Pupylation-based proximity labelling reveals regulatory factors in cellulose biosynthesis in Arabidopsis

doi:10.21203/rs.3.rs-3849556/v1

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Pupylation-based proximity labelling reveals regulatory factors in cellulose biosynthesis in Arabidopsis

https://doi.org/10.21203/rs.3.rs-3849556/v1

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Knowledge about how and where proteins interact provides a pillar for cell biology. Protein proximity-labelling has emerged as an important tool to detect protein interactions. Here, biotin-related proximity labelling approaches are by far the most commonly used, but may have labelling-related drawbacks. Here, we use pupylation-based proximity labelling (PUP-IT) as a tool for protein interaction detection in plants. We show that PUP-IT readily confirmed protein interactions for several known protein complexes across different types of plant hosts and that the approach increased detection of specific interactions as compared to biotin-based proximity labelling systems. To further demonstrate the power of PUP-IT, we used the system to identify protein interactions of the protein complex that underpin cellulose synthesis in plants. Apart from known complex components, we identified the ARF-GEF BEN1 (BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1). We show that BEN1 contributes to cellulose synthesis by regulating both clathrin-dependent and -independent endocytosis of the cellulose synthesis protein complex from the plasma membrane. Our results highlight PUP-IT as a powerful proximity labelling system to identify protein interactions in plant cells.

Biological sciences/Plant sciences/Plant cell biology

Biological sciences/Plant sciences/Plant molecular biology

Protein-protein interactions (PPIs) are at the core of all cellular processes and have the capacity to infer functions of unknown proteins. PPI analyses are done through a plethora of methods, much of which are performed in heterologous hosts or in vitro¹. By contrast, the assessment of PPIs in native hosts is largely restricted to different affinity-based purification schemes or what is referred to as proximity labelling. Proximity labelling may be done by fusing a protein of interest, referred to as bait, with an enzyme that can label proteins in the vicinity of the bait with a certain substrate. The efficiency of this process depends on the expression level of the enzyme-tagged bait and the substrate, and on favorable conditions to catalyze the labelling. The most frequently used proximity labelling entails various versions of biotin ligases or peroxidases, including for example TurboID^{2, 3}. Some of the drawbacks of these systems include the need to add the substrate (e.g. biotin) externally, timing and temperature optimization to activate the substrate and the ability to control the labeling reaction. In addition, endogenous biotin and peroxidases may confound results in plant cells^{2, 3}. However, recent developments have produced a completely genetically encoded proximity labelling system using pupylation, called PUP-IT (Fig. 1a)⁴.

The PUP-IT system is based on the prokaryotic enzyme PafA, which activates, holds and catalyzes the attachment of a PUP(E) peptide to lysine residues of proteins in close vicinity of the PafA⁴. Pupylation is absent in eukaryotes, and thus PUP-IT may be used to label proteins without labelling background⁴. In plant science, PUP-IT has recently been used in transient protoplast assays and in stable Arabidopsis lines with only one bait^{5, 6}. Here, we tested the PUP-IT system on a broader scale by using cytosolic and integral membrane bait proteins in a range of plant systems, including Nicotiana benthamina transient assays, Arabidopsis PSB-D suspension cells and Arabidopsis thaliana stable transgenic lines. We also added different tags on the Pup(E), e.g. FLAG-Pup(E) and StrepII-FLAG-Pup(E) providing several strategies for affinity-based protein purification of Pup(E)-tagged proteins. Finally, we provide a versatile cloning strategy with inducible- or ubiquitous promoters driving PafA and Pup(E) on the same vector backbone, which allows implementation of the system via a single transformation event.

We first tested the PUP-IT system using the scaffold protein RECEPTOR FOR ACTIVATED C KINASE1 (RACK1) as bait in transient expression assays in N. benthamiana leaves. RACK1 has a plethora of interactors⁷, mainly associated with protein translation and ribosome functions. RACK1 has three paralogs in Arabidopsis, of which RACK1A (AtRACK1a) is the most studied⁷. We therefore selected AtRACK1a and one of the N. benthamiana homologs (Fig. S1a), referred to as NbRACK1, as baits fused to PafA in our PUP-IT approach, with GFP fused to PafA as control (Fig. 1b). We introduced a flexible linker between the PafA and the RACK1 to minimize interference of the PafA on the native bait function. We based the linker length and content (glycine-serine linkers) on the Alphafold2 predicted structure of PafA and AtRACK1 to ensure flexibility and thus functionality of the linked proteins (Fig. S1b)^{8, 9, 10, 11}. We infiltrated the agrobacteria carrying the constructs into N. benthamiana leaves, performed subsequent enrichment of FLAG-PUP-labelled proteins and undertook mass-spectrometry (MS) analyses to identify target proteins of the different RACK1s (Fig. S1c-e). We next screened for proteins detected exclusively, or highly enriched, in the RACK1-PafA samples compared to GFP-PafA, and calculated fold enrichment and statistics to identify proteins that potentially interacted with RACK1-PafA. From this approach, we found 254 putative interactors of the AtRACK1a and 259 of the NbRACK1 compared to the GFP control (Fig. 1c) (Table S1). Importantly, we found that 154 of these proteins overlapped between the AtRACK1a and NbRACK1 samples (Fig. 1c), corroborating our expectation that the RACK1s in Arabidopsis and N. benthamiana engage with similar proteomes. To assess what processes were enriched among these proteins we next undertook Gene Ontology (GO) analyses and found that terms related to protein translation, RNA binding and ribosome function were among the most enriched for the proteins in line with the known activity of RACK1 (Fig. 1d). To further corroborate these inferences, we looked at overlap between the PUP-IT identified interactors and known interactors. To do this, we identified Arabidopsis homologs of the potential RACK1 interactors from the N. benthamiana PUP-IT experiment using BLAST and selected the best score hits. We then used these homologs to search the RACK1 interactome via the STRING database (https://string-db.org/). Out of the 154 proteins enriched from our RACK1 experiments, we found 51 proteins closely connected to RACK1 in STRING (Fig. 1e, network type: physical subnetwork, active interaction sources: experiments). In addition, several other interacting proteins formed smaller connected satellite units. It is important to note that although the BLAST analyses may have identified true orthologs between Arabidopsis and N. benthamiana, it is also possible that some proteins are part of more complex protein families, which may lead to an underestimation of positive hits.

We next tested the PUP-IT approach in another commonly used system for proteomics in plant biology; Arabidopsis PSB-D suspension cells. Here, we chose to investigate the TPLATE complex (TPC), which is a key endocytic protein complex in plant cells containing eight subunits¹². We, therefore, fused the PafA with TPLATE via a GSL linker and combined it with the expression of StrepII-FLAG-Pup(E) under a dexamethasone (DEX)-inducible promoter on the same T-DNA (Fig. 1f). We first optimized the time points and concentrations of DEX treatment by detecting the StrepII-FLAG-Pup(E) labelled TPLATE bait (Fig. S2a-b). Next, we affinity-purified the pupylated proteins and probed the bead fractions with an antibody against AtEH1/Pan1, a subunit of the TPC. The co-purification of the AtEH1/Pan1 subunit served as a first proxy for complex incorporation of the tagged bait (Fig. S2c). We also tested whether we could elute the majority of StrepII-FLAG-Pup(E) labelled proteins with 50 mM biotin while minimizing Streptactin contamination, which could interfere with mass spectrometry analysis (Fig. S2d). In parallel with the biotin elution strategy, we also used an SDS elution procedure (Fig S2e). Subsequent proteomic analyses showed that PUP-IT has the capacity to specifically isolate the TPLATE complex as various subunits of the TPLATE complex were significantly enriched or even exclusively found in the DEX group (Fig. S2f-g) (Table S2). Furthermore, the absence of the FLAG-PUP(E) peptides in the control group indicates that the DEX-inducible system in PSB-D is not leaky and therefore allowed tight control over the reaction (Fig. S2f-g). Affinity Purification-Mass Spectrometry (AP-MS) and TurboID-based biotin-proximity labelling have previously been performed on TPLATE in the same system, which allowed us to compare PUP-IT to these approaches^{12, 13}. To do this, we undertook intensity-based absolute quantification (iBAQ)¹⁴ of TPLATE subunits normalized to TPLATE as measure of enrichment of the subunits for each of the different experiments. We found that the subunits were substantially more enriched in the PUP-IT-based approaches compared to both AP-MS and the TurboID assays, highlighting high specificity of PUP-IT in labelling known TPLATE interactors (Fig 1g; Table S2).

The above examples demonstrate the utility of PUP-IT as an effective proximity labelling system in plant biology. We next employed the PUP-IT system to investigate the membrane-based CELLULOSE SYNTHASE (CESA) complex (CSC), which underpins cellulose synthesis in plant cell wall biology¹⁵. CSC has many known interactors; however, mechanisms that regulate CESA activity and trafficking are still unclear, suggesting that we are missing important interactors. Here, we chose to fuse the PafA to the N-terminus of COMPANION OF CESA 1 (CC1; Fig. S3a), which is one of the central components of the CSC¹⁶ and transformed the construct either into N. benthamiana leaf cells (transient infiltration) or into cc1cc2 double mutant Arabidopsis plants (stable transformation). As the CSC is largely present at the plasma membrane, we used the plasma membrane-localized protein LOW TEMPERATURE INDUCED PROTEIN 6B (LTI6B) as control (Fig. S3a). We first analyzed the enriched proteins from the N. benthamiana infiltration and found that many of the known CESA complex proteins were enriched in the CC1 samples as compared to the LTI6B (Fig. 2a, Fig. S3b-c) (Table S3). For example, beyond CC1, we found N. benthamiana proteins corresponding to the main CESAs that make up the core of the complex (e.g. CESA1), as well as CESA INTERACTING1 (CSI1) that connects the CSC to underlying microtubules¹⁷. In addition, we found SHOU4-LIKE that regulates trafficking of the CSC, as well as the STRUBBELIG-RECEPTOR FAMILY6 (SRF6) that is involved in response to cellulose deficiency (Fig. 2a) (Table S3)^{18, 19}.

With these promising results, we next generated Arabidopsis stable transgenic lines usingeither a CC1 promoter- or Ubiquitin10 promoter (UBI10pro)-driven PafA-CC1 construct to transform cc1cc2 double mutant plants. We used plants expressing a UBI10pro-driven PafA-LTI6B construct as control. We screened independent transgenic lines to obtain suitable levels of PafA-LTI6B and PafA-CC1 activity, and then selected lines based on the ability of the PafA-CC1 to complement the cc1cc2 mutant phenotype (Fig. S3 d-f). Here, we scored the phenotype based on growth on media plates supplemented with the cellulose synthesis inhibitor isoxaben, which leads to severe growth retardation of cc1cc2 mutant roots¹⁶. We found that while the CC1 promoter-driven constructs partially complemented the isoxaben-related phenotypes of cc1cc2, the UBI10pro-driven constructs complemented the phenotypes better (Fig. S3 g-h). Based on these results, and since we used the UBI10pro-driven LTI6b expression as control, we chose to use the UBI10pro-driven PafA-CC1 expression for our PUP-IT experiments. To ensure that we both had sufficient material for enrichment assays, as well as active cellulose synthesis, we used six-day-old seedlings as material for the FLAG-PUP enrichment (Fig. S3i). Similar to the transient infiltration assays in N. benthamiana leaves, we again found the components of the core cellulose synthesis machinery including CESAs (1, 3 and 6) and CSI (1 and 3)²⁰, as well the SHOU4, SRF1 corresponding to their homologs SHOU4L and SRF6 identified in the N. benthamiana samples (Fig. 2a-b; Table S4). In addition, we found the subunits of TPLATE complex, which mediates CSC endocytosis, as well as the v-ATPase subunit DE-ETIOLATED3 (DET3) involved in CSC secretion and recycling at the trans-Golgi network (TGN)^{21, 22}. We note that despite using different organisms, conditions, transformation approaches and organs, we identified the key core proteins of the CSC using the PafA-CC1 in N. benthamiana and Arabidopsis. However, the CC1 interactomes differ across the comparison, possibly indicating that the interactomes of a given protein vary depending on development, environment and biological system.

While some of the components of the CSC have been identified based on forward genetic screens, many of the more recently identified components have been found via co-expression analyses²³. To see if any of the genes that encode identified proteins from the CC1 PUP-IT analyses were co-expressed with the CESA genes, we inspected their co-expression relationships. Here, we identified BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1 (BEN1)/ BREFELDIN A-INHIBITED GUANINE NUCLEOTIDE-EXCHANGE PROTEIN 5 (BIG5)/ Arabidopsis thaliana HopM interactor 7 (AtMIN7) as a promising candidate (Fig. S4a) (Table S3-4). In addition, several proteins previously reported to interact with BEN1, i.e. HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1), Brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) and Arabidopsis thaliana HopM interactor 10 (AtMIN10)²⁴, were also enriched in the Arabidopsis PafA-CC1 samples as compared to PafA-LTI6B (Fig. 2b). We therefore aimed to place BEN1 function in context of the CC1 and the CSC.

BEN1 is an ADP-ribosylation factor (ARF) guanine nucleotide exchange factor (ARF-GEF) involved in the trafficking of proteins between the TGN/Early Endosomes (EE) and the plasma membrane²⁵. To put the BEN1 localization in context to that of CC1, we co-expressed GFP-CC1 and BEN1-mCherry in stable Arabidopsis lines and observed that the fluorescent signals co-localized at the plasma membrane and in endomembrane compartments (Fig. 2c-d), in line with the function of both BEN1 and CC1 at the TGN^{25, 26}. These results indicated that the proteins may function at the same locations inside the cell. To confirm interactions between the two proteins, we first co-expressed BEN1-mCherry with the FLAG-Pup(E) and PafA-CC1 in N. benthamiana. These experiments showed that PafA-CC1 also could pupylate BEN1 in this transient infiltration system (Fig. 2e). To further corroborate interaction between BEN1 and CC1, we performed co-immunoprecipitation (co-IP) assays using N. benthamiana leaves. Here, we found that a GFP-CC1 could co-IP BEN1-mCherry as compared to GFP (Fig. 2f). To rule out that the BEN1-mCherry enrichment was due to the interaction to CC1 and not to spurious GFP-mCherry interactions, we show that neither expression of GFP nor GFP-CC1 with mCherry revealed any co-IPed protein bands in the size region of BEN1-mCherry (Fig. S4b). Interestingly, the molecular weight of the enriched BEN1-mCherry protein was substantially larger in the output of the co-IP experiment (Fig. 2f). One reason for this might be that the enriched BEN1-mCherry is poly-ubiquitinated, which has been reported for BEN1 in context of other PPIs²⁷. Poly-ubiquitinated proteins may be targeted for degradation by the proteasome, which may be inhibited by applying the molecule MG132²⁸. To assess whether the enriched BEN1-mCherry was poly-ubiquitinated, we therefore treated the samples with the proteasome inhibitor MG132 and probed the co-IPed BEN1-mCherry with an antibody that recognizes poly-Ubiquitin, with positive signal (Fig. S4c). In addition and consistent with the notion that the poly-ubiquitinated BEN1-mCherry may be degraded, we also found that the treatment of MG132 led to an increased abundance of the poly-ubiquitinated BEN1-mCherry (Fig. S4d).

We next further confirmed the interaction between BEN1 and CC1 using split-ubiquitin yeast two hybird (Y2H) and N. benthamiana-based Bi-molecular Fluorescence Complementation (BiFC) assays. In the Y2H, we found that both CC1 and its C terminal truncation (fused to NubG) interacted weakly, but consistently, with BEN1 (fused to Cub). However, when we removed also the N-terminal region, we observed no interactions between the CC1 and BEN1 (Fig. 2g-h, and Fig. S4e). In the BiFC assays, we observed strong fluorescent signals when co-expressing nYFP-CC1 or nYFP-CC1∆C with BEN1-cYFP, but detected only faint fluorescence when co-expressing nYFP-CC1∆N and BEN1-cYFP (Fig. 2i). We quantified the fluorescence from the BiFC by including a 35S-driven RFP on the same backbone as the BiFC constructs, confirming our qualitative observations above (Fig. 2j). These results were not due to any mislocalization of the truncated versions of the CC1 as all the constructs still clearly labelled the plasma membrane (Fig. 2i-j, and Fig. S4f). From these analyses, we concluded that the N-terminal part of CC1 is necessary for the interaction of the protein with BEN1. The N-terminal part of CC1 consists of an intrinsically disordered region (IDR)²⁹. Intriguingly, HopM1 (formerly hopPtoM) also interacts with BEN1 via an IDR in its N terminus, which promotes poly-ubiquitination and degradation of BEN1²⁷. Our results therefore highlight a related interaction mode and degradation scheme of BEN1 in relation of CC1 or HopM1. We speculate that CC1 and HopM1 either specifically recognize the poly-ubiquitinated BEN1, or recruits Ubiquitin ligase to BEN1 upon interaction, which in turn regulates the degradation of BEN1.

Given the general trafficking role of BEN1 at the TGN/EE, we next assessed how CC1 and BEN1 may functionally interact. We therefore grew ben1 and cc1cc2 mutant seedlings on media supplemented with the trafficking inhibitor BrefeldinA (BFA), a potent ARF-GEF inhibitor that leads to BFA-related endomembrane aggregates in the cytoplasm, also called BFA bodies³⁰. Notably, cc1cc2 mutant seedlings were more sensitive to the BFA conditions than wild type and showed similar defects in root elongation as that of the ben1 mutant (Fig. S5a-b)³¹. We next generated ben1cc1cc2 triple mutants to investigate possible genetic interactions. Interestingly, ben1cc1cc2 triple mutant plants displayed reduced rosette leaf expansion, which corresponded to a lower level of crystalline cellulose, as compared to wild-type (Fig. 3a-b; S5c). In addition, ben1cc1cc2 seedlings displayed increased sensitivity to the cellulose synthesis inhibitor isoxaben (Fig. 3c-d; S5d).

We next sought to investigate the cell biological relationship between CC1 and BEN1. To do this, we attempted to directly visualize the dynamic behavior of CC1 and the CSC in the ben1 mutant. We therefore introgressed Venus-CC1 and YFP-CESA6 into ben1 plants. Previous studies showed that mutations in BEN1 affect trafficking of, for example, PIN-FORMED (PIN) and BRASSINOSTEROID INSENSITIVE1 (BRI1), which led to reduced accumulation of the proteins in BFA bodies^{25, 32}. Consistent with these reports, we observed reduced accumulation of both Venus-CC1 and YFP-CESA6 in BFA bodies in the ben1 mutant compared to the wild type (Fig. 3e-f). To investigate whether the BFA bodies were fueled from Endoplasmic Reticulum/Golgi (i.e. newly synthesized proteins en route to the TGN and plasma membrane), we pretreated seedlings with the de novo protein synthesis inhibitor cycloheximide (CHX), and then with BFA. These seedlings still contained substantial BFA bodies per cell (Fig. 3g; Fig. S6a-c), indicating that BEN1 mainly is associated with trafficking of the CSC between the plasma membrane and the TGN/EE. To assess if the CSCs were unable to get to the plasma membrane, or if there were problems in CSC internalization, in the ben1 mutant, we performed wash-out experiments of BFA. Here, the BFA bodies disappeared at similar rates in the ben1 mutant and WT, indicating that BEN1 may be related to internalization of the CSCs and not CSC trafficking from the TGN to the plasma membrane (Fig. S6a-b). Interestingly, the endocytosis of CSC is complex and may include both clathrin-dependent and independent processes¹⁵. To attempt to separate these processes in context of BEN1, we co-treated seedlings with the clathrin-mediated endocytosis inhibitor ES9-17 and CHX, and then with BFA. We found that this combination still led to BFA body formation, albeit to a substantially lesser degree than without ES9-17 (Fig. 3g-h, Fig. S6c). These results highlight the clathrin-independent internalization of the CSCs, and indicate that BEN1 may contribute both to clathrin-dependent and -independent endocytosis of the CSC (Fig.3g-h, Fig. S6c). Finally, we checked YFP-CESA6 protein abundance and mRNA levels in ben1 to see if the trafficking defects may also impact CESA6 levels. However, we did not find any major differences between ben1 and WT control (Fig. S6d-e). Taken together, our results indicate that BEN1 regulates the trafficking of the CSC between the plasma membrane and the TGN/EE and that defects in the BEN1 therefore impact cellulose synthesis.

Protein interactions are essential to understand how proteins work in context to each other and to infer protein function. The implementation of new tools to study PPIs is therefore of substantial interest across all aspects of cell biology. We provide a new toolbox for PPI inferences, PUP-IT, in plant biology and highlight how this tool may be used to identify new components of important processes in plants.

We show that PUP-IT may be used in a range of different plant biological systems, including cell suspensions, transient infiltration assays and in stable transgenic plants. In addition, we supply several vector constructs where both the bait and the substrate can be included on a single backbone. The substrate may be modified with different tags to enrich the labelled proteins, which increases the versatility of the toolbox. It is, however, important to realize that the tag needs to be placed upstream of the PUP, as the PafA recognizes a phosphorylated PUP to then ligate onto Lysine residues on proteins close to the PafA^{33, 34}. We also compared PUP-IT against the highly popular TurboID. Here, we show that known interactors of TPLATE (used in the comparison as bait) were enriched to higher relative levels with PUP-IT as compared to TurboID and even against AP-MS. These results show that PUP-IT may have a higher specificity for close interactors, i.e. proteins forming protein complexes, than that of for example TurboID. We therefore envision that PUP-IT may become a preferred approach in the emerging field of protein proximity labelling.

Plant materials and growth conditions

T-DNA insertional lines for ben1 (SALK_013761C) were obtained from NASC (http://Arabidopsis.info/); the double mutant cc1cc2 (SAIL_838_F07, Gabi_654A12) and marker lines Venus-CC1 (cc1cc2), YFP-CesA6 (Col-0)¹⁶ were as described in previous studies. cc1cc2 was crossed to ben1 to generate the triple mutant cc1cc2ben1, and plants were genotyped in the F2 generation. The fluorescent marker line YFP-CESA6 (Col-0) was crossed to ben1, and YFP-CESA6ben1 was identified via genotyping ben1 and imaging of the YFP-CESA6. The fluorescent marker line Venus-CC1 (cc1cc2) was crossed to the triple mutant cc1cc2ben1, with Venus-CC1cc1cc2ben1 plants identified via genotyping of ben1and imaging of Venus-CC1. The generated marker lines were re-examined in the F3 generation to confirm no segregations of the fluorescent markers and homozygosity of mutations. Primers used to screen for homozygous insertion lines are present in Table S5.

Arabidopsis seedlings were grown in a growth chamber under long-day conditions (16 h light / 21°C and 8 h dark / 19°C) on solid 1/2 Murashige and Skoog (MS) media with 1% sucrose for phenotyping. Seedlings from solid 1/2 MS media were transferred to soil pots in a greenhouse under set conditions (16 h light / 21°C and 8 h dark / 19°C). Tobacco (Nicotiana benthamiana) used in this work were grown in the same greenhouse conditions for 3 to 4 weeks.

Plasmid construction

Primers used for all the constructs in this manuscript and codon optimized sequence of PafA and Pup(E) are presented in Table S5. Backbones and fragments were assembled with NEBuilder® HiFi DNA assembly mix (New England Biolabs) / In-fusion Master mix (Takara) or ligated using T4 ligase (Thermo Fisher Scientific/New England Biolabs) into corresponding cloning vectors.

To create the vectors that express the FLAG-Pup(E) and PafA fused proteins, fragments of FLAG-Pup(E) + ter3A were first assembled into the AscI site of pMDC32³⁵ to produce the destination vector pMDC32_35S::FLAG-Pup(E)::ter3A::attR1-attR2::terNOS. The UBI10 promoter and PafA were assembled into a pENTR vector. Then CDS of LTI6B and the pENTR backbone with UBI10pro::PafA were amplified and assembled. After that, an LR reaction was performed to integrate the linearized pENTR UBI10pro::PafA-LTI6B into the destination vector to produce the pMDC32_35S::FLAG-Pup(E)::ter3A_+_UBIpro::PafA-LTI6B::terNOS.

pENTR/TOPO with UBI10 promoter and multiple cloning sites was used as the backbone for the GFP/AtRACK1a/NbRACK1 vectors. pENTR/TOPO CC1pro::mNeonGreen-CC1::terHSP was used as the backbone for the CC1 vectors. HpaI or ApaLI (New England Biolabs) linearized pENTR of attL1-UBIpro::GFP/AtRACK1a/NbRACK1-PafA::terHSP-attL2 and attL1-UBIpro/CC1pro::PafA-CC1::terHSP-attL2 were integrated into pMDC32_35S::FLAG-Pup(E)::ter3A::attR1-attR2::terNOS, respectively, through LR reactions.

To create the pGGK-35S::GRLhG4 > > A-G, vector pGG-A-35S-B, pGGD-linker-E, pGG-E-35St-F, pGG-F-AarI_linker-G³⁶ and the pSW610 (pGG-B-GR-LhG4-D³⁷ ; addgene #115992) were assembled into a pGGK A-G vector via a Golden Gate reaction as previously described^{36, 38}. After AarI (Thermo Scientific™) digestion, an A-ccdB/CmR-G PCR fragment was inserted into the resulting vector via ligation using T4 DNA ligase to generate pGGK-35S::GRLhG4 > > A-ccdB-G. pGGK-35S::GRLhG4 > > p6xOP::StrepII-FLAG-PUP::tHSP18.2_+_35S::TPLATE-GSL-PafA::tHSP18.2 was created with two rounds of iterative Golden Gate assembly as previously described³⁸. In the first Golden Gate reaction pGGK-35S::GRLhG4 > > A-ccdB-G was combined with pSW180a - pOp6 (pGG-A-p6xOP-B)³⁷, pGGB-StrepII-C, pGGC-FLAG-PUP-D, pGG-D-Decoy_v2-E (D017), pGG-E-tHSP18.2M-F³⁹ and pGG-F-A-AarI-SacB-AarI-G-G³⁶. After AarI digestion, the resulting vector was combined with pGGA-35S-C⁴⁰, pGGC-TPLATE-D, pGGD-GSL-PafA-E, pGG-E-tHSP18.2M-F and pGG-F-linkerII-G³⁶ in a second Golden Gate reaction.

The CDS of CC1 and BEN1 (without stop codon) were amplified by PCR and subcloned into the entry vectors pUCL3L2 (Plasmid #114019, addgene) and pUCL1L4 (Plasmid #114018, addgene) respectively. Then pUCL1L4_BEN1 and pUCL3L2_CC1 were introduced into the pFRETgc-2in1-NC ⁴¹ (Plasmid #105121, addgene) vector by LR reaction to generate the pFRETgc_35S::EGFP_+_35S::BEN1-mCherry and pFRETgc_35S::EGFP-CC1_+_35S::BEN1-mCherry. A pUCL1L4 with dummy sequence was generated to produce pFRETgc_35S::EGFP_+_35S::mCherry and pFRETgc_35S::EGFP-CC1_+_35S::mCherry.

The above-generated pUCL1L4_BEN1, pUCL3L2_CC1 and extra vectors with truncated CC1 in pUCL3L2 were introduced into the pBiFCt-2in1-NC ⁴² (Plasmid #1051112, addgene) and pFRETgc-2in1-NC to examine BiFC and protein subcellular localization signals.

The CDS of CC1, truncated CC1, PIP2A (without stop codon) and BEN1 (without stop codon) were subcloned in the pENTR/DTOPO and then introduced into the destination vectors pNX32-DEST (Plasmid #105083, addgene) and pMetYC-DEST (Plasmid ##105081, addgene) through LR reaction for split Y2H assay.

PUP-IT protein expression in N. benthamiana, Arabidopsis thaliana PSB-D cells and Arabidopsis plants.

The Agrobacterium gv3101, transformed with either pMDC32_35S::FLAG-Pup(E)::ter3A _+_UBIpro::GFP/AtRACK1A/NbRACK1-PafA::terHSP, pMDC32_35S::FLAG-Pup(E)::ter3A _+_UBIpro::PafA-Lti6B::terNOS, or pMDC32_35S::FLAG-Pup(E)::ter3A _+_UBIpro/CC1pro::PafA-CC1::terHSP, was cultured and infiltrated into N. benthamiana leaves along with strain P19⁴³ (OD600 = 0.5 in infiltration buffer) as previously described⁴⁴. The tissues were collected 2 days after plants were grown in the greenhouse.

The pGGK-35S::GRLhG4 > > p6xOP::StrepII-FLAG-PUP::tHSP18.2_+_35S::TPLATE-GSL-PafA::tHSP18.2 construct was transformed into dark grown PSB-D suspension culture cells as described before⁴⁵. The cultures were harvested 3 days after subculturing. StrepII-FLAG-PUP(E) expression was induced with 1, 10, 50, 100 or 200 µM dexamethasone as indicated (stock solution: 50 mMol Dexamethasone in DMSO) for 1 or 24 hours as indicated. DMSO was used as a mock treatment.

The Agrobacterium gv3101 transformed with either pMDC32_35S::FLAG-Pup(E)::ter3A _+_UBIpro::PafA-Lti6B::terNOS or pMDC32_35S::FLAG-Pup(E)::ter3A_+_ CC1pro/UBIpro::PafA-CC1::terHSP was used to produce Arabidopsis transgenics through floral dip method⁴⁶. After hygromycin selection, positive transgenics were identified by western blot with FLAG antibody (see below). Seedlings were grown in liquid 1/2 MS media for six days in culture bottle to generate tissues for IP-MS.

SDS-PAGE and Western blot

Protein extractions from N. benthamiana leaves and Arabidopsis transgenics in 1× Laemmli loading buffer (BioRad) with reducing agent (100 mM DTT), were boiled for 10 min at 95°C. Protein were loaded on a 12% or 4–15% SDS–PAGE TGX gel (BioRad) and subsequently blotted onto a polyvinylidenedifluoride membrane (BioRad). Membranes were blocked in 5% skimmed milk in TBST overnight at 4°C on a shaking device. Next, the blots were incubated with primary antibodies α-FLAG (Merck, F3165, 1:2000), α-GFP (Chromotek, 3H9, 1:2000), α-YFP (Venus) (Merck, MABE1906, 1:1000) and secondary antibodies α-mouse-HRP (Agilent, P0260, 1:5000), α-rat-HRP (GE HealthCare, NA935V, 1:5000) in 5% skimmed milk for 1h at room temperature on a shaking device. For RFP (mCherry) detection, membranes were blocked for 2 h at room temperature, incubated with primary antibody α-RFP (mCherry) (Chromotek, 6G6, 1:2000) overnight at 4°C, and secondary antibody α-mouse-HRP (Agilent, P0260, 1:5000) for 1 h at room temperature on a shaking device. Same conditions for α-RFP were used for the α-Ubiquitin (Thermofisher, eBioP4D1, 1:500) western blots, with secondary antibody α-mouse-HRP (Agilent, P0260, 1:5000). Three washes with 1 x TBST for 5 mins each were taken between the change of the primary antibody to the secondary antibody, and another three washes for 5 mins each were taken before imaging the HRP signal under ChemiDoc

Protein extractions from PSB-D suspension culture cells in 1× Laemmli loading buffer (BioRad) with reducing agent (1× NuPage [Invitrogen] or 100 mM DTT), were boiled for 10 min at 95°C. Proteins were loaded on a 4–20% SDS–PAGE TGX gel (BioRad) and subsequently blotted on a polyvinylidenedifluoride membrane (BioRad). Membranes were blocked in 5% skimmed milk overnight at 4°C on a shaking device. Next, the blots were incubated with primary antibodies α-FLAG (Merck, F1804, 1:1000], α-TPLATE⁴⁷( 1:1000), α-AtEH1/Pan1⁴⁸( 1:2000) and secondary antibodies α-mouse-HRP (Cytiva, NA931, 1:10,000), α-rabbit-HRP (Cytiva,NA934, 1:10,000) in 3% skimmed milk for 1h at room temperature on a shaking device. Three washes for 5 mins each were taken between the change of the primary antibody to the secondary antibody, and before imaging the HRP signal under ChemiDoc.

Protein extraction and immunoprecipitation

Tissues from N. benthamiana multiple leaves (2–3 g) were sampled and ground in liquid nitrogen to a fine powder. Total proteins were extracted with extraction lysis buffer (1:3 [W/V]- material: buffer), [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, and 0.1% protease inhibitor cocktail (Roche)] for 30 minutes on ice, and centrifuged at 4°C at 12,000 rpm for 30 min. The supernatant was divided into three equal amounts for the following independent immunoprecipitation triplicates, then incubated each with 100 µl equilibrated anti-Flag antibody coupled magnetic beads (Thermo Scientific™, A36797) for 2 hours at 4°C. Beads were washed three times with extraction buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100 and 0.1% protease inhibitor cocktail (Roche)], then washed three times with wash buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA and 0.1% protease inhibitor cocktail (Roche)] and three times with 1 X PBS. After that the beads were stored at − 80°C until LC-MS/MS analysis.

A similar process was performed on tissues from Arabidopsis seedlings (1–2 g) with three independent transgenic lines mixed. The extraction lysis buffer was replaced by [50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 1% Triton X-100, 1% PVP and 0.1% protease inhibitor cocktail (Roche)], and the wash buffer was replaced by [50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 1% PVP and 0.1% protease inhibitor cocktail (Roche)].

Total protein extracts from liquid N2 ground PSB-D cells (3 g/replicate) were generated by adding extraction buffer (2:3 - buffer:material), (25 mM TRIS-HCl pH7.6, 15 mM MgCl₂, 150 mM NaCl, 15 mM pNO₂phenylPO₄, 60 mM B-glycerophosphate, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 1 um E64, 0.5 mM EDTA, 0.50% NP40, 5% ethyleneglycol, 0.1% protease inhibitor cocktail EDTA-free (Roche)] ) and incubated for 1 hour at 4°C. Subsequently, the lysate was cleared by 2 centrifugation steps at 20,000 g at 4°C. Then the lysate was incubated with equilibrated Strep-Tactin®XT 4Flow® high-capacity resin (100 ul beads/ replicate) for 2 hours at 4°C. Next, the beads were washed 3 times (10 column volumes total). Bound proteins were eluted using either 2 x 50 ul 50 mM biotin [in 50 mM NH₄CO₃] or 2 x 50 ul 10% SDS [in 50 mM Triethylammonium bicarbonate (TEAB) pH 8.5]. Subsequently, the supernatant was cleared by 1-minute centrifugation at 20,000 g. Protein samples eluted with SDS were cleaned up with S-Trap™ micro columns (Profiti). Protein samples eluted with biotin or SDS were digested in-solution with 1 ug Trypsin/LysC (Promega) overnight at 37°C, followed with an additional 0.5 ug Trypsin/LysC for 2 hours. Finally, digested proteins were cleaned up with C-18 Omix tips (Agilent). Digests containing the cleaved peptides were dried in a SpeedVac and stored at − 20°C until LC-MS/MS analysis.

Mass spectrometry and data analysis

Washed beads were incubated for 30 min with elution buffer 1 (2 M Urea, 50 mM Tris-HCl pH 7.5, 2 mM DTT, 20 µg/ml trypsin) followed by a second elution for 5 min with elution buffer 2 (2 M Urea, 50 mM Tris-HCl pH 7.5, 10 mM Chloroacetamide). Both eluates were combined and further incubated at room temperature overnight. Tryptic peptide mixtures were acidified to 1% TFA and loaded on Evotips (Evosep). Peptides were separated on 15 cm, 150 µM ID columns packed with C18 beads (1.9 µm) (Pepsep) on an Evosep ONE HPLC applying the ‘30 samples per day’ method, and injected via a CaptiveSpray source and ten µm emitter into a timsTOF pro mass spectrometer (Bruker) ran in PASEF mode⁴⁹.

Peptides were re-dissolved in 20 µl loading solvent A (0.1% trifluoroacetic acid in water/acetonitrile (ACN) (98:2, v/v)) of which 2 µl was injected for LC-MS/MS analysis on an Ultimate 3,000 RSLCnano system in-line connected to a Q Exactive HF Biopharma mass spectrometer (Thermo). Trapping was performed at 20 µl/min for 2 min in loading solvent A on a 5 mm trapping column (Thermo scientific, 300 µm internal diameter (I.D.), 5 µm beads). 250 mm Aurora Ultimate, 1.7 µm C18, 75 µm inner diameter (Ionopticks) kept at a constant temperature of 45°C. Peptides were eluted by a non-linear gradient starting at 1% MS solvent B reaching 26% MS solvent B (0.1% FA in acetonitrile) in 30 min, 44% MS solvent B in 38 min followed by a 5-minute wash at 56% MS solvent B starting at 40 minutes and re-equilibration with MS solvent A (0.1% FA in water) at a flow rate of 300 nl/min.

The mass spectrometer was operated in data-dependent mode and raw files were processed using the MaxQuant software (version 2.0.3.0)⁵⁰. Peak lists were searched against the proteome of released Nicotiana benthamiana⁵¹ or the proteome of Arabidopsis from Araport11plus database (www.arabidopsis.org/), combined with 262 common contaminants by the integrated Andromeda search engine. Additionally, the baits, PafA and FLAG-Pup(E) sequences were added. Mass tolerance on precursor ions was set to 4.5 ppm and on fragment ions to 20 ppm. Match between runs and MS1-based Label Free Quantification (LFQ) were on. The false discovery rate was 1% for both peptides (minimum length of 7 amino acids) and proteins.

The protein groups result file was uploaded in Perseus (version 2.0.3.0)⁵⁰. Reverse hits, contaminants and only identified by site identifications were removed. LFQ intensity values were log2 transformed. For comparison between two groups, identifications were filtered for 50% values in total mimicking the assumption that specific proteins would be only detected in one group. A binary analyse was first performed to screen proteins as unique that were detected in all replicates within an experiment group but no detection in the corresponded control. For relative comparison, missing values were imputed with values around the detection limit, randomly drawn from a normal distribution with a width equal to 0.3 and a downshift equal to 1.8. Then a two-sided t-test was performed with false positive by permutation-based FDR calculation, using thresholds FDR = 0.05 and S0 = 1. For comparation of proteins from AtRACK1 and NbRACK1, an extra filter of log₂(FC) > 1, -log₁₀(p-value) > 2 was applied. The significant candidate lists were generated through collection from proteins unique and relatively high abundance through t-test, then plotted in volcano summary by R.

Plant phenotyping

Seedlings of wild type (Col-0) and corresponding mutants were grown on 1/2 MS plates for 3 days, then transferred to the plates with 5 µM BFA for 7 days or 2 nM Isoxaben for 4 days (DMSO as control). The elongated root length was measured by FIJI-ImageJ and root tip were captured by KEYENCE (VHX-7000).

7 days seedlings were transferred into soil for 20 days in the greenhouse and pictured to record the growth of rosette leaves. 3 samples were harvested for each group, and each sample contained leaves from two pots. The crystalline cellulose quantification was conducted as described^{52, 53}.

Immunoprecipitation, BiFC and split yeast two hybrid assay

For FLAG-PUP labelling assay, pFRETgc 35S::EGFP_+_35S::BEN1-mCherry were co-expressed with pMDC32_35S::FLAG-Pup(E)_+_UBIpro::PafA-CC1 or not in N. benthamiana leaves, then proteins were immunoprecipitated by RFP-Trap Agarose (Chromotek, rta). For Co-IP, pFRETgc 35S::EGFP_+_35S::BEN1-mCherry, pFRETgc 35S:: EGFP-CC1_+_35S::BEN1-mCherry, pFRETgc 35S::EGFP_+_35S:: mCherry and pFRETgc 35S::EGFP-CC1_+_35S::mCherry were expressed in N. benthamiana leaves respectively, then proteins were immunoprecipitated by GFP-Trap Agarose (Chromotek, gta). The Immunoprecipitation was performed according to methods for Agarose Trap from Chromotek. Briefly, total proteins were extracted with buffer (1:3 [W/V]- material: buffer), [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 0.5% Triton X-100, and 0.1% protease inhibitor cocktail (Roche)] for 30 minutes on ice, and centrifuged at 4°C at 12,000 rpm for 30 min. The supernatant was diluted with buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, and 0.1% protease inhibitor cocktail (Roche)], then incubated with 20 µl equilibrated RFP/GFP-Trap Agarose for 2 hours at 4°C. Beads were washed three times with buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 0.05% Triton X-100 and 0.1% protease inhibitor cocktail (Roche)], After that, beads were boiled at 95°C for 10 mins with 1 x Laemmli loading buffer for western blot.

The pFRETgc-2in1-NC and pBiFCt-2in1-NC vectors with subcloned BEN1, CC1, or truncated CC1 were transformed into Agrobacterium gv3101. Transient expression in N. benthamiana and imaging were performed according to methods^{41, 42}.

The split yeast two hybrid assay was performed by employing an approach already developed⁵⁴. Briefly, the tested group of plasmids were co-transformed into the yeast strain NMY51. The transformed yeast strains were plated on SD/-Leu/-Trp medium and placed in a 30°C constant temperature incubator. For the interaction assay, colonies regrown on SD/-Leu/-Trp plate for three days were diluted in water and dropped on SD/-Leu/-Trp plate for 3 days, and on SD/-Leu/-Trp/-His/-Ade/ + 400 mM NaCl selection medium for 5–10 days to check growth. Experiments were repeated three times to evaluate survival ratios.

Drug treatments

Seedlings were grown on half MS plates vertically for 4–7 days. For BFA (Merck B6542-5MG or Sigma B7651-5MG) treatment, seedlings were placed in liquid 1/2 MS and treated with 50 µM BFA (stock solution 5 mg.ml^− 1 in DMSO) or DMSO for 30/90 min under constant agitation before observation at the spinning microscope. For “BFA + washes”, BFA treatment was followed by 3 washes of 20 min in half MS before observation.

In the case of cycloheximide treatment, seedlings in liquid 1/2 MS were pre-treated with 50 µM CHX (Sigma C7698-1G; stock solution 100 mM in DMSO) for 60 min before treatment with 50 µM CHX and 50 µM BFA for 90 min. Control seedlings were treated with 50 µM CHX for 150 min.

For clathrin-mediated endocytosis inhibition, seedlings in liquid 1/2 MS pre-treated with 50 µM CHX for 60 min were treated with Endosidin (Merck HY-131683 693806-53-0; stock solution 30mM in DMSO) 30 µM or equivalent amount of DMSO for 30min, followed by addition of 50 µM BFA for another 30 min.

Fluorescence imaging and analysis

The pictures were taken by Confocal Microscope 3i CSU-W SoRa Spinning Disk and processed by Fiji for the followed analysis.

The vector pFRETgc_35S::EGFP-CC1_+_35S::BEN1-mCherry was transformed into the Col-0 and selected by BASTA for positive transgenics. Images were taken to collect signals of EGFP (C1: 488/563, 30%, 100 ms) and mCherry (C2: 563/488, 30%, 200 ms). The time lapse imaging was taken to include a mCherry bleach step. A random line was drawn to define the region of interest to produce an intensity plot of co-efficient subcellular localization.

For imaging of N. benthamiana leaves, in BiFC, signals were collected of YFP (C1: 515/563, 20%, 200 ms) and RFP (C1: 563/515, 20%, 50 ms), under 40 X lens. For subcellular localization, signals were collected of EGFP (C1: 488/563, 20%, 50 ms) and mCherry (C2: 563/488, 30%, 200 ms) under 40X lens.

For BFA treatment, Venus-CC1 and YFP-CESA6 were excited by laser 515 nm (30%, 200 ms or 20%, 400ms), and observed under 60X lens with z-step of 0.3-1 µm. TdT-CESA6 was observed using the following settings: excitation 561/400ms/ step: 0,5 or 1µm, 60X lens. BFA bodies were quantify manually in Fiji on epidermal cells using z-stacks.

YFP-CESA6 detection and q-PCR.

Total proteins were extracted from 0.5 g seedlings of YFP-CESA6 marker lines in Col-0 and ben1 background grown for 2 weeks on ½ MS plates. YFP-CESA6 were immunoprecipitated by GFP-Trap Agarose (Chromotek, gta) according to the method above, and detected by α-YFP (Venus) (Merck, MABE1906) in western blot.

Total RNA was extracted using plant total RNA kit (SIGMA, SLCD4163), cDNA was generated from iScript cDNA Synthesis Kit (BIO-RAD) and q-PCR (BIO-RAD, SYBR SYBR® Green qPCR mix) was performed according to the manufacturer’s instructions with primers in table S5.

Acknowledgements

We acknowledge Prof Jürgen Kleine-Vehn for codon optimised templates of PafA and Pup(E). We would like to thank Liu Wang for sharing unpublished marker lines Venus-CC1cc1cc2. We thank Michael Kraus, Moritz Nowack, and Norbert Bollier for sharing unpublished golden gate building blocks. We thank Michaël Vandorpe for assisting with the golden gate cloning expression clones. We thank Geert De Jaeger and Jelle Van Leene for constructive discussions and the Interactomics Facility of the Center for Plant Systems Biology (VIB) for assisting with PSB-D cell culture transformation. We thank the Proteomics Research Infrastructure (PRI) at the University of Copenhagen (UCPH) supported by NNF19SA0059305 and the VIB proteomics core for the mass spectrometry data collection. We thank the Center for Advanced Bioimaging (CAB) at the UCPH for the imaging support. A.D.M. is supported by a PhD grant (1124621N) from the Research Foundation– Flanders (FWO). L.C. N. is funded by EMBO ALTF 629–2021. G.A.K. is funded by a DECRA Fellowship from the Australian Research Council (DE210101200). S.P. was funded by a Villum, three Novo Nordisk, and Danish National Research Foundation grants (25915, 19OC0056076, 20OC0060564, 23OC0086341, DNRF155, respectively).

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Pupylation-based proximity labelling reveals regulatory factors in cellulose biosynthesis in Arabidopsis

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Abstract

Figures

Introduction

Methods

Plant materials and growth conditions

Plasmid construction

SDS-PAGE and Western blot

Protein extraction and immunoprecipitation

Mass spectrometry and data analysis

Plant phenotyping

Immunoprecipitation, BiFC and split yeast two hybrid assay

Drug treatments

Fluorescence imaging and analysis

Declarations

Acknowledgements

References

Additional Declarations

Supplementary Files

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