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GBPL3 localizes to the nuclear pore complex and functionally connects the nuclear basket with the nucleoskeleton in plants [1]

['Yu Tang', 'Department Of Plant', 'Microbial Biology', 'University Of California', 'Berkeley', 'California', 'United States Of America', 'Innovative Genomics Institute', 'Man Ip Ho', 'School Of Life Sciences']

Date: 2022-11 

The nuclear basket (NB) is an essential structure of the nuclear pore complex (NPC) and serves as a dynamic and multifunctional platform that participates in various critical nuclear processes, including cargo transport, molecular docking, and gene expression regulation. However, the underlying molecular mechanisms are not completely understood, particularly in plants. Here, we identified a guanylate-binding protein (GBP)-like GTPase (GBPL3) as a novel NPC basket component in Arabidopsis. Using fluorescence and immunoelectron microscopy, we found that GBPL3 localizes to the nuclear rim and is enriched in the nuclear pore. Proximity labeling proteomics and protein-protein interaction assays revealed that GBPL3 is predominantly distributed at the NPC basket, where it physically associates with NB nucleoporins and recruits chromatin remodelers, transcription apparatus and regulators, and the RNA splicing and processing machinery, suggesting a conserved function of the NB in transcription regulation as reported in yeasts and animals. Moreover, we found that GBPL3 physically interacts with the nucleoskeleton via disordered coiled-coil regions. Simultaneous loss of GBPL3 and one of the 4 Arabidopsis nucleoskeleton genes CRWNs led to distinct development- and stress-related phenotypes, ranging from seedling lethality to lesion development, and aberrant transcription of stress-related genes. Our results indicate that GBPL3 is a bona fide component of the plant NPC and physically and functionally connects the NB with the nucleoskeleton, which is required for the coordination of gene expression during plant development and stress responses.

Funding: This work was supported by the National Institute of Food and Agriculture (HATCH project CA-B-PLB-0243-H), National Science Foundation (NSF-MCBDivision of Molecular and Cellular Biosciences 2049931), Hellman Fellows Fund, and startup funds from the Innovative Genomics Institute and University of California at Berkeley (to Y.G.). B.K. is funded by the Hong Kong Research Grant Council (GRF14121019, 14113921, AoE/M-05/12, and C4002-17G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The raw RNA-seq data reported in this study were deposited in the NCBI Gene Expression Omnibus with accession number GSE199667. The raw mass spectrometry data reported in this study were deposited to the ProteomeXchange Database with accession number PXD032906. All other relevant data are within the paper and its Supporting Information files.

In this study, we describe GBPL3, a guanylate-binding protein (GBP), as an NB-associated protein in plants. GBPL3 was highly enriched at the NE and probed by multiple NPC basket and nucleoskeleton components in our previous proteomics profiling. Consistently, we showed that GBPL3 confers physical interaction with components of both the NB and the nucleoskeleton. Fluorescence imaging coupled with immunogold-labeling electron microscopy revealed that GBPL3 is predominantly located at the NPC. Proximity labeling proteomics showed that GBPL3 not only interacts with NB and nucleoskeleton proteins but also is associated with a large number of chromatin remodelers, transcription regulators, and components of RNA splicing and processing machinery, potentially facilitating transcription regulation at the NB. The gbpl3 mutants display stunted growth and sterility. More importantly, the combination of different nucleoskeleton mutants with gbpl3 mutant resulted in distinct development- and stress-related phenotypes and aberrant transcription of stress-related genes, suggesting a role of GBPL3 in connection with different nucleoskeleton components to differentially contribute to gene expression regulation at the NPC basket.

Compared to animals and yeasts, the function of NB in plants is far less well understood. The Arabidopsis TPR homolog, NUA, has been shown essential for the total mRNA export. Compromised NUA function led to pleiotropic developmental phenotypes including early flowering, phyllotaxy defects, and reduced fertility [ 38 , 39 ]. The Arabidopsis FG repeats-containing nucleoporin Nup136 is likely a functional homolog of vertebrate Nup153 and has been reported to be involved in total mRNA export as well as microRNA biogenesis through its direct interaction with the TREX-2 complex [ 40 , 41 ]. Nup82 is a paralog of Nup136 and is plant specific. Nup82 and Nup136 interact with each other in the NB and are redundantly required for activation of SA-mediated pathogen resistance in Arabidopsis [ 42 ]. Nup136 and Nup82 are thought to share a common evolutionary history with a plant-specific nucleoskeleton protein KAKU4, and conserved motifs in these 3 proteins mediate physical interaction with CROWDED NUCLEIs (CRWNs), filamentous proteins that compose the nucleoskeleton in Arabidopsis [ 43 ]. This finding provides an intriguing molecular mechanism for the association between the NB and the nucleoskeleton in plants. Nevertheless, the functional significance of the NB-nucleoskeleton association and how the NPC basket is involved in transcriptional regulation remain largely unknown in plants.

Besides interacting with the chromatin, the NB is also tightly associated with the nucleoskeleton distributed underneath the nuclear envelope. Cryo-electron tomography and super-resolution imaging analyses revealed that the NPC basket makes close contact with lamin filaments in animals [ 33 – 35 ]. TPR and Nup153 were reported to bind directly with lamin proteins, which is important for the proper maintenance of NPC distribution, lamin structure, and chromatin architecture [ 36 , 37 ].

Among the principal NPC modules, the NB protrudes into the nucleoplasm and establishes intimate connections with inner nuclear peripheral structures. Accumulating evidence supports that the NB is a multifunctional platform that plays versatile roles beyond mediating cargo transport [ 16 ]. For example, the conserved NB scaffolding nucleoporin TPR (in animals) or Mlp1/Mlp2 (in yeasts) forms large coiled-coil (CC) homo/heterodimers that link the NPC with the underlying chromatin [ 17 , 18 ]. Chromatin tethering to the NB is thought to be critical for regulating transcription activity, a phenomenon known as gene gating [ 19 , 20 ]. In yeasts, Mlp proteins selectively recruit inducible genes by binding to their promoter regions and avoiding R-loop formation, a process that is critical for maintaining transcriptional memory and facilitating messenger ribonucleoprotein (mRNP) biogenesis [ 21 , 22 ]. It has been reported that a significant number of yeast transcription factors (TFs) and chromatin remodelers are able to target a chromosome region to the NPC [ 23 ], and core transcription machinery, such as the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex and the Mediator complex of RNA polymerase II, are enriched at the NB [ 24 , 25 ]. In metazoans, the NB-localized FG nucleoporin Nup153 regulates transcription and chromatin organization by interacting with chromatin architectural proteins and mediates their binding to cis-regulatory elements and topologically associating domains [ 26 ]. These and other findings collectively highlight the role of the NB in tethering the genome and regulating its activity. Moreover, TPR and Mlps are also capable of recruiting mRNPs and the TREX-2 (Transcription Elongating and RNA Export) mRNA export complex, presumably to facilitate mRNA processing and export following active transcription at the NB [ 27 – 29 ]. Supporting this notion, mutations in TPR and Mlp1/2p lead to nuclear retention of mis-spliced or aberrant mRNAs [ 30 – 32 ].

The nuclear pore complex (NPC) is a structurally conserved multiprotein assembly embedded within the nuclear envelope and forms the main gateway to allow the nucleocytoplasmic exchange of macromolecules. The NPC is constructed by 500 to 1,000 nucleoporin proteins (Nups) of approximately 40 different types in multiple copies [ 1 – 3 ], which are assembled into 4 principal NPC modules: the core scaffold, the transmembrane ring, the nuclear basket (NB), and the selective barrier [ 4 – 6 ]. The core scaffold forms the NPC central channel by stacking ring-shaped protein complexes, namely the inner ring complex (IRC) and outer ring complexes (ORC) that sandwich the IRC. The core scaffold makes direct contact with the transmembrane ring, which anchors the NPC to the pore membrane [ 7 , 8 ]. On the nuclear side, 8 flexible proteinaceous filaments project from the ORC and join at a distal ring to form the NB [ 9 – 11 ]. Various types of nucleoporins containing intrinsically disordered Phe-Gly (FG) repeat motifs cover the inside surface of the core scaffold and extend to the NB to form the selective barrier and enable karyopherin-mediated nuclear transport [ 12 – 15 ].

Results

GBPL3 is a candidate component of the nuclear basket/nucleoskeleton protein network Previously, we used subtractive proteomics and proximity labeling proteomics to profile the plant nuclear envelope (NE)-associated proteome in Arabidopsis [44,45]. Among the identified candidates, the GUANYLATE-BINDING PROTEIN-LIKE 3 (GBPL3) protein attracted our attention. Although GBPL3 contains no predictable transmembrane (TM) domains, it was one of the top-ranked NE-associated proteins identified by the subtractive proteomics analysis (fold change = 580 and p-value = 1.2 × 10−9) with a high peptide-spectrum match (PSM) score (S1A Fig), suggesting that GBPL3 is an NE protein candidate of high confidence and likely an abundant component of the plant NE. On the other hand, proximity labeling proteomics revealed that GBPL3 was probed by multiple inner nuclear membrane baits, including SUN1, MAN1, PNET2_A, and PNET2_B, by main nucleoskeleton proteins, including CRWN1 and KAKU4, and by the NPC basket nucleoporin Nup82 (Fig 1A). In contrast, outer nuclear membrane protein baits, including SINE1 and WIP1, did not probe GBPL3. This evidence suggests that GBPL3 is likely a protein associated with the inner nuclear membrane. PPT PowerPoint slide

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TIFF original image Download: Fig 1. GBPL3 is a nuclear envelope protein located at the NPC basket. (A) Bubble plot showing the reanalysis of the mass spectrometry data retrieved from previously published proximity labeling proteomics using different baits (PXD015919 and PXD026924). LFQ intensity values of GBPL3 protein are plotted. A heatmap of normalized PSM values of GBPL3 are shown on the right. For bait protein SUN1, SINE1, Nup82, and WIP1, biotin-treated NEAP1-BioID2 (Ctrl 1) and mock-treated YFP-BioID2 (Ctrl 2) were used as controls. For bait protein MAN1, PNET2_A, PNET2_B, CRWN1, and KAKU4, non-transformant plants (NT) (Ctrl 1) and mock-treated YFP-BioID2 (Ctrl 2) were used as controls. (B) Weighted gene coexpression heatmap of GBPL3 with all known genes of nucleoporin and nucleoskeleton in Arabidopsis. Mutual rank-based gene expression data using microarray and RNA-seq were retrieved from ATTED-II (version 11.0) and converted into network edge weights before visualizing into a clustered heatmap. (C) Prediction of CC domains and IDRs by Smart (http://smart.embl-heidelberg.de/) and D2P2 (https://d2p2.pro/), respectively. (D) Fluorescence imaging showing subcellular localization of GBPL3 protein in root cells. One-week-old pGBPL3-mEGFP-GBPL3 transgenic plants with higher expression (left panel) and lower expression (right panel) levels are used for imaging. Bars = 10 μm. (E) Z-stack imaging followed by projection of root cells expressing mEGFP-GBPL3 in a line with lower expression. Bar = 10 μm. (F) Anti-GFP immunogold labeling and transmission electron microscopy analysis of pGBPL3-mEGFP-GBPL3 transgenic seedlings with low expression. Root tip cells were imaged. Red arrows indicate gold particles, and blue arrowhead pairs indicate nuclear pores. Bar = 1 μm. All underlying data in Fig 1 can be found in S5 Data. CC, coiled-coil; IDR, intrinsically disordered region; INM, inner nuclear membrane; LFQ, label-free quantitation; NLS, nuclear localization signal; NPC, nuclear pore complex; ONM, outer nuclear membrane; PSM, peptide-spectrum match. https://doi.org/10.1371/journal.pbio.3001831.g001 To investigate the potential connection between GBPL3 and nuclear envelope components, we analyzed the transcriptional correlation of GBPL3 with genes that encode the nucleoskeleton and nucleoporins in Arabidopsis using ATTED-II [46]. The clustered heatmap showed that GBPL3 is strongly coexpressed and clustered into a small group with 4 nucleoskeleton genes CRWN1/2/3/4 and the NB gene NUA (Fig 1B). This result suggests a potential connection of GBPL3 with the nucleoskeleton and/or the NPC basket. GBPL3 is conserved among eukaryotes and is an ortholog of human GBPs with 2 paralogs (GBPL1 and GBPL2) in Arabidopsis (S1B Fig and S1 Data). Besides the canonical N-terminal GTPase domain and the C-terminal helical domain (GBP_C) preserved in all GBPs, many plant GBPL proteins, including GBPL3, evolved a unique C-terminal extension that is annotated as a CC region but is also predicted to be intrinsically disordered (Figs 1C and S1B). Compared to ordered CC domains, disordered CC domains were found overrepresented in human proteins that function in actin filaments, cell junctions, macromolecular complexes, and nucleolus [47]. The disordered CC region of GBPL3 was recently demonstrated to mediate liquid–liquid phase separation (LLPS) of the protein [48]. Interestingly, part of the GBPL3 CC domain (562–784 aa) was predicted to have a structural similarity with the human Lamin_A protein (42–215 aa) by SWISS-MODEL (https://swissmodel.expasy.org) (S1C Fig), and in both Arabidopsis and humans, disordered CC domains are prevalent in the scaffolding NB protein (AtNUA/HsTPR) and nucleoskeleton protein (AtCRWN1/HsLamin_A) (Fig 1C), suggesting that this feature may be important for the NB and nucleoskeleton function. Together, the strong coexpression and domain similarity with NB and nucleoskeleton components led us to hypothesize that GBPL3 is functionally related to them.

GBPL3 localizes to the NPC To validate this hypothesis, we first verified the subcellular localization of GBPL3. We generated Arabidopsis stable transgenic lines expressing mEGFP-GBPL3 fusion protein. Although the construct was driven by the GBPL3 native promoter, we were able to detect a difference in the mEGFP-GBPL3 expression level in independent transgenic lines based on immunoblots (S1D Fig), potentially due to different copy number or insertion loci of the transgene. Using confocal fluorescence microscopy, we found that in most transgenic lines with relatively low expression, mEGFP-GBPL3 is enriched at the nuclear membrane with some signal in the nucleoplasm (Fig 1D). However, in transgenic lines with high expression levels, mEGFP-GBPL3 stained the nuclear rim in a relatively weak manner but predominantly labeled bright and large droplet-like condensates at the nuclear periphery (Fig 1D), in agreement with the previous report that GBPL3 is capable of undergoing LLPS and forming biomolecular condensates upon overexpression [48]. These GBPL3 localization patterns were also observed in mCherry-GBPL3 lines, confirming that the observation is not tag specific (S1E Fig). Because transgenic lines displaying both localization patterns can fully complement gbpl3 mutant phenotypes (see below), we conclude that GBPL3 is functionally localized to the nuclear periphery but forming large condensates is not essential for GBPL3 function, at least under normal conditions. However, we cannot exclude that LLPS of GBPL3 may still occur at a level beyond detection by fluorescence microscope in low expression lines. When we performed z-stack imaging of root cells using low expression lines, small punctate structures were captured at the nuclear surface (Fig 1E), similar to those formed by some plant nucleoporins (e.g., CPR5 and Nup155) [49], suggesting that GBPL3 is enriched in the NPC. To confirm this, we performed immunogold labeling and transmission electron microscopy using mEGFP-GBPL3 transgenic lines with low expression. In nearly all samples examined, we found that gold particles are predominantly associated with the inner nuclear membrane, close to nuclear envelope openings indicative of nuclear pores (Figs 1F and S1F). Together, these results support that GBPL3 localizes at the inner nuclear membrane and is highly enriched at the NPC.

GBPL3 physically interacts with both the nuclear basket and the nucleoskeleton To understand the function of GBPL3 at the nuclear periphery, we performed proximity labeling proteomics using GBPL3 as bait to profile the in vivo GBPL3 proxitome. GBPL3 was fused with TurboID, an engineered promiscuous biotin ligase that mediates efficient biotinylation of proximal proteins. The fusion construct was driven by the GBPL3 native promoter, and we showed separately that this promoter can drive ubiquitous expression of GUS in Arabidopsis seedlings (except in the hypocotyl) (S2A Fig). For proximity labeling, we chose 1-week-old pGBPL3-GBPL3-TurboID-3HA transgenic seedlings from a line with relatively low GBPL3 expression and treated them with free biotin for 4 h followed by biotin depletion. The resulting biotinylated protein collection was affinity purified using streptavidin-coated beads. Two biological replicates were used, and wild-type non-transgenic plants were sampled in parallel as control. Immunoblot using 2% affinity-purified samples confirmed inducible and efficient protein biotinylation by GBPL3-TurboID, including GBPL3 itself (S2B Fig). Samples were then digested with trypsin on beads and purified before being subjected to label-free quantitative mass spectrometry (LFQMS), from which we collected both label-free quantitation (LFQ) intensity and PSM values. By applying fold-change > 4 and p-value < 0.01 as cutoffs compared to control samples, we identified 243 and 216 significantly enriched proteins using LFQ and PSM data, respectively (Fig 2A and S2 Data). We merged the 2 datasets and obtained 270 unique protein candidates, of which more than 70% are present in both datasets. PPT PowerPoint slide

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TIFF original image Download: Fig 2. GBPL3 physically interacts with NPC basket and nucleoskeleton components. (A) Volcano plots of candidates identified by proximity labeling proteomics using GBPL3 as bait. Analyses based on the LFQ intensity and PSM data are shown on the left and right sides, respectively. The overlap of candidates identified by both analyses is shown in the middle. Non-transformants (WT) treated with biotin were used as control, and 2 biological replicates are included in the analysis. Significantly enriched proteins were selected using PSM > 4, fold-change > 4, and p-value < 0.01 as cutoffs and represented by red dots. The nuclear basket and nucleoskeleton components were text labeled. Underlying data can be found in S5 Data. (B) Heatmap showing normalized and averaged PSM values of all nucleoskeleton and nucleoporin proteins identified by GBPL3, Nup82, and KAKU4 using proximity labeling proteomics. Blue and green dots indicate p-value < 0.01 and 0.05, respectively. Nup82 and KAKU4 data were retrieved from PXD015919 and PXD026924, respectively. (C) A model diagram showing that the nuclear basket and the nucleoskeleton comprise a protein meshwork underneath the NPC and the position of GBPL3 at a more distal region of the NB. (D) Transient protein expression of GBPL3 and NPC basket components Nup136/Nup82 and nucleoskeleton components CRWN1/CRWN3 in N. benthamiana. The upper 2 panels show individual protein expression, and the lower 4 panels show mCherry-GBPL3 coexpression with CRWN1-YFP, CRWN3-GFP, Nup136-YFP, or Nup82-GFP. Bars = 10 μm. (E) Yeast-two-hybrid analysis using GBPL3-C (GBP_C domain and IDRs) and Nup82 as the bait and Nup136, Nup82, CRWN1, CRWN2, CRWN3 as the prey. Zygote yeasts were grown on DDO (SD/–Leu/–Trp) and QDO (SD/–Leu/–Trp/–His/–Ade) media. Empty vectors were used as negative controls. (F) BiFC assay between GBPL3 and CRWN1, KAKU4, Nup136, or Nup82. Indicated BiFC constructs were transiently expressed in N. benthamiana leaf epidermal cells. Free-mCherry was coexpressed to label the nuclei. Bars = 10 μm. BiFC, bimolecular fluorescence complementation; LFQ, label-free quantitation; NB, nuclear basket; NPC, nuclear pore complex; PSM, peptide-spectrum match. https://doi.org/10.1371/journal.pbio.3001831.g002 Among the significantly enriched candidates, we found all 5 known nucleoskeleton proteins in Arabidopsis (CRWN1, CRWN2, CRNW3, CRWN4, and KAKU4). Remarkably, GBPL3 also specifically probed all reported NB components (NUA, Nup136, Nup82, Nup50a, Nup50b, and Nup50c) but barely labeled other NPC constituents (Fig 2B). This result provides strong support that GBPL3 is part of the intimately associated NB and nucleoskeleton protein meshwork underneath the NPC. Consistently, the FG basket nucleoporin Nup82 probed GBPL3, nucleoskeleton proteins, and other basket nucleoporins (Fig 2B). However, Nup82 also probed a significant number of core scaffold nucleoporins, suggesting that compared to Nup82, GBPL3 may polarly localize to a more distal region of the NB away from the NPC core (Fig 2C). To confirm the association between GBPL3 with the NPC basket and the nucleoskeleton, we transiently coexpressed GBPL3 with the NB nucleoporin Nup136 and Nup82 or nucleoskeleton protein CRWN1 and CRWN3 in N. benthamiana. Overexpression of Nup136 or Nup82 alone led to the formation of condensates at the nuclear periphery (Fig 2D), consistent with a recent report [43]. Those condensates overlapped with GBPL3 condensates (Fig 2D), suggesting that GBPL3 can form condensates at the NPC basket. On the other hand, CRWN1 and CRWN3 did not form condensates when expressed alone; however, they were recruited to GBPL3 condensates upon coexpression (Fig 2D). To determine whether GBPL3 directly interacts with Nup82/Nup136 or CRWNs, we performed yeast two-hybrid (Y2H) assays. While Nup136 displayed auto-activation as bait, we found that GBPL3 C-terminus, including the GBP_C and the disordered CC domain, is sufficient to interact with CRWN2 and weakly interact with Nup82 in yeasts (Fig 2E). The NB scaffolding nucleoporin NUA was not included in the Y2H assay because we were not able to obtain the full-length cDNA of the gene due to its large size. Lastly, we performed bimolecular fluorescence complementation (BiFC) assay and confirmed the in vivo association between the full-length GBPL3 and Nup82/Nup136/CRWNs, which occurs in condensate structures (Fig 2F). Together, these results led us to conclude that GBPL3 physically associates with both the NB and the nucleoskeleton in plants.

GBPL3 can recruit the transcription apparatus and RNA processing machinery Besides NB and nucleoskeleton components, gene ontology (GO) analysis revealed that GBPL3 proxitome is highly enriched in proteins that function in chromatin modification and organization, transcription initiation and regulation, and RNA splicing and processing (Fig 3A), which account for over 65% of the identified GBPL3 proxitome (177/270). To better categorize the function of these proteins, we built a protein–protein interaction network of these 177 GBPL3 interactors using the STRING database, which further classified them into 14 protein complexes or functional groups with 2 main molecular functions: transcription (83 proteins) and RNA processing (94 proteins) (Fig 3B). The transcription-related category includes chromatin remodelers, histone modifiers, transcription initiator factor II (TFII) proteins, key transcription coactivators—the Mediator complex and the SAGA complex, as well as TFs and co-regulators that regulate major phytohormone responses, including auxin, gibberellin, cytokinin, salicylic acid, and jasmonic acid. The RNA processing category contains RNA helicases, mRNA splicing machinery, and other RNA processing proteins. Notably, these proteins are not enriched in the Nup82 proxitome (S3A Fig), suggesting that the GBPL3-defined NB region may be specifically enriched with molecular apparatus that is involved in active chromatin remodeling, transcription activation/repression, and RNA processing. PPT PowerPoint slide

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TIFF original image Download: Fig 3. GBPL3 is capable of recruiting transcription regulators and RNA processing machinery. (A) GO enrichment analysis of GBPL3 proxitome. GO terms under the 2 major molecular functions identified (transcription and RNA processing) are presented in a bubble plot. Underlying data can be found in S5 Data. (B) GBPL3 PPI network that contains 177 GBPL3 proximal proteins involved in transcription and RNA processing identified in (A). Cytoscape was used to construct and visualize the GBP3 PPI network. GBPL3 interactors were clustered and color-coded manually based on published protein functions. Node sizes represent scores of PPIs retrieved from the STRING database (https://string-db.org/). (C) Transient expression of selected GBPL3 proximal proteins involved in transcription regulation and RNA processing in N. benthamiana. The left column shows the individual expression of GFP-tagged GBPL3 proximal proteins, and the right 3 columns show coexpression with mCherry-GBPL3. CELL DIVISION CYCLE (CDC5), a putative TF, was included as a negative control. Bars = 10 μm. GO, gene ontology; PPI, protein–protein interaction; TF, transcription factor. https://doi.org/10.1371/journal.pbio.3001831.g003 To confirm the association of GBPL3 with these transcription and RNA processing factors, we selected 11 well-characterized candidates, which have been reported to participate in the regulation of plant development and responses to stress-related phytohormones and various environmental stimuli [50–60]. They include chromatin remodelers ACTIN-RELATED PROTEIN 4 (ARP4), ANTHESIS PROMOTING FACTOR 1 (APRF1), and PWWP DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1), transcription regulators TBP-ASSOCIATED FACTOR 5 (TAF5), ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1), ABA-RESPONSIVE KINASE SUBSTRATE 2 (AKS2), C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1), and TOPLESS-RELATED 1 (TPR1), and mRNA binding or processing proteins F OLIGOURIDYLATE BINDING PROTEIN 1C (UBP1C), SUPPRESSORS OF MEC-8 AND UNC-52 1 (SMU1), and SNW/SKI-INTERACTING PROTEIN (SKIP). We fused these proteins with mEGFP and transiently expressed them in N. benthamiana. When expressed alone, 6 of the fusion proteins formed spontaneous nuclear condensates while the other 5 diffused in the nucleus (Fig 3C). However, all of them colocalized with GBPL3-containing nuclear condensates upon coexpression with mCherry-GBPL3 (Fig 3C), suggesting that GBPL3 is capable of recruiting those chromatin remodelers, transcription regulators, and mRNA processing machinery to the nuclear periphery. These recruitment events were observed in the form of condensates with a wide range of sizes during transient overexpression (Fig 3C). Therefore, it is reasonable to speculate that GBPL3 may recruit those factors to the NB through LLPS. In line with this idea, most of these proteins contain intrinsically disordered domains predicted by multiple algorithms (S3B Fig). Together, these results support a hypothesis that the NPC basket region in plant cells is associated with active transcription reprogramming coupled with mRNA processing, and NB-associated GBPL3 appears to be sufficient for recruiting relevant molecular apparatus for this process.

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