Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

The use of proximity-dependent biotin labelling (PDL) approaches coupled with mass spectrometry recently greatly advanced the identification of protein-protein interactions and study of protein complexation. PDL is based on the expression of a promiscuous biotin ligase (PBL), e.g. BirA* or a peroxidase fused to a bait protein of interest. In the presence of biotin as substrate, PBL enables covalent biotin labelling of proteins in the vicinity of the PBL-fused bait in vivo, allowing the subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact during purification. To date, PDL has not been extensively used in plants. Here we present the results of a systematic multi-lab study applying a variety of PDL approaches in several plant systems under various conditions and bait proteins. We show that TurboID is the most promiscuous variant for PDL in plants and establish protocols for its efficient application. We demonstrate the applicability of TurboID in capturing membrane protein interactomes using the Lotus japonicus symbiotically active receptor kinases RLKs NOD FACTOR RECEPTOR 5 (NFR5) and LRR-RLK SYMBIOTIC RECEPTOR-KINASE (SYMRK) as test-cases. Furthermore, we benchmark the efficiency of various PBLs using the octameric endocytic TPLATE complex and compare PDL with one-step AP-MS approaches. Our results indicate that different PDL approaches in plants may differ in signal-to-noise ratio and robustness. We present a straightforward strategy to identify both non-biotinylated as well as biotinylated proteins in plants in a single experimental setup. Finally, we provide initial evidence that this technique has potential to infer structural information of protein complexes. Our methods, tools and adjustable pipelines provide a useful resource for the plant research community.


Abstract 27
The use of proximity-dependent biotin labelling (PDL) approaches coupled with mass 28 spectrometry recently greatly advanced the identification of protein-protein interactions and 29 study of protein complexation. PDL is based on the expression of a promiscuous biotin ligase 30 (PBL), e.g. BirA* or a peroxidase fused to a bait protein of interest. In the presence of biotin 31 as substrate, PBL enables covalent biotin labelling of proteins in the vicinity of the PBL-fused 32 bait in vivo, allowing the subsequent capture and identification of interacting and neighbouring 33 proteins without the need for the protein complex to remain intact during purification. To date, 34 PDL has not been extensively used in plants. Here we present the results of a systematic multi-35 lab study applying a variety of PDL approaches in several plant systems under various 36 conditions and bait proteins. We show that TurboID is the most promiscuous variant for PDL 37 in plants and establish protocols for its efficient application. We demonstrate the applicability 38 of TurboID in capturing membrane protein interactomes using the Lotus japonicus 39 symbiotically active receptor kinases RLKs NOD FACTOR RECEPTOR 5 (NFR5) and LRR-40 RLK SYMBIOTIC RECEPTOR-KINASE (SYMRK) as test-cases. Furthermore, we 41 benchmark the efficiency of various PBLs using the octameric endocytic TPLATE complex 42 and compare PDL with one-step AP-MS approaches. Our results indicate that different PDL 43 approaches in plants may differ in signal-to-noise ratio and robustness. We present a 44 straightforward strategy to identify both non-biotinylated as well as biotinylated proteins in 45 plants in a single experimental setup. Finally, we provide initial evidence that this technique 46 has potential to infer structural information of protein complexes. Our methods, tools and 47 adjustable pipelines provide a useful resource for the plant research community. 48 49 INTRODUCTION mutations. The N-terminal DNA-binding domain was deleted to reduce its size (28 versus 35 84 kDa), which also slightly impacted on its labelling efficiency by reducing it ~2-fold. The first 85 and second-generation PBLs required approximately 18 to 24 h of labelling (and sometimes 86 even much longer) to produce detectable levels of protein biotinylation, while the TurboID 87 variants required a labelling time only in the range of 1 h or less in the various eukaryotic, non-88 plant systems tested so far [9]. 89 PDL has its own intrinsic advantages and limitations. In the presence of biotin, the bait-90 PBL fusion protein labels proximal proteins without the activation by a conditional trigger, 91 thereby keeping track of all interactions that occurred over a time-period. The ability for 92 selective capture makes the method generally insensitive to protein solubility or protein 93 complexation, with obvious applicability to membrane proteins and cytoskeletal constituents, 94 a major advantage over alternative approaches. Nevertheless, the identity of a candidate 95 interactor does not immediately imply a direct or indirect interaction with the bait but could 96 merely reflect close proximity [estimated to be ~10 to 15 nm [10]]. Furthermore, true 97 interactors (false negatives) are missed if they lack accessible primary amines. 98 So far PBLs have successfully been used in yeast [11], protozoa [12], amoebae [13], 99 embryonic stem cells [14], and xenograft tumors [15] to map a wide range of interactomes in 100 both small-scale (i.e. single bait protein) and large-scale network mapping approaches (i.e. the 101 protein interaction landscape of the centrosome-cilium interface and the organization of 102 mRNA-associated granules and bodies (mRNP complexes)) [16,17]. 103 In plants, PBLs have not been extensively used. So far, four papers describe the 104 application of PDL in plants [18][19][20][21]. In these first trials, overexpression of a first-generation 105 BirA* was used combined with long labelling times, very high biotin levels and relatively poor 106 labelling efficiencies. These results, combined with the so far non-extensive use of PDL in 107 plants could suggest that BioID variants used so far show reduced activity in plant tissues due 108 to suboptimal working temperatures with reference to their temperature-activity profiles. 109 Here, we report a systematic multi-lab study of different PDL approaches in various 110 plant systems. We provide guidelines for the use of PDL in various frequently used plant 111 models suggesting most relevant shortcomings and contingencies. Furthermore, we benchmark 112 our PDL methods studying the TPLATE protein complex. We foresee that the methods, tools 113 and produced materials will greatly benefit the research community. 114 115 116

Increased PBL-mediated biotin labelling efficiencies upon biotin administration in planta 119
In non-plant systems, supplementation of biotin is important for efficient proximity biotin 120 ligation with all the PBLs tested so far. In contrast, plants synthesize biotin endogenously and 121 thus the intracellular pool of biotin might be high enough for the PBL. In fact, free biotin has 122 been shown to accumulate in the cytosol of plant mesophyll cells to a high concentration of ca. 123 11 μM [22], while for example in yeast this is more than 10-fold lower [23]. Considering that 124 the Km of BirA* for biotin is 0.3 µM, this could, in theory, lead to efficient PDL even in the 125 absence of exogenous biotin supplementation. 126 Figure S1. GFP expression in tomato hairy root cultures produced with rhizogenic Agrobacterium. Fluorescence micrograph images of eGFP expression were obtained from primary hairy roots (~14 days) transformed rhizogenic Agrobacterium with the following expression constructs; Pro35S::eGFP-BirA*, Pro35S::eGFP-BioID2, Pro35S::eGFP-TurboID, and Pro35S::eGFP-miniTurboID. The scale bars are 500 μm. Images are representative for the four to ten independent roots selected for subcultivation and showing expression of the marker per construct. Addition of 50 μM exogenous biotin to two-weeks old hairy root cultures for 2 or 24 h was used for labelling. Arrowheads indicate the expected size of the cis-biotinylation signal. (B) Comparison of biotinylation activity in four PBL hairy root cultures from wild-type tomato expressing eGPF.BirA* (~66 kDa), eGPF.BioID2 (~56 kDa), eGPF.Turbo (~64 kDa) and eGPF.miniTurbo (~57 kDa). Gray regions in intense black areas represent saturation of the streptavidin-s680 signal and is most prominent in case of self-biotinylation activity. This is a representative experiment repeated twice and two independent root cultures were analyzed per combination.
As a control for non-bait specific biotinylation, PBL-fused eGFP was used. 136 Biotinylation, was evident as smears in streptavidin-HRP Western blot. This smear depicts 137 biotinylation of other proteins than PBLs, and will be referred to as "trans-biotinylation". As a 138 proxy of PBL activity, we used the cis-biotinylation efficiency (i.e. auto-or self-biotinylation 139 level of PBL fusions) as readout (Figure 1). Manifold faster kinetics for TurboID and 140 mTurboID over BioID and BioID2 could be observed (Figure 1). This is in line with the 141 previously reported lower catalytic activities of the latter PBLs, especially at the growth 142 conditions used (i.e. cultivation of hairy roots was performed at 22-25°C) [9]. Noteworthy, 143 only residual trans-biotinylation was observed when no exogenous biotin was added. 144 Therefore, the addition of surplus of (free) biotin seems to function as an inducing agent of 145  -BirA* (~66 kDa), eGPF-BioID2 (~56 kDa), eGPF-Turbo (~64 kDa) and eGPF-miniTurbo (~57 kDa). Overlapping signal as indicated with a black arrow denote enzyme-catalysed cis-biotinylation. Gray bands in intense black areas represent saturation of the streptavidin-s680 signal and is most prominent in case of auto-biotinylation activity. Two infiltrated tobacco leaf segments/leaves were analyzed per setup and the experiment was repeated twice with similar results. under various conditions. In this case, biotin was infiltrated directly into leaf tissue 24 h after 155 transfection and harvested 24 h post-biotin infiltration (Supplemental Figure 2A). We 156 confirmed that also in this system, the highest cis-biotinylation level was observed in the case 157 of TurboID, and supplementation of biotin was important for the efficient detection of cis-158 biotinylation (Supplemental Figure 2B). Furthermore, the overall biotinylation output in 159 tobacco leaves increased when biotin concentration increased from 50 μM to 1 mM 160 (Supplemental Figure 2B). 161 We confirmed that the R118G mutation is responsible for promiscuous labelling in 162 plants, as the wild-type BirA showed no trans-biotinylation (in the presence of 50 µM 163 exogenous biotin; Supplemental Figure 3A). Furthermore, a temperature shift from 22°C to 164 28 o C increased cis-and trans-biotinylation for both BioID and TurboID, suggesting that 165 temperature control, at least to some extent, could also be used to modulate PDL in plants 166 (Supplemental Figure 3A, see also below). 167

168
Noteworthy, the effect of temperature on TurboID activity was less apparent compared to that 169 of BioID, consistent with the temperature-dependency of the two enzymes [9]. Interestingly, 170 similar to GFP-TurboID expressed in the hairy root cultures, biotinylation reaches its highest 171 cis-biotinylation level already 2 h after biotin administration in N.benthamiana (Supplemental 172   We first co-expressed the symbiotic receptors NFR5 and SYMRK as C-terminal GFP 197 fusion proteins with a cytosolic  Western Blot analysis revealed the successful expression and pull-down of all three proteins 199 ( Figure 2A). When probing for biotinylation levels, we exclusively detected self-biotinylation 200 in case of cytosolic TurboID-GFP (Figure 2A). However, prolonged exposure of the blot 201 resulted in weak detectable bands in case of NFR5-GFP and SYMRK-GFP indicating the 202 presence of some background trans-biotinylation. To test whether trans-biotinylation of known 203 interacting proteins can be observed, we generated an NFR5-TurboID and co-expressed it with 204 SYMRK-GFP. Here, we not only detected a clear cis-biotinylation of NFR5-TurboID but also 205 trans-biotinylation of SYMRK ( Figure 2B). These data show that specific trans-biotinylation 206 occurs within a known receptor complex. 207 To test for specificity in the assay, we co-expressed NFR5-TurboID additionally with 208 the functionally unrelated transmembrane RLK BRI1, which was previously shown to not 209 interact with the NFR5/SYMRK complex using co-IP [25]. While we detected strong BRI1 210 expression, this protein was only weakly trans-biotinylated by NFR5-TurboID indicating some 211 unspecific labelling or that the protein is in proximity to the complex ( Figure 2B). To broaden 212 this, we also co-expressed the transmembrane proteins FLS2, EFR and LTI6b with NFR5-213 TurboID. While no bands were detected for EFR and LTI6b, we observed some weak signal 214 for FLS2 indicating that this receptor may locate in close proximity to NFR5 ( Figure 2B). 215 Prolonged exposure of the blots yielded some weak signal for all membrane proteins as also 216 observed for GFP-TurboID (data not shown). However, these signals were orders of 217 magnitude lower than those detected for NFR5/SYMRK. This assay was further optimized by 218 temporally limiting the reaction. We could show that trans-biotinylation efficiently occurs 219 within 15 min after applying exogenous biotin, demonstrating that specificity is maintained by 220 minimizing the availability of the substrate (Supplemental Figure 4). 221 It should also be considered that in addition to ectopic expression of the constructs, the weak 222 dimerization potential of GFP here or other protein tags with similar properties may result in 223 potentially unspecific trans-biotinylation. 224 Taken together these data clearly show that TurboID-mediated PDL can be efficiently 225 used for membrane proteins. It can be advantageous over other methods such as co-226 immunoprecipitation as it does not require any optimization of the solubilization conditions 227 and it provides the possibility to detected transiently protein complex constituents. 228 Figure S4. Temporally limiting the reaction results in weak but specifically detectable bands in case of NFR5-TurboID and SYMRK-GFP. Biotin was applied for 15 or 30 min. IP= immunoprecipitation; WB= Western Blot.

Application of PDL in Arabidopsis thaliana cell cultures using the TPLATE complex as a 229
case study 230 Next, we surveyed the efficiency of trans-biotinylation for a stable multi-subunit plant protein 231 complex. As a test case, we took the plasma membrane-associated octameric TPLATE 232 complex ( In order to compare the effect of temperature on the biotinylation efficiency to 245 identification of proteins from isolated protein complexes, we focused on the other seven 246 TPLATE complex members and compared their abundance and fold changes after streptavidin-247 purification as deduced from label-free protein quantification (LFQ; [28]) to the control setup 248 (35S::GFP-BioID) ( Figure 3A). The fold change difference with respect to the control for the 249 Figure 3. Detection of TPC subunits with TPLATE-BioID is optimal at 28° C. (A) Experimental setup to look for enriched TPC subunits in biotin treated transformed Arabidopsis cell cultures. (B) Fold change abundance of the TPC subunits and statistical significance (-log(p-value)) compared to control. Fold change and p-values were calculated from the average LFQ intensities for 3 technical replicates of TPLATE-BioID w.r.t.versus GFP-BioID at similar temperature. Cell cultures were incubated with 50µM biotin at 25°-35°C for 24 h before harvesting. The TPC subunits are detected at all 4 temperatures without major differences. At 28°C and 30°C, the overall detection of several of the other subunits shows increased robustness (p-value) compared to both the lower (25°C) and higher (35°C) temperatures.
other TPC subunits was subunit dependent and not dramatically different between the different 250 temperatures. The highest overall fold change difference for the different subunits, combined 251 with the optimal robustness of the identification (p-value) was detected at 28 o C ( Figure 3B, 252 Supplemental Data Set 1), indicating that this temperature presents the optimal trade-off 253 between biotinylation efficiency of BioID and perturbation of physiological processes due to 254 elevated temperatures. 255 256

Various PBLs affect biotinylation of TPC subunits differently 257
The introduction of a flexible linker has been successfully used to extend the labelling radius 258 of PBLs [8], which is estimated to be about 10 to 15 nm [10]. This increased labelling radius 259 may be desirable when the protein of interest is significantly larger than the labelling radius of 260 the PBL alone, and/or when the goal is to map the constituency of a larger protein complex or 261 discrete subcellular region. We thus compared the efficiencies of various PBLs and assessed 262 their biotinylation radius by inserting a long flexible linker. For this, Arabidopsis cultures 263 expressing C-terminal fusions of TPLATE with BioID or BioID2 were assessed, with and 264 without a 65 aa linker similar to the one that was reported before [5]. As controls, we generated 265 GFP fused to BioID or BioID2 without additional linker (Supplemental Figure 5).  Figure 5B and D). 306 In order to compare the different PBL modules, we processed the cell cultures for LC-307 MS/MS and focused on the relative levels of the various TPC subunits compared to the control 308 setup. Our first mass spec results following streptavidin pull down and on-bead digestion 309 identified all known subunits of the TPC. Given that this is a robust multi-subunit complex [27] 310 and that we identify only non-biotinylated peptides with our on-bead digestion protocol, we 311 assumed that the subunits we detect are a combination of direct biotinylation as well as co-312 immunoprecipitation of the complex as a whole. To test this, we adapted our protocol ( Figure  313 4A) and included protein extraction and stringent washing steps with a buffer containing 8M 314 urea and 2% SDS to unfold proteins captured by the beads and to be able to remove unspecific 315 protein binders. We also included the TPLATE-linkerBioID setup treated with 2 mM biotin for 316 24 h to assess if increased biotin concentration improves TPC subunit detection. 317

318
In agreement with the higher stringency of the isolation procedure, the smallest TPC subunit, 319 LOLITA, which could be robustly detected using AP-MS [27] and could be detected without 320 being denatured prior to binding to streptavidin beads, was no longer detected (Figure 4, 321 Supplemental Data Set 2). LFQ revealed that the remaining seven TPC subunits, including 322 the bait TPLATE, could be detected using BioID, linkerBioID, linkerBioID2 and 323 linkerTurboID. The TASH3 and TWD40-2 subunits could however hardly be detected using 324 BioID2, which might be caused by the reduced expression level of the bait observed in these 325 cultures (Supplemental Figure 5). Increasing the concentration of biotin to 2mM had an 326 adverse effect on the detection of the TPC subunits as only the bait itself could be identified. It 327 is likely that increasing biotin concentrations causes residual free biotin to accumulate in the 328 protein extract, even after protein desalting to deplete free biotin, thereby occupying the 329 streptavidin binding sites on the beads (saturated at >9 µM of biotin). We tested this "saturation 330 hypothesis" using N. benthamiana leaves and protein precipitation to completely remove 331 residual biotin, showing that even low concentration of residual biotin can saturate the 332 streptavidin beads and incapacitate detection (Supplemental Figure 7). Hence, special care 333 should be taken to avoid excess of residual free biotin during streptavidin-capture. 334 It should be noticed that the fold change by which the other TPC subunits were detected with 337 TurboID was only similar or even sometimes lower (e.g. AtEH2) compared to the other BioID 338 forms ( Figure 4). This was caused by the fact that TPC subunits were identified more in the 339 TurboID control samples, resulting in the lower relative fold changes. All individual TPC 340 subunits were detected with more than 20 unique peptides using the GFP-linkerTurboID 341 whereas TWD40-2 was the only TPC subunit detected in other GFP-PBLs, which explains its 342 overall low fold change (Supplemental Table 3). Nevertheless, TurboID identified the other 343 TPC subunits more robustly compared to the other PBLs. So, although in our case, TurboID 344 showed to be superior to all others in identifying the other TPC subunits, the lower signal/noise 345 ratio of TurboID, due to its increased activity, might work as a disadvantage to observe 346 differences between bait proteins and control samples, which might even be enhanced if the 347 proteins are targeted to specific subcellular locations. 348 349 Figure S7. Exogenous application of biotin can exceed the binding capacity of streptavidin beads. Blot on the left: input and IP with streptavidin using 25 or 50 ul of beads. Note that 2 x more beads increased the recovery of the input signal, suggesting that the beads are saturated. Blot on the right: IP with 25 ul of streptavidin beads but in this case the supernatant was precipitated using ammonium acetate to remove excess biotin. Green arrowheads mark the position of the BL.

PDL and APMS 353
We compared the stoichiometry by which the different TPC subunits are detected using PDL 354 using our stringent washing protocol with our one step IgG pull down protocol using the GS rhino 355 TAP tag. To do this, we normalized the LFQ intensities for each TPC subunit to TPLATE and 356 compared the values of TPLATE-linkerBioID, TPLATE-linkerBioID2 and TPLATE-357 linkerTurboID with those coming from tandem affinity tag (TAP)-fused TPLATE (TPLATE-358 GS rhino ). Compared to the bait protein (TPLATE), the other TPC subunits are detected with 359 small differences in relative abundance between the different subunits in one-step-purification 360 MS as well as with PDL which showed the same trend. (Figure 5

, Supplemental Data Set 3). 361
Our results, comparing BioID, BioID2 and TurboID, each fused to TPLATE and having a long 362 linker in-between reveal that TurboID allows identifying the other subunits with higher 363 enrichment compared to the other PBLs. The smallest subunit, LOLITA, could only be 364 identified via TAP-MS, which points out that this subunit is not biotinylated although it 365 harbours 11 lysine residues. Our results furthermore reveal that, except for LOLITA, all TPC 366 subunits, which are part of a protein complex in the range of 1MDa can be identified using our 367 stringent wash protocol as proxy for biotinylation. For example, using TPLATE as bait and 368 using a long linker sequence linking it to the PBL, the TASH3 subunit was detected with 15 369 peptides instead of 2 in the absence of the linker (Supplemental Table 3). 370

Supplemental Table 3: Cell cultures expressing different TPLATE-PBLs identifies TPC subunits with different amount of non-biotinylated peptides. The table shows the amount of non-biotinylated peptides identified for TPC subunits in GFP and TPLATE-BioIDs PBL cell cultures. Preparation of samples for LC-MS/MS
involved the use of a buffer containing 8M Urea and 2% SDS. Cell cultures were incubated with either 50µM or 2mM biotin for 24 hours before harvesting. This table shows higher amount of peptides detected for all TPC subunits in case of linkerTurboID constructs than other BioIDsPBL. Also, it show higher amounts of peptides detected in TurboID control experiments (GFP-linkerTurboID vs. GFP-BioID and GFP-BioID2) suggesting increased promiscuity of TurboID.

Identification of biotinylated peptides allows identifying structural relationships between 372 complex subunits 373
The interaction between biotin-streptavidin is strong enough to be retained even under harsh 374 conditions, e.g, in reductive buffers (Supplemental Figure 7). Thus, biotinylated peptides are 375 expected to be retained on the streptavidin beads even under stringent washing. Following 376 stringent washing under denaturing conditions, on bead digest will release non-biotinylated 377 proteins, which can subsequently be identified using LC-MS. This approach, however, does 378 not provide direct evidence for biotinylation and it relies on the assumption that only 379 biotinylated proteins remain bound to the beads after the washing steps. To acquire direct proof 380 of biotinylation MS-based identification of biotinylated peptides is required. subunits towards biotinylation at their C-terminal parts (Figure 6). It is tempting to speculate 406 that the observed distribution of biotinylated peptides, as well as their absence, reflect the 407 proximity of the domains as well as structural constraints with respect to the bait protein and 408 Figure S8. The biotin-streptavidin interaction is retained under harsh conditions. Different extraction buffers were used for testing the binding affinity of biotin-labelled proteins with streptavidin from equal amount of plant protein material: 1. 50mM HEPES, 150 mM NaCl, 0.5% NP40, 10% Glycerol, 1mM PMSF; 2. 50mM Tris/HCl, 150 mM NaCl, 0.5% NP40, 10% Glycerol, 1mM PMSF; 3. 1x PBST, 1mM PMSF; 4. 2x Laemmli sample buffer (65 mM Tris-HCl, pH 6.8, 20% (w/v) glycerol, 2% SDS, 0.01% bromophenol blue, 10mM DTT). We provide a comprehensive comparison of various PBL based proximity labelling strategies 414 in plants and show that TurboID is the most promiscuous one, and that this also sometimes 415 leads to a lower signal to noise ratio. We also provide guidelines and approaches for 416 interactome capture in various plant systems specifically focusing on the ones that interact with 417 the plasma membrane. Furthermore, we show that for each bait/system conditions need to be 418 optimized independently. 419 We observed that in all three plant systems, using exogenous application of biotin 420 enhances PDL output but might not be a strict requirement for the successful application of  intermediate. We assume that various proteins may show variability in functioning as acceptors 431 of bioAMP (e.g. depending on the presence of accessible lysine residues). 432 PDL utilizing bacterial enzymes poses the question of whether these enzymes could 433 perform adequately in plants [8]. The activity optimum for BioID2 is 50ºC, whereas for BioID 434 this is 37ºC and thus BioID2 may be most adequate for use at higher temperature conditions. 435 Both temperatures are however far-off from the usual growth temperatures of most plant 436 species grown in temperate regions (e.g. Arabidopsis sp.). Both BioID2 and BioID show 437 reduced activity below 37ºC ([8] and our results herein). Furthermore, the lower temperature 438 optimum of TurboID (and mTurboID) [9] would imply that may function better at normal plant 439 growth temperature. In fact, we observed that TurboID activity is only increased by 2-fold from 440 22 o C to 28 o C. We, however, cannot rule out that the optimal temperature for PDL may vary 441 depending on the bait protein. At all tested temperatures, we observed that TurboID (and 442 mTurboID) outperforms other PBLs in terms of speed and promiscuity. Hence, TurboID might 443 be preferable when it comes to initial study of (transient) complex composition where the 444 generation of as much as possible specific biotinylation output in short time might be desirable. 445 However, the strong promiscuity of the control might also work as a disadvantage in 446 revealing specific interactions in cases where the reaction cannot be controlled that easily in 447 time or when both the bait and the control would be targeted to a confined intracellular space. 448 We provide evidence that our methods and conditions are applicable to plasma-449 membrane complexes. We showed that the interaction of the symbiotic RLKs NFR5 and 450 SYMRK can be identified by exploiting PDL and particularly the PBL TurboID. Furthermore, 451 the use of proper negative controls is imperative. However, even though the brassinosteroid 452 receptor BRI1 was not co-immunoprecipitated with the symbiotic receptors in a previously 453 published dataset [25], we detected weak biotinylation of this RLK and the immune-receptor 454 FLS2. While it could be interpreted as unspecificity within the PBL system, it should also be 455 considered, that PBL allows labelling of transient interactions or proximal proteins. As a 456 consequence, continuous unstable interactions accumulate to detectable amounts of proteins 457 and would thus allow their identification. As PDL using TurboID is capable of trans-458 biotinylation in the range of minutes (15 minutes under our experimental conditions), the 459 enrichment of unstable interactions would thus be more prominent. Therefore, putative 460 interactions identified by PBL still need to be verified using independent experimental systems 461 but comparisons between the different experimental systems should always reflect the technical 462 limitations of each approach. 463 By expanding our protocols and PBLs into Arabidopsis cell cultures, we could 464 reproduce the composition of the TPC except for one subunit. We show that the use of linkers 465 can be advantageous when it comes to identifying protein-protein interactions of multi-subunit 466 complexes. Furthermore, TPLATE-linkerBioID2 shows reduced cis-biotinylation compared to 467 TPLATE-linkerBioID in the presence of exogenous biotin but seems to function in the absence 468 of biotin suggesting that in plants, BioID2 can function in tissues where exogenous 469 supplementation of biotin may be slower, e.g. the vasculature. Furthermore, increased biotin 470 applications can lead to serious impediments when it comes to the identification of TPC 471 subunits as this can interfere with biotinylated protein binding on streptavidin slurries. Caution 472 is warrented to assure sufficient capture capacity of biotinylated proteins', since the amount of 473 beads needed for capture should be tested for each experimental setup/protocol. 474 Finally, by establishing a strategy for simultaneous identification of biotinylated and 475 non-biotinylated peptides we could provide evidence for the accessibility of different protein 476 parts to PDL. We show that EH1, EH2 and TML subunits are preferentially biotinylated at 477 their C-terminal parts, suggesting that their C-termini are in closer proximity to the C-terminal 478 end of TPLATE and/or some domains (even complex subunits) are not available for 479 biotinylation. We thus provide evidence that PDL approaches in plants may be able to provide 480 structural information of multi-subunit protein complexes and that this may be extended to the 481 topology of membrane proteins. 482 While this manuscript was on preparation, two additional works appeared in BioRxiv 483 making use of TurboID in plants. These

Cloning of the proximity label-tagged control constructs 509
For constructs used in hairy roots: Constructs encoding the full-length ORF of the PBL (e.g. 510 BirA* (pDEST-pcDNA5-BirA*-Flag C-term, a kind gift from the Gingras laboratory 511 (Couzens, Knight et al. 2013)), BioID2 (MCS-BioID2-HA, Addgene, Plasmid #74224 (Kim, 512 Jensen et al. 2016)), TurboID (V5-TurboID-NES_pCDNA3, Addgene, Plasmid #107169 513 (Branon, Bosch et al. 2018 This way, the following expression constructs were created; Pro35S::eGFP-BirA*, 530 Pro35S::eGFP-BioID2, Pro35S::eGFP-TurboID and Pro35S::eGFP-miniTurboID and 531 Pro35S::eGFP-BirA*(Deep) construct (in pKm43GW), with a C-terminally triple HA-tagged 532 BirA* fused to eGFP. 533 For constructs used in N. benthamiana: original BioID, BioID2 and TurboID DNA 534 sequences were taken from [5,9,10], codon optimized to Arabidopsis. The GOLDENGATE 535 compatible BirA, BirA*, BioID2 and TurboID were synthesized and codon optimized using 536 the codon optimization tool of Integrated DNA Technologies, Inc. The ORFs were synthesized 537 with BsaI overhands and were ligated to the Level1/2 vector pICSL86900 and pICSL86922, 538 as previously described [38]. The following expression vectors were used: Pro35S::BirA-Myc, 539 Pro35S::BirA*-myc, Pro35S::HF-BioID2-HA and Pro35S::superfolderGFP-TurboID-FLAG. 540 The genomic sequence of NFR5 and the coding sequence of BRI1 was synthesized with 541 BsaI overhangs for Golden Gate as Hairy roots: Seeds of tomato (Solanum spp.) cv. Moneymaker were surface-sterilized in 70% 565 ethanol for 10 min and in 3% NaOCl for 20 min (rinsing with sterile deionized water was 566 performed in between the two sterilization steps), and then rinsed 3 times 5 min each with sterile deionized water. The seeds were germinated on Murashige and Skoog (MS) tissue 568 culture medium [46] containing 4.3 g/L MS medium (Duchefa; catalog no. M0221.0050), 0.5 569 g/L MES, 20 g/L sucrose, pH 5.8, and 8 g/L agar (Difco; catalog no. 214530) in magenta boxes 570 (~50 ml). The pH of the medium was adjusted to 5.8 with KOH and autoclaved at 121°C for 571 20 min. The boxes were covered and placed in the dark at 4°C in a cold room for two days. 572 Subsequently, the boxes were transferred to a 24°C growth chamber (16 h light/8 h 573 photoperiod) for ~10 days until cotyledons were fully expanded and the true leaves just 574 emerged. Rhizogenic Agrobacterium (RAB) transformation was essentially performed as 575 described previously [47] with some minor modifications. More specifically, competent 576 rhizogenic Agrobacterium cells were transformed by electroporation (Shen and Forde 1989) 577 with the desired binary vector, plated on YEB medium plates with the appropriate antibiotics 578 (100 mg/L spectinomycin), and incubated for 3 to 4 d at 28°C. A transformed rhizogenic 579 Agrobacterium culture was inoculated from fresh plates into YEB liquid medium with the 580 appropriate antibiotics added and grown overnight at 28°C with shaking at 200 rpm. The RAB 581 culture was used to transform 20 to 40 tomato cotyledon halves. Using a scalpel, the cotyledons 582 were cut in half from ~10 days old tomato seedlings, transferred (adaxial side down) onto MS 583 liquid medium. The MS liquid was subsequently removed and the cotyledon halves 584 immediately immersed in a bacterial suspension at an optical density at 600 nm of 0.3 in MS 585 liquid medium for 20 min, then blotted on sterile Whatman filter paper and transferred (adaxial 586 side down) onto MS agar plates without antibiotics (4.3 g/L MS medium, 0.5 g/L MES, 30 g/L 587 sucrose, pH 5.8, and 8 g/L agar). The co-cultivation culture plates were closed with aeropore 588 tape. After 3 to 4 days of incubation at 22-25°C in the dark (Oberpichler, Rosen et al. 2008), 589 the cotyledons were transferred to MS agar plates with 200 mg/L cefotaxime (Duchefa; 590 catalogue no. c0111.0025) and 50 mg/L kanamycin and returned to 22-25°C. Typically, three 591 to five independent roots arise from each cotyledon. The expression of the eGFP marker of 592 antibiotic-resistant roots that emerged was monitored by means of fluorescent microscopic 593 imaging (Leica stereomicroscope and imaging DFC7000 T Leica microscope camera) and four 594 to ten independent roots showing expression of the marker were subcloned for each construct. 595 These roots were subsequently transferred to new selection plates with the same antibiotic 596 concentration for 3 rounds of subcultivation (~6 weeks) before antibiotics-free cultivation of 597 the hairy root cultures in liquid MS (in 50 ml Falcon tubes containing 10 to 30 ml MS medium 598 at 22-25°C and shaking at 300 rpm) and downstream analysis. After 3 rounds of cultivation, 599 root cultures were maintained and grown in antibiotics-free half-strength (½) Murashige and N. benthamiana: Wild-type tobacco (Nicotiana benthamiana) plants were grown under normal 602 light and dark regime at 25°C and 70% relative humidity1. 3-to 4-weeks old N. benthamiana 603 plants were watered from the bottom ~2h prior infiltration. Transformed Agrobacterium 604 tumefaciens strain C58C1 Rif R (pMP90), AGL1 Rif R ) or GV3101 Rif R harbouring the 605 constructs of interest were used to infiltrate tobacco leaves and used for transient expression of 606 binary constructs by Agrobacterium tumefaciens-mediated transient transformation of lower 607 epidermal leaf cells essentially as described previously [48]. Transformed Agrobacterium 608 tumefaciens were grown for ~20h in a shaking incubator (200 rpm) at 28°C in 5 mL of LB-609 medium (Luria/Miller) (Carl Roth) or yeast extract broth (YEB) medium (5 g/L beef extract, 1 610 g/L yeast extract, 5 g/L peptone, 0.5 g/L MgCl2, and 15 g/L bacterial agar), supplemented with 611 appropriate antibiotics (i.e. 100 g/L spectinomycin). After incubation, the bacterial culture was 612 transferred to 15 ml Falcon tubes and centrifuged (10 min, 5,000 rpm). The pellets were washed 613 with 5 mL of the infiltration buffer (10 mM MgCl2, 10 mM MES pH 5.7) and the final pellet 614 resuspended in the infiltration buffer supplemented with 100-150 μM acetosyringone. The 615 bacterial suspension was diluted with supplemented infiltration buffer to adjust the inoculum 616 concentration to a final OD600 value of 0.025-1.0. The inoculum was incubated for 2-3 h at 617 room temperature before injecting and delivered to tobacco by gentle pressure infiltration of 618 the lower epidermis leaves (fourth and older true leaves were used; and about 4/5-1/1 of their 619 full size) with a 1-mL hypodermic syringe without needle [49]. with infiltration buffer (no biotin) or alternatively, infiltration buffer supplemented with biotin 631 (stock solution dissolved in DMSO or water) and samples collected at the indicated times 632 points. Two infiltrated tobacco leaf segments/leaves were analyzed per combination. 633