SCFPC (fragment SCFP156- 212 239). SIZ1 was fused at its C-terminus to SCFPC. Expression of both SCFPN-tagged 213 SUMO1GG and SUMO1GG+SIM with SCFPC-tagged SCE1 or SIZ1 yielded in all four 214 combinations CFP reconstitution in relatively large punctate structures that were 215 exclusively found in the nucleus (as marked with Hoechst dye; Supplemental Figure 216 S3A.2), hereafter called nuclear bodies (NBs) (Fig. 1, A and B; Supplemental Fig. 217 S3). These NBs were absent for the BiFC combinations with SUMO1ΔGG or 218 SUMO1ΔGG+SIM; instead in combination with SCE1 their BiFC signal displayed a 219 uniform distribution in both the nucleus and cytoplasm (Supplemental Fig. S3A’), 220 while in combination with SIZ1 their signal was homogeneously spread only in the 221 nucleus (Supplemental Fig. S3B’). These findings suggest that SUMO conjugation 222 itself causes NB assembly in the case of the SUMO1∙SCE1 and SUMO1∙SIZ1 BiFC 223 pairs. 224 To confirm this notion, we examined if another Arabidopsis SUMO paralogue, 225 SUMO3, could trigger NB assembly in a BiFC interaction with SCE1 or SIZ1. We 226 reasoned that SUMO3 would not trigger NB assembly, as (i) it is a poor substrate in 227 vitro for SUMO conjugation compared to SUMO1 (Lois et al., 2003), (ii) it interacts 228 weakly with SCE1 in the Y2H assay (Supplemental Fig. S1E) and (iii) its 229 overexpression in Arabidopsis fails to increase the global level of SUMO1/2 230 conjugates, while overexpression of SUMO1/2GG or SUMO1/2ΔGG massively 231 increased these conjugate levels (Van den Burg et al., 2010). Indeed, SUMO3GG 232 failed to activate SCE1 or SIZ1 recruitment to NBs (Fig. 1, A and B and 233 Supplemental Fig. S3), while GFP-tagged SUMO3GG localized to the nucleus and 234 cytoplasm similar to GFP-SUMO1GG (Supplemental Fig. S1, D and F). Additional 235 proof that assembly of these SUMO NBs requires enzymatic activity came from 236 blocking sumoylation using the chemical inhibitor anacardic acid. Anacardic acid

8 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

237 directly binds to the E1 enzyme SAE1/2 and prevents formation of the E1~SUMO 238 intermediate (Fukuda et al., 2009); consequently, it also blocks SUMO transfer onto 239 the E2 protein (E2~SUMO thioester complex formation). In the presence of 240 anacardic acid, the CFP signal of the SUMO1GG∙SCE1 pair disappeared from pre- 241 existing NBs within 90 min after adding the inhibitor (Fig. 1C, Supplemental Fig. 242 S3C). Thus, the E1~SUMO complex is needed to retain high levels of the 243 SUMO1∙SCE1 pair in NBs, meaning that maintenance of these SUMO NBs is a 244 dynamic process.

245 Not only SUMO loading (E2~SUMO) but also the SUMO binding site 246 (E2∙SUMO) is critical for SCE1 to assemble in SUMO1∙SCE1 NBs 247 To further discern how these NBs are formed, different Arabidopsis SCE1 variants 248 were used that (i) cannot interact with SUMO or SIZ1, (ii) had lost their catalytic 249 activity (SCE1CAT1 and CAT2), or (iii) no longer recognized the ΨKxE SUMO acceptor 250 motif in SUMO substrates (SCE1CAT2) (Mazur et al., 2017) (Fig. 1D; Supplemental 251 Table S1). Studies with the human and yeast SUMO E2 enzymes had identified that 252 the residues Arg14, Arg18 and His20 of SCE1 determine together the binding of 253 SUMO1 to the second non-covalent binding site (the E2∙SUMO complex) (Bencsath 254 et al., 2002). Likewise, the residues Pro69 and Pro106 of the human Ubc9 (SUMO 255 E2) are essential for the SUMO E2 enzyme to interact with the PIAS family of SUMO 256 E3 ligases (closest homolog of Arabidopsis SIZ1) (Mascle et al., 2013). These five 257 residues are strictly conserved in Arabidopsis SCE1 and other plant homologues of 258 SCE1 (Supplemental Fig. S4), and also for Arabidopsis SCE1 these residues are 259 essential for its interaction with SUMO1 or SIZ1 in the Y2H assay (Mazur et al., 260 2017). The SCE1CAT1 mutant still interacted with SUMO1; however, SCE1CAT2 had 261 lost both its catalytic activity and its non-covalent interaction with SUMO1 (Mazur et 262 al., 2017). See Supplemental Table S1 for an overview of the mutants tested. 263 Introduction of these mutations in SCE1 did not change its subcellular 264 localization in planta, i.e. each variant showed a uniform distribution in both the 265 nucleus and cytoplasm when transiently expressed as GFP-fusion in N. 266 benthamiana (Supplemental Fig. S2D). Both SCE1CAT1 and SCE1SUM1 appeared to 267 accumulate to higher protein levels in planta (Supplemental Fig. S2E), possibly 268 indicating that the stability of wild type SCE1 is negatively impacted by its own

9 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

269 enzymatic activity and/or its non-covalent association with SUMO1. Importantly, 270 SCE1SIZ1 still interacted with SUMO1GG in NBs, but SCE1CAT1, SCE1CAT2 and 271 SCE1SUM1 were not recruited to such NBs while they still interacted with SUMO1GG 272 in the BiFC assay (Fig. 1E and Supplemental Fig. S3E). These findings thus indicate 273 that besides SUMO loading (E2~SUMO thioester complex) also the non-covalent 274 SUMO binding (E2∙SUMO) is critical for SCE1 to assemble in SUMO1∙SCE1 NBs.

275 The SUMO∙SCE1 interface contributes to the SCE1-SIZ1 interaction

276 In contrast to the SUMO1GG∙SCE1/SIZ1 couples, we noted that the SCE1∙SIZ1 BiFC 277 complex did not aggregate in NBs but rather resided in small nuclear speckles 278 spread across the nucleus (Fig. 1F and Supplemental Fig. S3F). We already 279 showed that the SCE1 residues Pro70 and Pro106 (Supplemental Table S1, 280 SCE1SIZ1) are essential for SCE1 and SIZ1 to interact in the Y2H assay, while 281 mutating the catalytic site (SCE1CAT1 and SCE1CAT2) did not impair their Y2H 282 interaction (Mazur et al., 2017). Also in the BiFC assay, both the SCE1SUM1 and 283 SCE1SIZ1 variants failed to interact with SIZ1, while the catalytic-dead variants 284 (SCE1CAT1 and SCE1CAT2) still interacted with SIZ1. This suggests that SUMO 285 loading in the catalytic site of SCE1 (SCE1CAT1 and CAT2; E2~SUMO1) is not required 286 for SCE1 and SIZ1 to interact, while the non-covalent interaction (SCE1SUM1; 287 E2∙SUMO1) strengthens this interaction in both the Y2H and BiFC assays (Fig 1F; 288 Mazur et al., 2017). Apparently, the non-covalently bound SUMO acts as a glue in 289 the SCE1-SIZ1 complex or it alters the SCE1 conformation such that it enhances 290 the interaction between SCE1 and SIZ1. 291 Next, we examined whether the BiFC pairs SUMO1GG∙SCE1 and 292 SUMO1GG∙SIZ1 physically co-localized in one and the same NBs. Thereto, we 293 performed multicolour BiFC (mcBiFC) (Gehl et al., 2009), where SUMO1GG was 294 expressed as fusion protein with SCFPC, SCE1 was fused at its N-terminus to 295 SCFPN and SIZ1 was fused at its C-terminus to the Venus residues 1-173 (VenusN). 296 Reconstitution of both fluorophores was examined using optical filters that separate 297 CFP (for SCFPC-SUMO1GG with SCFPN-SCE1) from the chimeric VenusN-SCFPC 298 signal (for SCFPC-SUMO1GG with VenusN-SIZ1). This mcBiFC experiment revealed 299 a complete overlap between the SUMO1GG∙SCE1 and SUMO1GG∙SIZ1 signals in 300 NBs (Fig. 1G).

10 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

301 As the SCE1∙SIZ1 BiFC pair alone did not form large distinct NBs (Fig. 1F), 302 we tested if the levels of free SUMO1GG could have been limiting, thus preventing 303 the aggregation of the SCE1∙SIZ1 protein complex into enlarged nuclear structures. 304 We expressed the SCE1∙SIZ1 pair together with YFP-tagged SUMO1GG or 305 SUMO1ΔGG (negative control). The YFP-SUMO1GG signal overlapped with the 306 SCFP signal, but only in a few cells the SCE1∙SIZ1 BiFC signal shifted from 307 speckles/puncta to enlarged NBs. YFP-SUMO1ΔGG localized as well to the nucleus, 308 but overall it did not co-localize with the SCE1∙SIZ1 BiFC pair in nuclear specks 309 (Supplemental Fig. S5). Thus, the SUMO1-dependent NB enlargement is foremost 310 seen when SUMO is trapped in a BiFC interaction with SCE1 or SIZ1. Possibly, the 311 BiFC system stabilizes a transient protein-protein interaction, which then allows 312 formation of enlarged SUMO NBs. Alternatively, the presence of the intact YFP tag 313 attached to SUMO1GG might cause steric hindrance in the ternary complex.

314 SUMO chain formation potentially stimulates formation and enlargement of 315 SUMO NBs

316 As in mammalian cells SUMO chain formation promotes formation of Promyelocytic 317 Leukemia (PML) NBs (Hattersley et al., 2011), we assessed whether the formation 318 of SUMO NBs depends SUMO chain formation in plants. To block SUMO chain 319 formation, we engineered SUMO1 variants in which Lys9, Lys10 or all seven Lys 320 were replaced by Arg (Supplemental Table S1, SUMO1K9R, SUMO1K10R, 321 SUMO1K9R+K10R and SUMO1K7ØR). Although Lys10 is the main site for SUMO chain 322 elongation, Lys9 can serve as an alternative site (Colby et al., 2006). Therefore, we 323 also created the SUMO1K9R+K10R double mutant. To test if these KtoR mutants can 324 still recruit SCE1 to NBs, they were expressed as BiFC pair with SCE1. Two days 325 after Agrobacterium infiltration, the NBs formed were reduced in size and number 326 for the KtoR mutants compared to wild type SUMO1, while the diffuse nuclear signal 327 of SCFP had increased (Supplemental Fig. S6A). The SUMO1K7Ø∙SCE1 pair failed 328 entirely to form NBs two days post infiltration, while SUMO1K9R+K10R∙SCE1 displayed 329 a reduced number of NBs at this time point. These data agree with the proposed 330 role of SUMO chains in NB formation. However, three days post infiltration NBs 331 were found for each KtoR SUMO1 mutant (Supplemental Fig. S6A), albeit the 332 number of cells that contained NBs was less for SUMO1K7Ø∙SCE1 (40-50% for

11 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

333 SUMO1K7Ø compared to 70–100% for the other combinations). Even though, we 334 cannot rule out that these Lys mutations influence protein translation at this time, 335 these data suggest that SUMO chain formation potentially stimulates the targeting 336 of the SUMO1∙SCE1 complex to NBs, affecting both their initial formation and 337 subsequent enlargement.

338 SUMO1GG∙SCE1 NBs show complete co-localization with COP1 NBs

339 Importantly, COP1 co-localizes with SIZ1 in NBs (Kim et al., 2016). Using the Y2H 340 assay, we confirmed that COP1 directly interacts with SIZ1 and not with Arabidopsis 341 SCE1, SUMO1 or SUMO3 (Fig. 2A). We then assessed whether sumoylation of 342 COP1 is needed to co-localize with SUMO1GG∙SCE1 in NBs. Similar to SIZ1 (Kim 343 et al., 2016), RFP-COP1 co-localized strongly with the SUMO1GG∙SCE1 BiFC pair 344 in NBs (Fig. 2, B and C and Supplemental Fig. S6C). Importantly, both the 345 SUMO1ΔGG∙SCE1 and SUMO1GG∙SCE1CAT1 pairs (Fig. 2, B and C) still failed to 346 localize to NBs regardless of RFP-COP1 overexpression. Thus, co-localization of 347 SCE1 and COP1 in NBs requires again SCE1 catalytic activity. Co-expression of 348 RFP-COP1 with different SUMO1KtoR∙SCE1 BiFC pairs revealed that all these 349 SUMO1KtoR∙SCE1 variants strongly co-localized with COP1 in NBs irrespective of 350 the lysine mutations (Supplemental Fig. S6B). Next, we tested if the COP1 SUMO 351 acceptor site (Lys193) is important for this co-localization. Introduction of the K193R 352 mutation (COP1SUMO) reduced the overlap between the COP1 and SUMO1∙SCE1 353 NBs (Fig. 3, B and C, Supplemental Fig. S7A), but it did not suppress the COP1- 354 SIZ1 protein-protein interaction in the Y2H assay (Fig. 3, A and D) suggesting that 355 residues other than Lys193 promote the interaction between the E3 ligases SIZ1 356 and COP1. 357 Structural studies revealed that substrates of COP1 interact with it via VP 358 peptide motifs that dock in the central groove of the COP1 WD-40 propeller head 359 (Uljon et al., 2016). The mutation Q529E (the causal mutation in the cop1-9 allele, 360 hereafter COP1SUBSTRATE) is positioned in this VP-binding groove and was shown to 361 disrupt the binding of different COP1 substrates (Holm et al., 2002). Importantly, this 362 COP1SUBSTRATE variant fails to form NBs when expressed as a GFP-fusion protein 363 (Stacey et al., 1999). COP1 also interacts with the DBB1-CUL4 E3 ubiquitin ligase

12 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

364 complex via two WDRX motifs; these WDRX motifs are also located in this WD-40 365 domain near the VP-binding groove (Chen et al., 2010). 366 We used these COP1 variants to test whether the capacity of COP1 to bind 367 substrates or CUL4 (i) determines its targeting to NBs and (ii) related its recruitment 368 to SUMO NBs. First, we tested if these COP1 variants still interacted with SIZ1 in 369 the Y2H assay (Fig. 3D). Except for COP1SUMO, all the other mutants failed to 370 interact with SIZ1 (Fig. 3D). When fused to RFP, COP1SUMO and COP1RING still 371 formed NBs in N. benthamiana, while the variants COP1SUBSTRATE, COP1CUL4,1 and 372 COP1CUL4,1 and 4,2 failed to localize to NBs in planta (Fig. 3C, Supplemental Fig. S7B). 373 Mutations in the WD-40 domain thus suppressed formation of COP1 NBs. To 374 quantify the degree of co-localisation between the COP1 variants and the 375 SUMO1GG∙SCE1 BiFC signal, the RFP/CFP pixel intensities were depicted in a 376 scatter plot. This yielded a strong positive correlation between the signal intensities 377 of wild type RFP-COP1 and the SUMO1GG∙SCE1 CFP signal (Pearson’s R = 0.882). 378 The localization of RFP-COP1RING also correlated strongly with the SUMO1GG∙SCE1 379 CFP signal (Pearson’s R = 0.866). The degree of co-localization was less for RFP- 380 COP1SUMO (Pearson’s R = 0.490), while localization of the variants RFP- 381 COP1SUBSTRATE, -COP1CUL4,1 and -COP1CUL4,1 and 2 did not correlate with the 382 SUMO1GG∙SCE1 signal (Fig. 3B, and Supplemental Fig. S7B; Pearson’s R = 0.188, 383 0.177 and 0.148, respectively). 384 RFP-COP1 also co-localized with the SUMO1∙SIZ1 pair in NBs (Fig. 4A). 385 Their co-localization in NBs required an intact substrate-binding pocket in COP1 386 (COP1SUBSTRATE). Similar to the SUMO1∙SCE1 BiFC pair, the SUMO acceptor site 387 in COP1 (COP1SUMO) contributed to the overlap between the SUMO1∙SIZ1 and 388 RFP-COP1 signals in NBs, while disruption of COP1 ubiquitin ligase activity 389 (COP1RING) had no apparent effect on targeting of the SUMO1∙SIZ1 complex to 390 these COP1 NBs (Fig. 4A). Thus, the substrate binding pocket of COP1 appears to 391 be the main determinant for both COP1 recruitment to NBs and its binding with SIZ1.

392 SIZ1 contains two VP domains important for COP1 binding 393 To map the COP1-binding site in SIZ1, a series of SIZ1 mutants was prepared. In 394 yeast both the PHD and PINIT domain are essential for ScSIZ1-directed 395 sumoylation of the substrate PCNA (Proliferating cell nuclear antigen) (Yunus and

13 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

396 Lima, 2009). Loss-of-function mutations in these two domains in Arabidopsis SIZ1 397 did not compromise the interaction with COP1 in the Y2H assay. Disruption of the 398 SP-RING domain, which is needed to recruit the E2~SUMO thioester into a complex 399 with its substrate PCNA (Yunus and Lima, 2009), impaired the interaction between 400 SIZ1 and COP1 (Fig. 4, B and C). SIZ1 also contains two VP motifs (VP1:Val251 401 and VP2:Val725/728), which could explain why SIZ1 is recognized by COP1 as a 402 substrate. Both motifs were mutated by replacing Val for Asp residues. Mutating 403 VP1 did not suppress the SIZ1-COP1 interaction, while mutating VP2 reduced this 404 interaction. Furthermore, mutating both VP motifs disrupted the interaction entirely 405 (Fig. 4, B and C). Thus, the SP-RING domain of SIZ1 contributes to the interaction 406 with COP1, but the VP motifs combined are essential for this interaction. 407 To determine if SCE1 and SIZ1 can already reside in COP1 NBs independent 408 of their SUMO-BiFC interaction, we co-expressed GFP-tagged SCE1 or SIZ1 409 together with RFP-tagged COP1 or COP1SUMO. For both proteins (SCE1 and SIZ1), 410 the GFP signal was enriched in RFP-COP1 NBs (Fig. 5A). Moreover, their targeting 411 to COP1 NBs was significantly less when they were co-expressed with RFP- 412 COP1SUMO (Figs. 5, A and B) and these latter GFP-SIZ1/RFP-COP1SUMO NBs also 413 had an amorphous shape (asterisk in Fig. 5A). Combined, these data suggest that 414 the SUMO acceptor site in COP1 contributes to the recruitment of SCE1 and SIZ1 415 to COP1 NBs.

416 Recruitment of SCE1 to COP1 NBs requires the SIZ1 gene in Arabidopsis 417 As the native proteins are still present in the N. benthamiana BiFC experiments, we 418 shifted to particle bombardment in Arabidopsis leaf epidermal cells. Using the 419 Arabidopsis mutants cop1-4, siz1-2, and sumo1;amiR-SUMO2, we genetically 420 inferred whether the endogenous proteins are required for (co-)localization of SCE1 421 and COP1 in NBs. The different constructs where shifted to high expression vectors 422 suitable for particle bombardment (Walter et al., 2004) in which SCE1 and 423 SUMO1GG/SUMO1ΔGG were tagged at their N-terminus with the BiFC halves NYFP 424 and CYFP, respectively. Similar to the N. benthamiana data, the SUMO1GG∙SCE1 425 combination formed NBs in Arabidopsis, while SUMO1∆GG∙SCE1 interacted but this 426 combination displayed a uniform BiFC signal across the nucleus (Fig. 6A). Targeting 427 of SUMO1GG∙SCE1 to NBs did not change when we the BiFC protein complex was

14 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

428 expressed in the genotypes cop1-4 or siz1-2, confirming that SUMO conjugation is 429 the main force behind SUMO NB formation. To assess genetically if sumoylation 430 activity is essential for COP1 NB formation, GFP-COP1 was expressed in wild type 431 Arabidopsis (Col-0), siz1-2 and sumo1;amiR-SUMO2 using particle bombardment. 432 Irrespective of these three genetic backgrounds, GFP-tagged COP1 localized to 433 NBs in the Arabidopsis plants (Fig. 6B). This corroborates our notion that SUMO 434 conjugation activity per se is not the main driving force for COP1 aggregation in 435 NBs. To demonstrate that the co-localization of SCE1 and COP1 in NBs depends 436 on SIZ1, RFP-SCE1 and GFP-COP1 were transiently co-expressed in wild type 437 Arabidopsis and siz1-2 using particle bombardment. As expected, in wild type 438 bombarded Arabidopsis rosettes RFP-SCE1 was enriched in GFP-COP1 NBs, 439 while in the siz1-2 background RFP-SCE1 was absent from these COP1 NBs (Fig. 440 6, B and C). Thus, recruitment of SCE1 and COP1 to one and the same NB requires 441 the endogenous SIZ1 protein to be present.

442 Simultaneous recruitment of CRY1/2 and SUMO1∙SCE1 to COP1 NBs 443 CRY1 and -2 are blue light receptors that inhibit COP1 activity indirectly via the SPA 444 proteins or directly, respectively, after blue light exposure (Wang et al., 2001; Liu et 445 al., 2011; Holtkotte et al., 2017). Fluorescent protein fusions of CRY2 localize to 446 NBs after blue light exposure (Mas et al., 2000). As, at least to our knowledge, 447 neither CRY1 nor CRY2 are sumoylated, their subcellular localization might 448 correlate with COP1, but not with the SUMO conjugation enzymes. CRY1-GFP and 449 CRY2-GFP were co-expressed with RFP-COP1, RFP-SCE1 and SIZ1-RFP in N. 450 benthamiana cells. CRY2-GFP localized in our system in NBs and CRY1-GFP 451 localized to the whole nucleus. As expected, both CRY1 and CRY2 were recruited 452 to mRFP-COP1 NBs (Supplemental Fig. 8, A and B). SIZ1-mRFP or mRFP-SCE1 453 co-expressed with CRY1/CRY2-GFP were evenly distributed in the nucleus and did 454 neither alter CRY1- nor CRY2-GFP localization. Importantly, SCE1 and SIZ1 were 455 not recruited to CRY2-GFP NBs. Next, the SUMO1GG∙SCE1 BiFC pair was co- 456 expressed with CRY1-mRFP or CRY2-mRFP. Neither CRY1-mRFP nor CRY2- 457 mRFP was recruited to the SUMO1GG∙SCE1 NBs (Fig. 7). However, when YFP- 458 COP1 was co-expressed in the same cells, all components co-localized to the same

15 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

459 NBs. Thus, CRY2 NBs and SUMO1GG∙SCE1 NBs are different entities that coincide 460 in COP1 NBs.

461 Discussion

462 Here we characterized the assembly of the ternary complex between SUMO1, 463 SCE1 and SIZ1 in NBs. SCE1 activity (i.e. SCE1~SUMO thioester) and the non- 464 covalent association of SUMO with SCE1 are both required to target this complex 465 to NBs (Fig. 1, A and E). Strikingly, COP1 fully co-localizes with these SUMO NBs. 466 COP1 is a master regulator of skoto- and thermomorphogenesis that translocates 467 from the cytosol to the nucleus at night-time and at high temperature (28oC) 468 conditions (Höcker, 2017; Park et al., 2017). Once nuclear localized, COP1 targets 469 a set of substrates, mainly transcription factors (TFs) that act as positive regulators 470 of light signaling, for degradation. We found that mutating the COP1 SUMO acceptor 471 lysine (Lys193) reduced the overlap between COP1 and the BiFC pair 472 SCE1∙SUMO1 in NBs albeit that both still predominantly localized to NBs (Fig. 3, B 473 and C, Supplemental Fig. S7A). In support, GFP-tagged SIZ1 and SCE1 proteins 474 showed reduced co-localization with COP1 NBs when co-expressed with a COP1 475 variant that lacks this acceptor lysine (K193R, COP1SUMO) compared to wild type 476 COP1 (Fig. 5). Moreover, COP1 interacted physically with SIZ1 and not with SCE1, 477 SUMO1 or SUMO3 in the Y2H assay. Importantly, COP1 is a SIZ1-dependent 478 sumoylation substrate and its sumoylation enhances its biochemical activity, 479 resulting in ubiquitylation and degradation of SIZ1 and other substrates, which in 480 turn results in enhanced plant growth (Kim et al., 2016; Lin et al., 2016; Kim et al., 481 2017). Structural studies had exposed that COP1 substrates dock in a central 482 groove of WD-40 propeller head of COP1 via short VP peptide motifs (Uljon et al., 483 2016). We identified two of such VP motifs in SIZ1 that together control SIZ1 binding 484 to COP1 (Fig. 4, B and C). Genetically, targeting of SCE1 to COP1 NBs also 485 requires a functional SIZ1 gene in Arabidopsis (Fig. 6, B and C). Combined, these 486 data signify that SIZ1 is recognized by COP1 as a bona fide substrate, likely via its 487 two VP motifs, and that in turn SIZ1 can recruit SCE1 to COP1 NBs.

488 Substrate binding drives COP1 to NBs independent of SIZ1

16 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

489 Structural studies had exposed that the C-terminal half of COP1, including its WD- 490 40 domain, is important for substrate binding, but also for its localization to NBs 491 (Stacey et al., 1999; Stacey and von Arnim, 1999; Holm et al., 2002; Uljon et al., 492 2016). In particular, the amino acid change G524Q found in the cop1-9 allele is 493 located at the central substrate-binding groove of the WD-40 propeller head and this 494 mutation prevents COP1 targeting to NBs (Stacey et al., 1999; Uljon et al., 2016, 495 Fig. 3B). In contrast, sumoylation of COP1 at Lys193 and the ubiquitin ligase activity 496 of its RING domain (Stacey et al., 1999; Stacey and von Arnim, 1999) are not critical 497 for COP1 aggregation in NBs (Fig. 3B). Using particle-bombardment in Arabidopsis 498 mutants, we established genetically that COP1 resides in NBs independent of SIZ1 499 (Fig. 6B). As well, the BiFC pair SCE1∙SUMO1GG still aggregated in NBs when 500 transiently expressed in siz1-2 or cop1-4 (a strong COP1 allele with little residual 501 COP1 activity remaining). Thus, recruitment of COP1 or SUMO∙SCE1 to NBs does 502 not depend on the physical interaction between COP1 and SIZ1, while their collision 503 in the same NBs requires SIZ1 as bridge protein.

504 Sumoylation and blue light signalling coincide in COP1 bodies.

505 The blue light receptors CRY1 and CRY2 inhibit COP1 activity in a blue-light 506 dependent manner (Wang et al., 2001). The interaction of CRY2 and COP1 is direct, 507 while CRY1 uses SPA1 as a bridge protein (Wang et al., 2001; Liu et al., 2011; 508 Holtkotte et al., 2017). Directly after blue light exposure, CRY2 is rapidly degraded. 509 COP1 is only partially responsible for this light-dependent degradation of CRY2 510 (Shalitin et al., 2002). We observed that in the absence of YFP-COP1 511 overexpression neither CRY1 nor CRY2 co-localize with SCE1, SIZ1 or the SUMO- 512 SCE1 BiFC complex (Fig. 7 and Supplemental Fig. S8). However, when COP1 is 513 present all these proteins are targeted to the same NBs. This suggests that the first 514 steps in the blue light response are sumoylation independent, thereafter all 515 components are apparently dragged to the same NBs for downstream responses. 516 As CRY1 is not and CRY2 is for its degradation only partially dependent on COP1, 517 their action might, however, affect or be affected by COP1 sumoylation. As 518 sumoylation enhances COP1 activity (Lin et al., 2016), CRY1 or CRY2 might either 519 prevent COP1 sumoylation or make COP1 insensitive to CRY1 or CRY2 520 interference.

17 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

521 Enzymatic activity of SCE1 drives localization of SUMO1∙SCE1 to NBs

522 Our data on the molecular interactions between SUMO1, SCE1, and SIZ1 support 523 that they adopt a similar ternary complex in Arabidopsis, as previously reported for 524 their yeast and human counterparts (Bencsath et al., 2002; Bernier-Villamor et al., 525 2002; Reverter and Lima, 2005; Mascle et al., 2013; Sekhri et al., 2015; Mazur et 526 al., 2017). Recruitment of this ternary complex to NBs depends tightly on their 527 intermolecular interactions in N. benthamiana and Arabidopsis. Furthermore, we 528 established that the SIM binding cleft around Phe32 (Colby et al., 2006) is 529 functionally conserved in Arabidopsis SUMO1 and that it enforces the interaction of 530 SUMO with SCE1/SIZ1 (Fig. 1A). Mutations in the SIM did not suppress assembly 531 of the SUMO∙SCE1 and SUMO∙SIZ1 NBs, while conjugation-deficient variants of 532 SUMO1 failed to aggregate in these SUMO NBs (Fig. 1, A and B). Thus, a key factor 533 for SUMO NB assembly is conjugation activity. Two lines of evidence argue that the 534 SUMO-SCE1/SIZ1 NBs are not an artefact of the BIFC system or caused by 535 unspecific aggregation of (misfolded) proteins, but rather require active formation 536 and/or maintenance. First, pre-existing NBs disappear within 90 minutes after 537 inhibition of the SUMO E1 enzyme by anacardic acid. Second, NB assembly 538 requires the catalytic site of SCE1 to be intact. 539 Our data suggest that SUMO chain formation is not essential but might 540 promote formation of the SUMO1∙SCE1 NBs (Supplemental Fig. S6A). The non- 541 covalent association of SUMO with SCE1 was reported to be important for SUMO 542 chain formation (Knipscheer et al., 2007). Our data support a mechanistic model in 543 which thioester (E2~SUMO) formation is essential, while the non-covalent- 544 association of SUMO (E2∙SUMO) has a dual role in the redistribution of Arabidopsis 545 SUMO1 and SCE1 to NBs. This is reminiscent to the mechanism by which SUMO 546 drives NB formation in yeast and human cells, e.g. Pc2 (Polychrome 2) in Polycomb 547 group bodies and PML bodies (Yang and Sharrocks, 2010; Jentsch and Psakhye, 548 2013). Not only the SUMO conjugation machinery localizes to NBs (this study), but 549 also a substantial number of Arabidopsis SUMO substrates and several SUMO 550 proteases have been reported to localize to nuclear speckles/bodies/puncta. For 551 example, the SUMO protease OTS2 (OVERLY TOLERANT TO SALT2) and the 552 putative SUMO-targeted Ubiquitin E3 ligases STUbL1 and STUbL4 were reported 553 to localize to undefined nuclear puncta, resembling the NBs here seen (Conti et al.,

18 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

554 2008; Elrouby et al., 2013). Future studies should address if all these bodies reflect 555 one and the same nuclear structure.

556 Coincidental or functional: the overlap between COP1 and SIZ1 substrates?

557 This study strengthens the notion that COP1 and sumoylation go hand-in-hand in 558 controlling each other’s activities while simultaneously targeting shared substrates. 559 Interestingly, many of the COP1 substrates and interactors are also (putative) 560 SUMO conjugation targets (phyB, DELLAs, HFR1 (LONG HYPOCOTYL IN FAR 561 RED 1), LAF1 (LONG AFTER FAR-RED LIGHT 1), HY5 (ELONGATED 562 HYPOCOTYL 5), and HYL (HY5 HOMOLOGUE)) (Ballesteros et al., 2001; Seo et 563 al., 2003; Conti et al., 2014; Sadanandom et al., 2015; Tan et al., 2015; Mazur et 564 al., 2017). Jointly, these COP1 substrates control at the transcriptional and post- 565 translational level the activity of the PIFs, which in turn control the light and 566 temperature-sensitive growth pathways (Paik et al., 2017). Moreover, the TF ABI5 567 (ABA-INSENSITIVE 5), a known interactor of SIZ1 and a SUMO substrate, was also 568 found to translocate to COP1-containing NBs in the presence of the regulator AFP 569 (ABI FIVE INTERACTING PROTEIN) (Lopez-Molina et al., 2003; Miura et al., 2009). 570 Disruption of a putative SUMO acceptor site in the TF LAF1 prevented its 571 translocation to nuclear speckles (Ballesteros et al., 2001), while the TF HFR1 was 572 shown to interact with the bacterial SUMO protease XopD (Xanthomonas outer 573 protein D from the bacterium Xanthomonas) in nuclear speckles (Tan et al., 2015). 574 Translocation of HFR1 and ABI5 to NBs was in each case associated with their 575 degradation, which provides again a link between COP1 and sumoylation in the 576 regulation of nuclear processes. Considering that SIZ1 controls both skoto- and 577 thermomorphogenesis (Lin et al., 2016; Hammoudi et al., 2018), the recruitment of 578 SIZ1 to COP1 bodies might suggest the existence of a (transient) SUMO 579 conjugation wave in these bodies with unknown physiological and biochemical 580 consequences in a normal diurnal dark/light cycle. As previously shown, SIZ1 is 581 needed for hypocotyl elongation in both darkness and/or high temperature 582 conditions and the siz1-2 mutation delays and weakens significantly the 583 transcription response of substantial proportion of the PIF4 genomic targets (Lin et 584 al., 2016; Hammoudi et al., 2018). Thus, in addition to having a role in plant

19 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

585 immunity, SIZ1-dependent sumoylation rapidly sees the light as an important 586 positive regulator of dark/high temperature-induced growth responses.

587 Materials and methods

588 Construction mutants and vectors for yeast two-hybrid interactions

589 All molecular techniques were performed using standard methods (Sambrook and 590 Russel, 2001). Primers (synthesized by Eurofins genomics) are listed in 591 Supplemental Table S2. Information on clones with primers can be found in 592 Supplemental Table S3. Primers containing the attB1 and attB2 recombination sites 593 were used to amplify the CDS sequences (see Supplemental Table S2). The PCR 594 products were cloned into the pDONR207 or pDONR221 using BP Clonase II 595 (Thermo Fischer) and checked by sequencing. The inserts were transferred to 596 destination vectors using Gateway LR Clonase II (Thermo Fischer) and the clones 597 were re-sequenced. For the GAL4 BD/AD-fusion yeast-two-hybrid constructs, the 598 cDNA clones were introduced in pDEST22/pDEST32 (Thermo Fischer). As the 599 interactions of SUMO1∙SCE1 were weak in the pDEST system (due to low 600 expression levels, Supplemental Fig. S1B), these proteins were expressed with 601 pGBKT7/pGADT7 (Clontech). CRY1 (G12079) and CRY2 (G19559) cDNA clones 602 were obtained from the Arabidopsis Biological Research Centre. For in planta 603 protein localization, the CDS were introduced in the destination plasmids pGWB452 604 (N-terminal GFP tagging), pGWB442 (N-terminal YFP tagging) or pGWB655 (N- 605 terminal mRFP-tagging) (Nakagawa et al., 2007). For BiFC studies, we used the 606 destination vectors pSCYNE(R) (N-terminus SCFP3A), pSCYCE(R) (C-terminus of 607 SCFP3A) and pVYNE (N-terminus of Venus) (Gehl et al., 2009) or pESPYNE- 608 Gateway (N-terminus of YFP)/ pESPYCE-Gateway (C-terminus of YFP) (Walter et 609 al., 2004; Schütze et al., 2009).

610 Plant protein isolation and detection using immunoblotting

611 To detect GFP-tagged proteins in Nicotiana benthamiana, total protein was 612 extracted from ground leaf material in 2x v/w extraction buffer (8M urea, 100mM Tris 613 pH 6.8, 2% SDS, 10 mM DTT), incubated on ice for 15 min, centrifugated at 13,000g 614 for 20 min at 4oC and then separated on 10% SDS-PAGE, blotted to PVDF

20 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

615 membranes and after blocking with 5% milk in PBS detected using monoclonal 616 antibodies (diluted 1:1000) directed against GFP (Chromotec, #029762). The 617 secondary antibody goat anti-rat IgG conjugated to Horseradish peroxidase 618 (ThermoFisher #31470) was used at a dilution of 1:10,000. The proteins were 619 visualized using enhanced chemiluminescence (ECL, home-made recipe). Equal 620 loading of the protein samples was confirmed by Ponceau staining of the blots.

621 Protein isolation from yeast and detection using immunoblotting

622 To obtain a total protein lysate from yeast, the protocol from Yeast Protocols 623 Handbook was followed (Clontech; Yeast Protocols Handbook: www.clontech.com/ 624 images/pt/PT3024-1.pdf). Additional details are described in Mazur et al. (2017).

625 Transient expression of protein in N. benthamiana using agro-infiltration

626 Transient expression of proteins was performed as described by Ma et al. (2012). 627 In brief, Agrobacterium GV3101 cells containing the desired constructs were

628 infiltrated into 4-5-week-old N. benthamiana leaves (OD600=1.0 for each construct). 629 To suppress gene silencing, an A. tumefaciens strain GV3101 carrying pBIN61 630 containing the P19 silencing suppressor of Tomato busy shunt virus (TBSV)

631 (Voinnet et al., 2003), was co-infiltrated with the samples (OD600=0.5). Protein 632 accumulation was examined 2-3 days post-infiltration. 633 634 Transient expression of proteins in N. benthamiana with anacardic acid 635 treatment 636 Four-week-old N. benthamiana plants were used for agroinfiltration. Three days 637 post-agroinfiltration, 100 μM anacardic acid in 1% DMSO or only 1% DMSO (as a 638 negative control) were directly injected into N. benthamiana leaves transiently 639 expressing the BiFC constructs. One and half hours after anacardic acid infiltration, 640 the treated leaf discs were collected and fluorescence was analysed using a Zeiss 641 LSM510 confocal laser microscope. Two independent biological experiments were 642 carried out and a minimum of 50 nuclei for each sample and treatment were 643 observed.

644 Transient expression using particle bombardment of Arabidopsis

21 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

645 Complete Arabidopsis rosettes of 4-5-week-old plants grown in 11 hrs light/13 hrs 646 dark (22oC) were placed on 1% agar containing 85 μM benzimidazole (Sigma- 647 Aldrich) and kept in the growth chamber until bombardment. Transformation by 648 particle bombardment was performed as described previously (Schweizer et al., 649 1999; Shirasu et al., 1999). In brief, 1 μm diameter gold particle were coated with 650 2.5 μg of each type of plasmid. The PDS1000/HE particle gun with Hepta-adaptor 651 (Bio-Rad) was used according to manufacturer’s protocol using a 900 psi. rupture 652 disk (Bio-Rad). After bombardment, Petri dishes were sealed with medical tape and 653 returned to the growth chamber for 2-3 days before inspection. Plates were kept in 654 the dark from the evening before inspection till loading on the confocal microscopy 655 to retain COP1 in the nucleus.

656 GAL4 yeast two-hybrid protein-protein interaction assay

657 The protocol for yeast two-hybrid was followed as described in (de Folter and 658 Immink, 2011) and further details are described in Mazur et al. (2017).

659 Confocal microscopy

660 N. benthamiana and Arabidopsis leaves were analyzed 2-3 days post infiltration with 661 A. tumefaciens or particle bombardment, respectively. The Hoechst 33342 (Sigma- 662 Aldrich) chromatin stain and FM4-64 (ThermoFisher) membrane dye and endocytic 663 tracer were syringe infiltrated into the leaves at a final concentration of 1 μg/ml and 664 50 µM in water, respectively, and stored in the dark in a Petri dish on wet paper. 665 FM4-64 could be directly imaged, Hoechst 33342 was incubated for at least an hour 666 before imaging. Accumulation of the tagged proteins was examined in leaf 667 epidermal cells using a Zeiss LSM510 or Nikon A1 confocal laser-scanning 668 microscope. Images at the LSM510 were taken with C-Apochromat 40x water 669 immersive objective with a numerical aperture (NA) of 1.2. At the A1 images were 670 taken with a Plan Fluor 40x oil immersive DIC lens (NA=1.3). SCFP, GFP, chimeric 671 VenusN-SCFPC, YFP and RFP labelled samples were excited with 458, 488, 488, 672 514 nm or 568 nm diode lasers. At the LSM510, GFP and the VenusN-SCFPC 673 chimera were detected using a 520-555 nm BP filter (BP520-555), SCFP (BiFC) 674 with BP470-500, RFP with BP585-615. At the Nikon A1, SCFP was detected with 675 BP468-502; YFP/GFP with BP500-550, and RFP with BP570-620. At the Nikon A1,

22 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

676 for simultaneous Hoechst 33342, SCFP and mRFP imaging, Hoechst 33342 was 677 excited with the 402 nm diode laser and detected between 425-475 nm, SCFP was 678 excited with the 488 nm diode laser and detected between 500-550 nm and mRFP 679 was excited with the 561 nm diode laser and detected between 570-620 nm. FM4- 680 64 was excited with the 514 nm diode laser and detected between 570 and 620 nm. 681 Bright field images were recorded with the transmitted light photomultiplier detector. 682 On both microscopes, co-expressed fluorophores or dyes were excited 683 consecutively to limit bleed through of emission signals between detection channels. 684 For all observations, the pinhole was set at 1 Airy unit. Images were processed 685 using ImageJ (NIH). 686 687 Supplemental Data 688 689 Supplemental Figure S1. Arabidopsis SUMO1 interacts specifically via a non- 690 covalent SIM-like interaction with SCE1 in Arabidopsis. 691 Supplemental Figure S2. The ternary complex between Arabidopsis SUMO1, 692 SCE1 and SIZ1 is stabilized by the substrate-binding pocket (CAT2) and non- 693 covalent interactions between the different proteins. 694 Supplemental Figure S3. The SUMO1GG-SCE1 and SUMO1GG-SIZ1 BiFC 695 complex localize to the nucleus in nuclear bodies. 696 Supplemental Figure S4. Alignment of the SCE1 protein sequences of different 697 monocot and dicot plant species showing that SCE1 protein is extremely conserved 698 at the sequence level. 699 Supplemental Figure S5. Co-expression of SCE1∙SIZ1 as BiFC pair with an 700 excess of YFP-SUMO1GG drives an increased pool of the SCE1∙SIZ1 BiFC pair to 701 small puncta. 702 Supplemental Figure S6. SUMO chain formation accelerates localization of the 703 SUMO1∙SCE1 BiFC pair in nuclear bodies. 704 Supplemental Figure S7. Quantification of the co-localization of the SUMO1∙SCE1 705 BiFC pair with COP1. 706 Supplemental Figure S8. Neither mRFP-SCE1 nor SIZ1-mRFP are recruited to 707 CRY2 NBs.

23 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

708 Supplemental Table S1 SUMO1, COP1, SIZ1 and SCE1 mutants here used. 709 Supplemental Table S2 Sequences of oligonucleotides. 710 Supplemental Table S3 Clone information.

711 Acknowledgments

712 We kindly acknowledge L. Tikovsky and H. Lemereis for horticultural assistance, 713 the van Leeuwenhoek Centre for Advanced Microscopy, Section Molecular 714 Cytology (SILS-University of Amsterdam) for use of their microscopy facilities, V. 715 Hammoudi for SIZ1SP-RING. We thank D. Baulcome (University of Cambridge, UK), 716 I. Dikic (Goethe University, Frankfurt am main, Germany), Dongqing Xu (Peking 717 University, Beijing), J. Haas (Ludwig-Maximilians-University of Munich, Germany), 718 J. Kudla (WWU Muenster, Germany), Klaus Harter (ZMBP Tübingen, Germany), T. 719 Nagakawa (Shimane University, Japan), Seong Wook Yang (University of 720 Copenhagen, Denmark), Jin Bo Lin (Chinese Academy of Sciences, Beijing, 721 China), Dae-Jin Yun (Gyeongsang National University, Korea) and Ute Höcker 722 (Universität zu Köln) for sharing plasmids and seeds. We thank the ABRC for 723 sending us the CRY1 and CRY2 cDNA clones. The Netherlands Scientific 724 Organisation (ALW-VIDI grant 864.10.004 to HvdB) and the Topsector T&U 725 program Better Plants for Demands (grant 1409-036 to HvdB), including the 726 partnering breeding companies, supported this work; FM is financially supported by 727 Keygene N.V. (The Netherlands). 728

24 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

729 Figure Legends 730 731 Fig. 1 SUMO nuclear body formation depends on SCE1 conjugation activity. 732 A, Nuclear localization pattern of the SUMO1∙SCE1 and SUMO3∙SCE1 BiFC pairs. 733 Top diagram depicts residues mutated and/or deleted in SUMO1 (GG, mature 734 SUMO; SIM, F32A+I34A; ΔGG, deletion of the C-terminal diGly motif). 735 B, Similar to (A); nuclear localization pattern of the SUMO1/3-SIZ1 BiFC pairs. 736 C, Nuclear localization pattern of the SUMO1∙SCE1 couple in response to inhibition 737 of SUMO conjugation using anacardic acid (100 μM in 1% DMSO) 1.5hrs post 738 infiltration. 739 D, Diagram depicting SCE1 mutants and their substitutions. CAT1; catalytic Cys 740 residue mutated; CAT2; binding pocket for the ψKxE SUMO acceptor motif mutated; 741 SUM1; non-covalent association of SUMO disrupted; SIZ1; SIZ1-binding disrupted. 742 E, Nuclear localization pattern of the SUMO1∙SCE1 BiFC pair; SCE1CAT1, SCE1CAT2, 743 SCE1SUM1 do not accumulate in NBs in a BiFC interaction with SUMO1GG. 744 F, Nuclear localization pattern of the SCE1∙SIZ1 BiFC pair; SCE1SUM1 and SCE1SIZ1 745 fail to interact with SIZ1. 746 G, Multicolour BiFC of SUMO1GG, SCE1 and SIZ1 showing complete co-localization 747 in NBs. The micrographs show the nuclear signal of the reconstituted 748 SCFPN/SCFPC (SCE1∙SUMO1GG) and VenusN/SCFPC (SIZ∙SUMO1GG) 749 fluorophores and their merged signals. The two chimeric BiFC couples differ in their 750 excitation and emission spectra. The BiFC pairs in A-F were fused to two halves of 751 SCFP (SCFPN+SCFPC) with the orientation of the fusions indicated. Scale bar = 20 752 µm. All micrographs were taken in N. benthamiana epidermal leaf cells 2-3 dpi 753 Agrobacterium expressing the indicated constructs; nuclei are outlined with white 754 lines. The Supplemental figure S3 depicts for the panels A-F an overlay of the DIC 755 and CFP images of the nuclei shown and a zoom-out (A’-F’) depicting the BiFC 756 signal in the entire cell. 757 758 Figure 2. The SUMO1∙SCE1 BiFC pair co-localizes with COP1 in nuclear 759 bodies when catalytically-active. 760 A, Yeast two-hybrid assay expressing COP1 as GAL4 BD fusion protein and the 761 SUMO (machinery) proteins were fused to the GAL4 AD-domain. Yeast growth was

25 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

762 scored 3 days after incubation on selective media at 30°C (-LW, -LWH, -LWH+ 1mM 763 3AT, -LWHA). 764 B and C, Nuclear localization pattern of the SUMO1∙SCE1 BiFC pair in cells 765 overexpressing RFP-COP1. Only wild type SCE1 (WT) with SUMO1GG (GG) co- 766 localizes as BiFC couple with COP1 in NBs. B: ΔGG, conjugation-deficient SUMO 767 variant; C: CAT1, Catalytically-dead SCE1. Micrographs show from top-to-bottom 768 the reconstituted BiFC signal, RFP-COP1 and their merged signals. Conditions 769 were identical to Fig 1. Scale bar = 10 µm. 770 771 Figure 3. The COP1-SIZ1 interaction in NBs depends on both the substrate 772 binding pocket and the SUMO acceptor site in COP1. 773 A, Diagram depicting COP1 variants and the protein-protein interaction domain 774 disrupted (left side, red text) with their mutations shown: RING, RING Zn2+-finger 775 binding domain mutant; SUMO, loss of SUMO acceptor site; SUBSTRATE, 776 mutation in the substrate binding groove; CUL4, mutations in the CUL4-binding 777 ‘WDRX’ motifs. 778 B, Nuclear localization pattern of the SUMO1∙SCE1 BiFC couple in cells 779 overexpressing functional mutants of RFP-COP1. Micrographs depict from top-to- 780 bottom the BiFC CFP signal, RFP-COP1, and their combined signals. Bottom row 781 depicts a scatter plot of the RFP/CFP signal intensities per pixel and their Pearson’s 782 correlation coefficient (R). In particular, the COP1 SUMO acceptor site (COP1SUMO) 783 promotes colocalization of SCE1∙SUMO1 and COP1 in the same NBs. Conditions 784 were identical to Fig 1. Scale bar = 10 µm. 785 C, Normalized intensity profiles depict the fluorescence signal intensities for SCFP: 786 (BiFC pair) and RFP-COP1: co-expression of (1) WT COP1 or (2) COP1SUMO; the 787 profiles follow the white arrows depicted in panel B. Note the profiles of RFP- 788 COP1SUMO and SUMO∙SCE1 CFP signals poorly overlap in (2). 789 D, Mapping of the SIZ1-interaction site in COP1 using the yeast 2-hybrid assay. The 790 COP1 variants were fused to the GAL4 BD; SIZ1 was fused to the GAL4 AD-fusion. 791 Yeast growth was scored 3 days after incubation on selective medium at 30°C. 792 793 Figure 4. Formation of the multiparty COP1+SUMO1∙SIZ1 NBs depends on the 794 COP1 substrate binding pocket that apparently recruits SIZ1 via VP-motifs.

26 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

795 A, Nuclear localization pattern of the SUMO1∙SIZ1 BiFC couple in cells 796 overexpressing RFP-COP1 variants. The COP1 substrate-binding pocket is 797 essential for COP1 NB formation, while co-localization of the SUMO1GG∙SIZ1 BiFC 798 pair with RFP-COP1 depends on both the SUMO acceptor site (SUMO) and the 799 substrate binding pocket of COP1 (SUBSTRATE). See Fig. 4A for details on the 800 COP1 variants. Micrographs show from top-to-bottom the BiFC signal, RFP-COP1, 801 and their merged signals. Conditions were identical to Fig 1. Scale bar = 10 µm. 802 B, Diagram depicting SIZ1 variants and the protein-protein interaction domain 803 disrupted (left side) by the mutations introduced: PHD and PINIT, both reduced 804 substrate binding; SP-RING, no interaction with SCE1; VP1 and VP2, putative 805 interacting motifs for the COP1 substrate binding groove. 806 C, Mapping of the COP1-interaction site in SIZ1 using the yeast 2-hybrid assay. The 807 COP1-SIZ1 interaction depends on an intact SP-RING and VP2 domain in SIZ1. 808 Similar to Fig. 3, SIZ1 variants were fused to GAL4 AD-fusion, while COP1 was 809 fused to GAL4 BD; Yeast growth was scored after 3 days at 30°C. 810 811 Figure 5. Co-localization of GFP-tagged SCE1/SIZ1 with RFP-COP1 NBs is 812 compromised when the SUMO acceptor site is mutated in COP1. 813 A, Nuclear localization pattern of GFP-tagged SCE1 and SIZ1 is modulated by loss 814 of the SUMO acceptor site (Lys193Arg, COP1SUMO) in RFP-COP1. Remarkably, 815 GFP-SIZ1 forms together with RFP-COP1SUMO amorphous NBs (marked with *). 816 Micrographs from top-to-bottom: GFP, RFP and their merged signals. Scale bar = 817 10 µm. 818 B, Quantification of the average GFP signal intensity in the NBs per nucleus divided 819 by the average fluorescence signal in the nucleus (with the data of 3 biological 820 replicates pooled with at least 5 nuclei per replica). Total number of nuclei analyzed 821 is shown. Significant differences were detected using an unpaired Student’s T-test 822 assuming unequal variances; ** p < 0.01, * p < 0.05. Conditions were identical to 823 Fig. 1. 824 825 Figure 6. SUMO conjugation triggers also NB formation in Arabidopsis, which 826 depends genetically on SIZ1 for recruitment to COP1 NBs.

27 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

827 A, Nuclear localization pattern of SCE1∙SCE1 BiFC pair in Arabidopsis (wild type, 828 siz1-2 or cop1-4) using particle bombardment. The BiFC constructs and the used 829 Arabidopsis genotypes are indicated at the top; to detect the transformed cells, 830 mCherry was co-bombarded. Again SUMO1GG∙SCE1 is effectively targeted to NBs 831 in Arabidopsis independent of the SIZ1 and COP1 genes. The micrographs show 832 from top to bottom the YFP, RFP and the merged signal; nuclei are outline with a 833 white line. 4-week-old Arabidopsis rosettes were bombarded. Two days post 834 bombardment, plant material was transferred to a dark box and 1 day later the 835 fluorescence signals were examined. 836 B, Nuclear localization pattern of GFP-COP1 and RFP-SCE1 in Arabidopsis (wild 837 type, siz1-2 or sumo1;amiR-SUMO2) using particle bombardment. To detect the 838 transformed cells, either mCherry was co-bombarded or RFP-SCE1 was used. 839 COP1 localizes to NBs independent of SIZ1 or SUMO conjugation. Micrographs 840 with nuclei with GFP-COP1 containing NBs are shown. Scale bar = 10 µm. 841 842 Figure 7. CRY1-mRFP or CRY2-mRFP and the SUMO1∙SCE1 BiFC complex, 843 meet in COP1 NBs. 844 Micrographs of cells transiently expressing the SCFPN-SCE1 and SCFPC- 845 SUMO1GG with or without YFP-COP1 and either CRY1-mRFP or CRY2-mRFP 3 846 days after Agrobacterium infiltration. Note that neither CRY1-mRFP nor CRY2- 847 mRFP are recruited to SCE1∙SUMO1GG NBs. Note that SCE1∙SUMO1GG and 848 CRY1-mRFP or CRY2-mRFP are recruited together to YFP-COP1 NBs. Scale bar 849 = 10 µm 850

28 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

851 References

852 Ballesteros ML, Bolle C, Lois LM, Moore JM, Vielle-Calzada JP, Grossniklaus U, 853 Chua NH (2001) LAF1, a MYB transcription activator for phytochrome A 854 signaling. Genes Dev 15: 2613-2625 855 Bencsath KP, Podgorski MS, Pagala VR, Slaughter CA, Schulman BA (2002) 856 Identification of a multifunctional binding site on Ubc9p required for Smt3p 857 conjugation. J Biol Chem 277: 47938-47945 858 Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for 859 E2-mediated SUMO conjugation revealed by a complex between ubiquitin- 860 conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345-356 861 Bruderer R, Tatham MH, Plechanovova A, Matic I, Garg AK, Hay RT (2011) 862 Purification and identification of endogenous polySUMO conjugates. EMBO Rep. 863 12: 142-148 864 Castaño-Miquel L, Segui J, Manrique S, Teixeira I, Carretero-Paulet L, Atencio F, 865 Lois LM (2013) Diversification of SUMO-activating enzyme in Arabidopsis: 866 implications in SUMO conjugation. Mol Plant 6: 1646-1660 867 Chen H, Huang X, Gusmaroli G, Terzaghi W, Lau OS, Yanagawa Y, Zhang Y, Li J, Lee 868 JH, Zhu D, Deng XW (2010) Arabidopsis CULLIN4-damaged DNA binding 869 protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1- 870 SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and 871 flowering time. Plant Cell 22: 108-123 872 Cheong MS, Park HC, Bohnert HJ, Bressan RA, Yun DJ (2010) Structural and 873 functional studies of SIZ1, a PIAS-type SUMO E3 ligase from Arabidopsis. Plant 874 Signal Behav 5: 567-569 875 Cheong MS, Park HC, Hong MJ, Lee J, Choi W, Jin JB, Bohnert HJ, Lee SY, Bressan 876 RA, Yun DJ (2009) Specific domain structures control abscisic acid-, salicylic 877 acid-, and stress-mediated SIZ1 phenotypes. Plant Physiol 151: 1930-1942 878 Colby T, Matthai A, Boeckelmann A, Stuible HP (2006) SUMO-conjugating and 879 SUMO-deconjugating enzymes from Arabidopsis. Plant Physiol 142: 318-332 880 Conti L, Nelis S, Zhang CJ, Woodcock A, Swarup R, Galbiati M, Tonelli C, Napier R, 881 Hedden P, Bennett M, Sadanandom A (2014) Small Ubiquitin-like Modifier 882 protein SUMO enables plants to control growth independently of the 883 phytohormone Gibberellin. Dev Cell 28: 102-110 884 Conti L, Price G, O'Donnell E, Schwessinger B, Dominy P, Sadanandom A (2008) 885 Small ubiquitin-like modifier proteases OVERLY TOLERANT TO SALT1 and -2 886 regulate salt stress responses in Arabidopsis. Plant Cell 20: 2894-2908 887 de Folter S, Immink RG (2011) Yeast protein-protein interaction assays and screens. 888 Methods Mol Biol 754: 145-165 889 Delker C, Sonntag L, James GV, Janitza P, Ibanez C, Ziermann H, Peterson T, Denk 890 K, Mull S, Ziegler J, Davis SJ, Schneeberger K, Quint M (2014) The DET1- 891 COP1-HY5 pathway constitutes a multipurpose signaling module regulating 892 plant photomorphogenesis and thermomorphogenesis. Cell Rep 9: 1983-1989 893 Elrouby N, Bonequi MV, Porri A, Coupland G (2013) Identification of Arabidopsis 894 SUMO-interacting proteins that regulate chromatin activity and developmental 895 transitions. Proc Natl Acad Sci U S A 110: 19956-19961 896 Flotho A, Melchior F (2013) Sumoylation: a regulatory protein modification in health 897 and disease. Annu Rev Biochem 82: 357-385

29 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

898 Fukuda I, Ito A, Hirai G, Nishimura S, Kawasaki H, Saitoh H, Kimura K, Sodeoka M, 899 Yoshida M (2009) Ginkgolic Acid Inhibits Protein SUMOylation by Blocking 900 Formation of the E1-SUMO Intermediate. Chem Biol 16: 133-140 901 Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape 902 specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11: 861-871 903 Gehl C, Waadt R, Kudla J, Mendel RR, Hansch R (2009) New GATEWAY vectors for 904 high throughput analyses of protein-protein interactions by bimolecular 905 fluorescence complementation. Mol Plant 2: 1051-1058 906 Guo L, Giasson BI, Glavis-Bloom A, Brewer MD, Shorter J, Gitler AD, Yang X (2014) 907 A cellular system that degrades misfolded proteins and protects against 908 neurodegeneration. Mol cell 55: 15-30 909 Hammoudi V, Fokkens L, Beerens B, Vlachakis G, Chatterjee S, Arroyo-Mateos M, 910 Wackers PFK, Jonker MJ, van den Burg HA (2018) The Arabidopsis SUMO E3 911 ligase SIZ1 mediates the temperature dependent trade-off between plant 912 immunity and growth. PLoS Genet 14: e1007157 913 Hammoudi V, Vlachakis G, Schranz ME, van den Burg HA (2016) Whole-genome 914 duplications followed by tandem duplications drive diversification of the 915 protein modifier SUMO in Angiosperms. New Phytol 211: 172-185 916 Hattersley N, Shen L, Jaffray EG, Hay RT (2011) The SUMO protease SENP6 is a direct 917 regulator of PML nuclear bodies. Mol Biol Cell 22: 78-90 918 Hecker CM, Rabiller M, Haglund K, Bayer P, Dikic I (2006) Specification of SUMO1- 919 and SUMO2-interacting motifs. J Biol Chem 281: 16117-16127 920 Hendriks IA, D'Souza RC, Yang B, Verlaan-de Vries M, Mann M, Vertegaal AC 921 (2014) Uncovering global SUMOylation signaling networks in a site-specific 922 manner. Nat Struct Mol Biol 21: 927-936 923 Höcker U (2017) The activities of the E3 ubiquitin ligase COP1/SPA, a key repressor 924 in light signaling. Curr Opin Plant Biol 37: 63-69 925 Holm M, Ma LG, Qu LJ, Deng XW (2002) Two interacting bZIP proteins are direct 926 targets of COP1-mediated control of light-dependent gene expression in 927 Arabidopsis. Genes Dev 16: 1247-1259 928 Holtkotte X, Ponnu J, Ahmad M, Höcker U (2017) The blue light-induced interaction 929 of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light 930 signaling. PLoS Genet 13: e1007044 931 Jentsch S, Psakhye I (2013) Control of nuclear activities by substrate-selective and 932 protein-group SUMOylation. Annu Rev Genet 47: 167-186 933 Kaur K, Park H, Pandey N, Azuma Y, De Guzman RN (2017) Identification of a new 934 small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy. J 935 Biol Chem 292: 10230-10238 936 Kim JY, Jang IC, Seo HS (2016) COP1 controls abiotic stress responses by modulating 937 AtSIZ1 function through its E3 Ubiquitin ligase activity. Front Plant Sci 7: 1182 938 Kim JY, Song JT, Seo HS (2017) COP1 regulates plant growth and development in 939 response to light at the post-translational level. J Exp Bot 68: 4737-4748 940 Knipscheer P, van Dijk WJ, Olsen JV, Mann M, Sixma TK (2007) Noncovalent 941 interaction between Ubc9 and SUMO promotes SUMO chain formation. EMBO J 942 26: 2797-2807 943 Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA 944 (2009) High temperature-medated adaptations in plant architecture require 945 the bHLH transcription factor PIF4. Current Biology 19: 408-413

30 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

946 Lamoliatte F, McManus FP, Maarifi G, Chelbi-Alix MK, Thibault P (2017) 947 Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using 948 sequential peptide immunopurification. Nat Commun 8: 14109 949 Lau OS, Deng XW (2012) The photomorphogenic repressors COP1 and DET1: 20 years 950 later. Trends Plant Sci 17: 584-593 951 Lin XL, Niu D, Hu ZL, Kim DH, Jin YH, Cai B, Liu P, Miura K, Yun DJ, Kim WY, Lin R, 952 Jin JB (2016) An Arabidopsis SUMO E3 ligase, SIZ1, negatively regulates 953 photomorphogenesis by promoting COP1 activity. PLoS Genet 12: e1006016 954 Liu B, Zuo Z, Liu H, Liu X, Lin C (2011) Arabidopsis cryptochrome 1 interacts with 955 SPA1 to suppress COP1 activity in response to blue light. Genes Dev 25: 1029- 956 1034 957 Lois LM, Lima CD, Chua NH (2003) Small ubiquitin-like modifier modulates abscisic 958 acid signaling in Arabidopsis. Plant Cell 15: 1347-1359 959 Lopez-Molina L, Mongrand S, Kinoshita N, Chua NH (2003) AFP is a novel negative 960 regulator of ABA signaling that promotes ABI5 protein degradation. Genes Dev 961 17: 410-418 962 Mas P, Devlin PF, Panda S, Kay SA (2000) Functional interaction of phytochrome B 963 and cryptochrome 2. Nature 408: 207-211 964 Mascle XH, Lussier-Price M, Cappadocia L, Estephan P, Raiola L, Omichinski JG, 965 Aubry M (2013) Identification of a non-covalent ternary complex formed by 966 PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation. J Biol 967 Chem 288: 36312-36327 968 Mazur MJ, Spears BJ, Djajasaputra A, van der Gragt M, Vlachakis G, Beerens B, 969 Gassmann W, van den Burg HA (2017) Arabidopsis TCP Transcription Factors 970 Interact with the SUMO Conjugating Machinery in Nuclear Foci. Front Plant Sci 971 8: 2084 972 Miller MJ, Barrett-Wilt GA, Hua Z, Vierstra RD (2010) Proteomic analyses identify a 973 diverse array of nuclear processes affected by small ubiquitin-like modifier 974 conjugation in Arabidopsis. Proc Natl Acad Sci USA 107: 16512-16517 975 Miura K, Lee J, Jin JB, Yoo CY, Miura T, Hasegawa PM (2009) Sumoylation of ABI5 976 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid 977 signaling. Proc Natl Acad Sci USA 106: 5418-5423 978 Murtas G, Reeves PH, Fu YF, Bancroft I, Dean C, Coupland G (2003) A nuclear 979 protease required for flowering-time regulation in Arabidopsis reduces the 980 abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates. Plant Cell 981 15: 2308-2319 982 Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, 983 Tanaka K, Niwa Y, Watanabe Y, Nakamura K, Kimura T, Ishiguro S (2007) 984 Improved Gateway binary vectors: high-performance vectors for creation of 985 fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 986 71: 2095-2100 987 Paik I, Kathare PK, Kim JI, Huq E (2017) Expanding Roles of PIFs in Signal Integration 988 from Multiple Processes. Mol Plant 10: 1035-1046 989 Park YJ, Lee HJ, Ha JH, Kim JY, Park CM (2017) COP1 conveys warm temperature 990 information to hypocotyl thermomorphogenesis. New Phytol 215: 269-280 991 Perry JJ, Tainer JA, Boddy MN (2008) A SIM-ultaneous role for SUMO and ubiquitin. 992 Trends Biochem Sci 33: 201-208

31 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

993 Quint M, Delker C, Franklin KA, Wigge PA, Halliday KJ, van Zanten M (2016) 994 Molecular and genetic control of plant thermomorphogenesis. Nature Plants 2: 995 15190 996 Reverter D, Lima CD (2005) Insights into E3 ligase activity revealed by a SUMO- 997 RanGAP1-Ubc9-Nup358 complex. Nature 435: 687-692 998 Rytz TC, Miller MJ, McLoughlin F, Augustine RC, Marshall RS, Juan YT, Charng YY, 999 Scalf M, Smith LM, Vierstra RD (2018) SUMOylome Profiling Reveals a Diverse 1000 Array of Nuclear Targets Modified by the SUMO Ligase SIZ1 during Heat Stress. 1001 Plant Cell 30: 1077-1099 1002 Sadanandom A, Adam E, Orosa B, Viczian A, Klose C, Zhang CJ, Josse EM, Kozma- 1003 Bognar L, Nagy F (2015) SUMOylation of phytochrome-B negatively regulates 1004 light-induced signaling in Arabidopsis thaliana. Proc Natl Acad Sci USA 112: 1005 11108-11113 1006 Sambrook J, Russel DW (2001) Molecular Cloning: A Laboratory Manual,. Cold Spring 1007 Harbor Laboratory Press Volume 1 1008 Saracco SA, Miller MJ, Kurepa J, Vierstra RD (2007) Genetic analysis of SUMOylation 1009 in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is 1010 essential. Plant Physiol 145: 119-134 1011 Schütze K, Harter K, Chaban C (2009) Bimolecular fluorescence complementation 1012 (BiFC) to study protein-protein interactions in living plant cells. Methods Mol 1013 Biol 479: 189-202 1014 Schweizer P, Christoffel A, Dudler R (1999) Transient expression of members of the 1015 germin-like gene family in epidermal cells of wheat confers disease resistance. 1016 Plant J 20: 540-552 1017 Sekhri P, Tao T, Kaplan F, Zhang XD (2015) Characterization of amino acid residues 1018 within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear 1019 localization. Biochem Biophys Res Commun 458: 128-133 1020 Sekiyama N, Ikegami T, Yamane T, Ikeguchi M, Uchimura Y, Baba D, Ariyoshi M, 1021 Tochio H, Saitoh H, Shirakawa M (2008) Structure of the small ubiquitin-like 1022 modifier (SUMO)-interacting motif of MBD1-containing chromatin-associated 1023 factor 1 bound to SUMO-3. J Biol Chem 283: 35966-35975 1024 Seo HS, Yang JY, Ishikawa M, Bolle C, Ballesteros ML, Chua NH (2003) LAF1 1025 ubiquitination by COP1 controls photomorphogenesis and is stimulated by 1026 SPA1. Nature 423: 995-999 1027 Shalitin D, Yang H, Mockler TC, Maymon M, Guo H, Whitelam GC, Lin C (2002) 1028 Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent 1029 phosphorylation. Nature 417: 763-767 1030 Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P (1999) Cell- 1031 autonomous complementation of mlo resistance using a biolistic transient 1032 expression system. Plant J 17: 293-299 1033 Song J, Zhang Z, Hu W, Chen Y (2005) Small ubiquitin-like modifier (SUMO) 1034 recognition of a SUMO binding motif: a reversal of the bound orientation. J Biol 1035 Chem 280: 40122-40129 1036 Stacey MG, Hicks SN, von Arnim AG (1999) Discrete domains mediate the light- 1037 responsive nuclear and cytoplasmic localization of Arabidopsis COP1. Plant Cell 1038 11: 349-364 1039 Stacey MG, von Arnim AG (1999) A novel motif mediates the targeting of the 1040 Arabidopsis COP1 protein to subnuclear foci. J Biol Chem 274: 27231-27236

32 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1041 Streich FC, Jr., Lima CD (2016) Capturing a substrate in an activated RING E3/E2- 1042 SUMO complex. Nature 536: 304-308 1043 Tan CM, Li MY, Yang PY, Chang SH, Ho YP, Lin H, Deng WL, Yang JY (2015) 1044 Arabidopsis HFR1 Is a potential nuclear substrate regulated by the 1045 Xanthomonas type III effector XopD(Xcc8004). PLOS One 10: e0117067 1046 Tatham MH, Matic I, Mann M, Hay RT (2011) Comparative proteomic analysis 1047 identifies a role for SUMO in protein quality control. Sci Signal 4: rs4 1048 Tomanov K, Nehlin L, Ziba I, Bachmair A (2018) SUMO chain formation relies on the 1049 amino-terminal region of SUMO-conjugating enzyme and has dedicated 1050 substrates in plants. Biochem J 475: 61-74 1051 Tomanov K, Zeschmann A, Hermkes R, Eifler K, Ziba I, Grieco M, Novatchkova M, 1052 Hofmann K, Hesse H, Bachmair A (2014) Arabidopsis PIAL1 and 2 promote 1053 SUMO chain formation as E4-type SUMO ligases and are involved in stress 1054 responses and sulfur metabolism. Plant Cell 26: 4547-4560 1055 Uljon S, Xu X, Durzynska I, Stein S, Adelmant G, Marto JA, Pear WS, Blacklow SC 1056 (2016) Structural Basis for Substrate Selectivity of the E3 Ligase COP1. 1057 Structure 24: 687-696 1058 Ulrich HD (2008) The fast-growing business of SUMO chains. Mol Cell 32: 301-305 1059 Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signaling. Plant 1060 Physiol 158: 52-60 1061 Van den Burg HA, Kini RK, Schuurink RC, Takken FLW (2010) Arabidopsis Small 1062 Ubiquitin-like Modifier paralogs have distinct functions in development and 1063 defense. Plant Cell 22: 1998-2016 1064 Vertegaal ACO (2007) Small ubiquitin-related modifiers in chains. Biochem Soc Trans 1065 35: 1422-1423 1066 Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression 1067 system in plants based on suppression of gene silencing by the p19 protein of 1068 tomato bushy stunt virus. Plant J 33: 949-956 1069 Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Nake C, Blazevic D, 1070 Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization 1071 of protein interactions in living plant cells using bimolecular fluorescence 1072 complementation. Plant J 40: 428-438 1073 Wang H, Ma L-G, Li J-M, Zhao H-Y, Deng XW (2001) Direct Interaction of Arabidopsis 1074 Cryptochromes with COP1 in Light Control Development. Science 294: 154-158 1075 Xiong R, Wang A (2013) SCE1, the SUMO-conjugating enzyme in plants that interacts 1076 with NIb, the RNA-dependent RNA polymerase of Turnip mosaic virus, is 1077 required for viral infection. J Virol 87: 4704-4715 1078 Yang SH, Sharrocks AD (2010) The SUMO E3 ligase activity of Pc2 is coordinated 1079 through a SUMO interaction motif. Mol Cell Biol 30: 2193-2205 1080 Yunus AA, Lima CD (2006) Lysine activation and functional analysis of E2-mediated 1081 conjugation in the SUMO pathway. Nat Struct Mol Biol 13: 491-499 1082 Yunus AA, Lima CD (2009) Structure of the Siz/PIAS SUMO E3 ligase Siz1 and 1083 determinants required for SUMO modification of PCNA. Mol Cell 35: 669-682 1084 Zhu J, Zhu S, Guzzo CM, Ellis NA, Sung KS, Choi CY, Matunis MJ (2008) Small 1085 ubiquitin-related modifier (SUMO) binding determines substrate recognition 1086 and paralog-selective SUMO modification. J Biol Chem 283: 29405-29415 1087 1088

33 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1089

34 bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. FIGURE 1 A F32A/I34A ΔG92/G93 SIM ΔGG SUMO1 1 93 SCFPC-SCE1 N SCFP -: SUMO1 SUMO3 GG ΔGG GG+SIM ΔGG+SIM GG

CFP

B SIZ1-SCFPC SCFPN-: SUMO1 SUMO3 GG ΔGG GG+SIM ΔGG+SIM GG

CFP

C SCFPC-SCE1 SCFPN-SUMO1: GG ΔGG DMSO Anarcadic acid DMSO Anarcadic acid

CFP

C94S D R14E/R18E/H20D P70A CAT1 P106A SUM1 SIZ1 SIZ1 SCE1 1 160 CAT2 CAT2 Y87A/S89A/C94S D129A E SCFPC-SUMO1 SCFPN-SCE1: WT CAT1 CAT2 SUM1 SIZ1

CFP

F SIZ1-SCFPC N SCFP -SCE1: WT CAT1 CAT2 SUM1 SIZ1

CFP

G SCFPC-SUMO1GG SCFP N - SCE1 SIZ1-VenusN

CFP chimeric

(em. 480 nm) (em. 515 nm) merge bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1. SUMO nuclear body formation depends on SCE1 conjugation activity. A, Nuclear localization pattern of the SUMO1∙SCE1 and SUMO3∙SCE1 BiFC pairs. Top diagram depicts residues mutated and/or deleted in SUMO1 (GG, mature SUMO; SIM, F32A+I34A; ΔGG, deletion of the C-terminal diGly motif). B, Similar to (A); nuclear localization pattern of the SUMO1/3-SIZ1 BiFC pairs. C, Nuclear localization pattern of the SUMO1∙SCE1 couple in response to inhibition of SUMO conjugation using anacardic acid (100 μM in 1% DMSO) 1.5hrs post infiltration. D, Diagram depicting SCE1 mutants and their substitutions. CAT1; catalytic Cys residue mutated; CAT2; binding pocket for the ψKxE SUMO acceptor motif mutated; SUM1; non-covalent association of SUMO disrupted; SIZ1; SIZ1-binding disrupted. E, Nuclear localization pattern of the SUMO1∙SCE1 BiFC pair; SCE1CAT1, SCE1CAT2, SCE1SUM1 do not accumulate in NBs in a BiFC interaction with SUMO1GG. F, Nuclear localization pattern of the SCE1∙SIZ1 BiFC pair; SCE1SUM1 and SCE1SIZ1 fail to interact with SIZ1. G, Multicolour BiFC of SUMO1GG, SCE1 and SIZ1 showing complete co-localization in NBs. The micrographs show the nuclear signal of the reconstituted SCFPN/SCFPC (SCE1∙SUMO1GG) and VenusN/SCFPC (SIZ∙SUMO1GG) fluorophores and their merged signals. The two chimeric BiFC couples differ in their excitation and emission spectra. The BiFC pairs in A-F were fused to two halves of SCFP (SCFPN+SCFPC) with the orientation of the fusions indicated. Scale bar = 20 µm. All micrographs were taken in N. benthamiana epidermal leaf cells 2-3 dpi Agrobacterium expressing the indicated constructs; nuclei are outlined with white lines. The Supplemental figure S3 depicts for the panels A-F an overlay of the DIC and CFP images of the nuclei shown and a zoom-out (A’-F’) depicting the BiFC signal in the entire cell. bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 2

A B C RFP-COP1 RFP-COP1

C N BD-COP1 6&)3SCE1 6&)3SUMO1  N ** ** SCFP -SUMO1 GG у** -SCE1 WT CAT1 AD: HY SUMO1 SUMO3 6&( 6,= CFP -LW (BiFC)

-LWH

-LWH+3AT RFP (COP1) -LWHA LQWHUDFWLRQVWUHQJKW

CFP + RFP

/ECPMA'%"'8DA'7924$V7-.$',E/-'L=EM'?K!HK?=HEUAN'REOD'-45$'EJ'JP?HA=M'>K@EAN' RDAJ'?=O=HTOE?=HHT!=?OEQA"' 3$# JPL^_# _bZ%SdM]TO# L^^Ld# Pc[]P^^TYR# 5@A)# L^# 93=,# 46# Q`^TZY# []Z_PTY# LYO# _SP# DF>@# XLNSTYP]d!#[]Z_PTY^#bP]P#Q`^PO#_Z#_SP#93=,#36%OZXLTY&#JPL^_#R]Zb_S#bL^# ^NZ]PO#+#OLd^#LQ_P]#TYN`ML_TZY#ZY#^PWPN_TaP#XPOTL#L_#+(f5# %=H$#%=H:$#%=H:##)X># +3E$#%=H:3!&# 4# LYO# 5$# ?`NWPL]# WZNLWTeL_TZY# [L__P]Y# ZQ# _SP# DF>@)jD57)# 4T85# [LT]# TY# NPWW^# ZaP]Pc[]P^^TYR# C8A%5@A)&# @YWd# bTWO# _d[P# D57)# HE!# bT_S# DF>@)99# 99!# NZ% WZNLWTeP^#L^#4T85#NZ`[WP#bT_S#5@A)#TY#?4^/#k99$#NZYU`RL_TZY%OPQTNTPY_#DF>@# aL]TLY_0#5/#53E)$#5L_LWd_TNLWWd%OPLO#D57)&#>TN]ZR]L[S^#^SZb#Q]ZX#_Z[%_Z%MZ__ZX#_SP# ]PNZY^_T_`_PO# 4T85# ^TRYLW$# C8A%5@A)# LYO# _SPT]# XP]RPO# ^TRYLW^&# 5ZYOT_TZY^# bP]P# TOPY_TNLW#_Z#8TR#)&#DNLWP#ML]#2#)(#µX&# bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 3 A RING Coil NLS WD-40 D -LW -LWH -LWHA 1 45 94 128209 293 314 367 627 675 WT

RING C52S/C56S/C86S/C89A SUMO K193R SUBSTRATE G524Q CUL4,1 D534A/R536A CUL4,2 D576A/K578A CUL4,1+4,2 D534A/R536A/D576A/K578A EV

B SCFP N -SUMO1 + SCFP C -SCE1 RFP-COP1: WT SUMO RING SUBSTRATE CUL4,1

CFP (BiFC)

RFP (COP1)

CFP + 1 RFP 2

R = 0.882 R = 0.490 R = 0.866 R = 0.188 R = 0.177 Scatter plot + Pearson’s R

C 1 1 COP1 2 COP1SUMO bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

/ECPMA' &"' 8DA' -45$!71<$' EJOAM=?OEKJ' EJ' 3,N' @ALAJ@N' KJ' >KOD' ODA' NP>NOM=OA' >EJ@EJC'LK?GAO'=J@'ODA'7924'=??ALOKM'NEOA'EJ'-45$"' 3$# 6TLR]LX# OP[TN_TYR# 5@A)# aL]TLY_^# LYO# _SP# []Z_PTY%[]Z_PTY# TY_P]LN_TZY# OZXLTY# OT^]`[_PO# WPQ_# ^TOP$# ]PO# _Pc_!# bT_S# _SPT]# X`_L_TZY^# ^SZbY/# 1+.*=) C;?9# KY*#%QTYRP]# MTYOTYR#OZXLTY#X`_LY_0#24-/$#WZ^^#ZQ#DF>@#LNNP[_Z]#^T_P0#24'231&3)$#X`_L_TZY# TY#_SP#^`M^_]L_P#MTYOTYR#R]ZZaP0#(4,$$#X`_L_TZY^#TY#_SP#5F=,%MTYOTYR#hH6CIi#XZ_TQ^&# 4$# ?`NWPL]# WZNLWTeL_TZY# [L__P]Y# ZQ# _SP# DF>@)jD57)# 4T85# NZ`[WP# TY# NPWW^# ZaP]Pc[]P^^TYR# Q`YN_TZYLW# X`_LY_^# ZQ# C8A%5@A)&# >TN]ZR]L[S^# OP[TN_# Q]ZX# _Z[%_Z% MZ__ZX# _SP# 4T85# 58A# ^TRYLW$# C8A%5@A)$# LYO# _SPT]# NZXMTYPO# ^TRYLW^&# 4Z__ZX# ]Zb# OP[TN_^#L#^NL__P]#[WZ_#ZQ#_SP#C8A'58A#^TRYLW#TY_PY^T_TP^#[P]#[TcPW#LYO#_SPT]#APL]^ZYi^# NZ]]PWL_TZY#NZPQQTNTPY_# C!&#;Y#[L]_TN`WL]$#_SP#5@A)#DF>@#LNNP[_Z]#^T_P# 5@A)24-/!# []ZXZ_P^# NZWZNLWTeL_TZY# ZQ# D57)jDF>@)# LYO# 5@A)# TY# _SP# ^LXP# ?4^&# 5ZYOT_TZY^# bP]P#TOPY_TNLW#_Z#8TR#)&#DNLWP#ML]#2#)(#µX&# 5$#?Z]XLWTePO#TY_PY^T_d#[]ZQTWP^#OP[TN_#_SP#QW`Z]P^NPYNP#^TRYLW#TY_PY^T_TP^#QZ]#D58A/# 4T85# [LT]!# LYO# C8A%5@A)/# NZ%Pc[]P^^TZY# ZQ# )!# HE# 5@A)# Z]# *!# 5@A)DF>@0# _SP# []ZQTWP^# QZWWZb# _SP# bST_P# L]]Zb^# OP[TN_PO# TY# [LYPW# 4&# ?Z_P# _SP# []ZQTWP^# ZQ# C8A% 5@A)24-/#LYO#DF>@jD57)#58A#^TRYLW^#[ZZ]Wd#ZaP]WL[#TY# *!&## 6$#>L[[TYR#ZQ#_SP#D;K)%TY_P]LN_TZY#^T_P#TY#5@A)#`^TYR#_SP#dPL^_#*%SdM]TO#L^^Ld&#ESP# 5@A)#aL]TLY_^#bP]P#Q`^PO#_Z#_SP#93=,#460#D;K)#bL^#Q`^PO#_Z#_SP#93=,#36%Q`^TZY&# JPL^_#R]Zb_S#bL^#^NZ]PO#+#OLd^#LQ_P]#TYN`ML_TZY#ZY#^PWPN_TaP#XPOT`X#L_#+(f5&# bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4 A SCFP N -SUMO1 + SIZ1-SCFP C RFP-COP1: WT SUMO RING SUBSTRATE

CFP (BIFC)

RFP (COP1)

CFP + RFP

B C -LWH -LW -LWHA SAP PHD PINIT SP-RING VP2SIMNLS +1mM 3AT 1 35 115 166251 317 359 407 600 659 728 860 873 WT VP1 PHD C134Y PINIT I274A/I275A SP-RING C379A VP1 V251D VP2 V725D/V727D/V728D VP1/2 V251D V725D/V727D/V728D EV bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

/ECPMA''"'/KMI=OEKJ'KB'ODA'IPHOEL=MOT'-45$N7924$V71<$'3,N'@ALAJ@N'KJ'ODA' -45$'NP>NOM=OA'>EJ@EJC'LK?GAO'OD=O'=LL=MAJOHT'MA?MPEON'71<$'QE=':5!IKOEBN"'' 3$# ?`NWPL]# WZNLWTeL_TZY# [L__P]Y# ZQ# _SP# DF>@)jD;K)# 4T85# NZ`[WP# TY# NPWW^# ZaP]Pc[]P^^TYR#C8A%5@A)#aL]TLY_^&#ESP#5@A)#^`M^_]L_P%MTYOTYR#[ZNVP_#T^#P^^PY_TLW# QZ]#5@A)#?4#QZ]XL_TZY$#bSTWP#NZ%WZNLWTeL_TZY#ZQ#_SP#DF>@)99jD;K)#4T85#[LT]#bT_S# C8A%5@A)# OP[PYO^# ZY# MZ_S# _SP# DF>@# LNNP[_Z]# ^T_P# DF>@!# LYO# _SP# ^`M^_]L_P# MTYOTYR#[ZNVP_#ZQ#5@A)# DF4DEC3E7!&#DPP#8TR&#,3#QZ]#OP_LTW^#ZY#_SP#5@A)#aL]TLY_^&# >TN]ZR]L[S^#^SZb#Q]ZX#_Z[%_Z%MZ__ZX#_SP#4T85#^TRYLW$#C8A%5@A)$#LYO#_SPT]#XP]RPO# ^TRYLW^ZYOT_TZY^#bP]P#TOPY_TNLW#_Z#8TR#)&#DNLWP#ML]#2#)(#µX&# 4$# 6TLR]LX# OP[TN_TYR# D;K)# aL]TLY_^# LYO# _SP# []Z_PTY%[]Z_PTY# TY_P]LN_TZY# OZXLTY# OT^]`[_PO# WPQ_# ^TOP!# Md# _SP# X`_L_TZY^# TY_]ZO`NPO/# A:6# LYO# A;?;E$# MZ_S# ]PO`NPO# ^`M^_]L_P# MTYOTYR0# DA%C;?9$# YZ# TY_P]LN_TZY# bT_S# D57)0# GA)# LYO# GA*$# [`_L_TaP# TY_P]LN_TYR#XZ_TQ^#QZ]#_SP#5@A)#^`M^_]L_P#MTYOTYR#R]ZZaP&# 5$#>L[[TYR#ZQ#_SP#5@A)%TY_P]LN_TZY#^T_P#TY#D;K)#`^TYR#_SP#dPL^_#*%SdM]TO#L^^Ld&#ESP# 5@A)%D;K)# TY_P]LN_TZY# OP[PYO^# ZY# LY# TY_LN_# DA%C;?9# LYO# GA*# OZXLTY# TY# D;K)&# DTXTWL]#_Z#8TR&#+$#D;K)#aL]TLY_^#bP]P#Q`^PO#_Z#93=,#36%Q`^TZY$#bSTWP#5@A)#bL^#Q`^PO# _Z#93=,#460#JPL^_#R]Zb_S#bL^#^NZ]PO#LQ_P]#+#OLd^#L_#+(f5&# bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.



 )  ) )  
  







)


 ) RFP

 ) &)  
   )
)  ) ) ) ) ) !)  ) ) ) 

 &)
 &)
   
 (() )

 
  
        
 
  bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

/ECPMA' ("' -K!HK?=HEU=OEKJ' KB' 0/5!O=CCA@' 7-.$#71<$' REOD' 6/5!-45$' 3,N' EN' ?KILMKIENA@'RDAJ'ODA'7924'=??ALOKM'NEOA'EN'IPO=OA@'EJ'-45$"'' 3$#?`NWPL]#WZNLWTeL_TZY#[L__P]Y#ZQ#98A%_LRRPO#D57)#LYO#D;K)#T^#XZO`WL_PO#Md#WZ^^#ZQ#_SP# DF>@#LNNP[_Z]#^T_P# =d^).+3]R$#5@A)24-/!#TY#C8A%5@A)&#CPXL]VLMWd$#98A%D;K)# QZ]X^# _ZRP_SP]# bT_S# C8A%5@A)24-/# LXZ][SZ`^# ?4^# XL]VPO# bT_S# "!&# >TN]ZR]L[S^#Q]ZX#_Z[%_Z%MZ__ZX/#98A$#C8A#LYO#_SPT]#XP]RPO#^TRYLW^&#DNLWP#ML]#2#)(#µX&# 4$#B`LY_TQTNL_TZY#ZQ#_SP#LaP]LRP#98A#^TRYLW#TY_PY^T_d#TY#_SP#?4^#[P]#Y`NWP`^#OTaTOPO#Md# _SP# LaP]LRP# QW`Z]P^NPYNP# ^TRYLW# TY# _SP# Y`NWP`^# bT_S# _SP# OL_L# ZQ# +# MTZWZRTNLW# ]P[WTNL_P^#[ZZWPO#bT_S#L_#WPL^_#-#Y`NWPT#[P]#]P[WTNL!&#EZ_LW#Y`XMP]#ZQ#Y`NWPT#LYLWdePO#T^# ^SZbY&# DTRYTQTNLY_# OTQQP]PYNP^# bP]P# OP_PN_PO# `^TYR# LY# `Y[LT]PO# D_`OPY_i^# E%_P^_# L^^`XTYR#`YP\`LW#aL]TLYNP^0#""#[#1#(&()$#"#[#1#(&(-ZYOT_TZY^#bP]P#TOPY_TNLW#_Z#8TR&# bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/393272; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

/ECPMA')"'7924'?KJFPC=OEKJ'OMECCAMN'=HNK'3,'BKMI=OEKJ'EJ'+M=>E@KLNEN 'RDE?D' @ALAJ@N'CAJAOE?=HHT'KJ' "!$'BKM'MA?MPEOIAJO'OK'-45$'3,N"'' #3$#?`NWPL]#WZNLWTeL_TZY#[L__P]Y#ZQ#D57)jD57)#4T85#[LT]#TY#3]LMTOZ[^T^# bTWO#_d[P$# B@)99jD57)#T^#PQQPN_TaPWd#_L]RP_PO#_Z#?4^#TY# 3]LMTOZ[^T^#TYOP[PYOPY_#ZQ#_SP)2+5")LYO)(/0")RPYP^&#ESP#XTN]ZR]L[S^#^SZb#Q]ZX# _Z[#_Z#MZ__ZX#_SP#J8A$#C8A#LYO#_SP#XP]RPO#^TRYLW0#Y`NWPT#L]P#Z`_WTYP#bT_S#L#bST_P#WTYP&# ,%bPPV%ZWO#3]LMTOZ[^T^#]Z^P__P^#bP]P#MZXML]OPO&#EbZ#OLd^#[Z^_#MZXML]OXPY_$#[WLY_ XL_P]TLW#bL^#_]LY^QP]]PO#_Z#L#OL]V#MZc#LYO#)#OLd#WL_P]#_SP#QW`Z]P^NPYNP#^TRYLW^#bP]P PcLXTYPO& 4$#?`NWPL]#WZNLWTeL_TZY#[L__P]Y#ZQ#98A%5@A)#LYO#C8A%D57)#TY#3]LMTOZ[^T^# bTWO#_d[P$ B@#NZYU`RL_TZY&#>TN]ZR]L[S^#bT_S#Y`NWPT bT_S#98A%5@A)#NZY_LTYTYR#?4^#L]P#^SZbY&#DNLWP#ML]#2#)(#µX& SCFP N -SCE1 + SCFP C -SUMO1GG CRY1-RFP CRY2-RFP

Figure 7 bioRxiv preprint _ YFP-COP1 _ YFP-COP1 _ YFP-COP1 not certifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. CFP doi: https://doi.org/10.1101/393272

YFP ; this versionpostedAugust22,2018.

RFP The copyrightholderforthispreprint(whichwas

CFP+ YFP+ RFP

DIC bioRxiv preprint

/ECPMA'*"'-6;$!I6/5'KM'-6;%!I6/5'=J@'ODA'7924$V7-.$',E/-'?KILHAS 'IAAO'EJ'-45$'3,N"'

? 5 99 not certifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. >TN]ZR]L[S^#ZQ#NPWW^#_]LY^TPY_Wd#Pc[]P^^TYR#_SP#D58A %D57)#LYO#D58A %DF>@) #bT_S#Z]#bT_SZ`_#J8A%5@A)#LYO# doi: PT_SP]#5CJ)%XC8A#Z]#5CJ*%XC8A#+#OLd^#LQ_P]#3R]ZMLN_P]T`X#TYQTW_]L_TZY&#?Z_P#_SL_#YPT_SP]#5CJ)%XC8A#YZ]#5CJ*% https://doi.org/10.1101/393272 XC8A#L]P#]PN]`T_PO#_Z#D57)jDF>@)99#?4^&#?Z_P#_SL_#D57)jDF>@)99#LYO#5CJ)%XC8A#Z]#5CJ*%XC8A#L]P#]PN]`T_PO# _ZRP_SP]#_Z#J8A%5@A)#?4^&#DNLWP#ML]#2#)(#µX ; this versionpostedAugust22,2018. The copyrightholderforthispreprint(whichwas