Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation.

The nuclear pore complex (NPC) is vital for nucleocytoplasmic communication. Recent evidence emphasizes its extensive association with proteins of diverse functions, suggesting roles beyond cargo transport. Yet, our understanding of NPC's composition and functionality at this extended level remains limited. Here, through proximity-labelling proteomics, we uncover both local and global NPC-associated proteome in Arabidopsis, comprising over 500 unique proteins, predominantly associated with NPC's peripheral extension structures. Compositional analysis of these proteins revealed that the NPC concentrates chromatin remodellers, transcriptional regulators and mRNA processing machineries in the nucleoplasmic region while recruiting translation regulatory machinery on the cytoplasmic side, achieving a remarkable orchestration of the genetic information flow by coupling RNA transcription, maturation, transport and translation regulation. Further biochemical and structural modelling analyses reveal that extensive interactions with nucleoporins, along with phase separation mediated by substantial intrinsically disordered proteins, may drive the formation of the unexpectedly large nuclear pore proteome assembly.

Nuclear transport receptor KA120 regulates molecular condensation of MAC3 to coordinate plant immune activation.

The nucleocytoplasmic exchange is of fundamental importance to eukaryotic life and is mediated by karyopherins, a superfamily of nuclear transport receptors. However, the function and cargo spectrum of plant karyopherins are largely obscure. Here, we report proximity-labeling-based proteomic profiling of in vivo substrates of KA120, a karyopherin-ß required for suppressing autoimmune induction in Arabidopsis. We identify multiple components of the MOS4-associated complex (MAC), a conserved splicing regulatory protein complex. Surprisingly, we find that KA120 does not affect the nucleocytoplasmic distribution of MAC proteins but rather prevents their protein condensation in the nucleus. Furthermore, we demonstrate that MAC condensation is robustly induced by pathogen infection, which is sufficient to activate defense gene expression, possibly by sequestrating negative immune regulators via phase transition. Our study reveals a noncanonical chaperoning activity of a plant karyopherin, which modulates the nuclear condensation of an evolutionarily conserved splicing regulatory complex to coordinate plant immune activation.

A loyal "G"uard.

G proteins are conserved eukaryotic signal transducers that play crucial roles in plant development and responses to environmental stimuli. In this issue of Cell Host & Microbe, Ma et al. (2022) discover a plant-specific family of kinases that act as bona fide nuclear effectors for G-protein signaling during plant immune activation.

GBPL3 localizes to the nuclear pore complex and functionally connects the nuclear basket with the nucleoskeleton in plants.

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.

Structural analysis of receptor-like kinase SOBIR1 reveals mechanisms that regulate its phosphorylation-dependent activation.

Plant leucine-rich repeat (LRR) receptor-like kinases (RLKs) and LRR receptor-like proteins (RLPs) comprise a large family of cell surface receptors that play critical roles in signal perception and transduction. Both LRR-RLKs and LRR-RLPs rely on regulatory LRR-RLKs to initiate downstream signaling pathways. BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) and SUPPRESSOR OF BIR1-1 (SOBIR1) are important and extensively studied regulatory LRR-RLKs with distinct functions. Although the regulatory mechanism of BAK1 activation has been studied in detail, the activation mechanism of SOBIR1 remains poorly understood. Here, the crystal structures of the catalytically inactive kinase domain of SOBIR1 (SOBIR1-KD) from Arabidopsis thaliana were determined in complexes with AMP-PNP and Mg2+. The results show that SOBIR1-KD contains a uniquely long ß3-αC loop and adopts an Src-like inactive conformation with an unusual architecture at the activation segment, which comprises three helices. Biochemical studies revealed that SOBIR1 is transphosphorylated by BAK1 following its autophosphorylation via an intermolecular mechanism, and the phosphorylation of Thr529 in the activation segment and the ß3-αC loop are critical for SOBIR1 phosphorylation. Further functional analysis confirmed the importance of Thr529 and the ß3-αC loop for the SOBIR1-induced cell death response in Nicotiana benthamiana. Taken together, these findings provide a structural basis for the regulatory mechanism of SOBIR1 and reveal the important elements and phosphorylation events in the special stepwise activation of SOBIR1-KD, the first such processes found in regulatory LRR-RLKs.

Towards understanding inner nuclear membrane protein degradation in plants.

The inner nuclear membrane (INM) hosts a unique set of membrane proteins that play essential roles in various aspects of the nuclear function. However, overaccumulation or malfunction of INM protein has been associated with a range of rare genetic diseases; therefore, maintaining the homeostasis and integrity of INM proteins by active removal of aberrantly accumulated proteins and replacing defective molecules through proteolysis is of critical importance. Within the last decade, it has been shown that INM proteins are degraded in yeasts by a process very similar to endoplasmic reticulum-associated degradation (ERAD), which is accomplished by retrotranslocation of membrane substrates followed by proteasome-dependent proteolysis, and this process was named inner nuclear membrane-associated degradation (INMAD). INMAD is distinguished from ERAD by specific INM-localized E3 ubiquitin ligases and proteolysis regulators. While much is yet to be determined about the INMAD pathway in yeasts, virtually no knowledge of it exists for higher eukaryotes, and only very recently have several critical regulators that participate in INM protein degradation been discovered in plants. Here, we review key molecular components of the INMAD pathway and draw parallels between the yeast and plant system to discuss promising directions in the future study of the plant INMAD process.

Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

A glossary of plant cell structures: Current insights and future questions.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

The emerging role of biomolecular condensates in plant immunity.

Biomolecular condensates are dynamic nonmembranous structures that seclude and concentrate molecules involved in related biochemical and molecular processes. Recent studies have revealed that a surprisingly large number of fundamentally important cellular processes are driven and regulated by this potentially ancient biophysical principle. Here, we summarize critical findings and new insights from condensate studies that are related to plant immunity. We discuss the role of stress granules and newly identified biomolecular condensates in coordinating plant immune responses and plant-microbe interactions.

PNET2 is a component of the plant nuclear lamina and is required for proper genome organization and activity.

The interaction between chromatin and the nuclear lamina (NL) is intrinsically important to the establishment of three-dimensional chromatin architecture and spatiotemporal regulation of gene expression. However, critical regulators involved in this process are poorly understood in plants. Here, we report that Arabidopsis PNET2 and its two homologs are bona fide inner nuclear membrane proteins and integral components of the NL. PNET2s physically interact with the plant nucleoskeleton and engage nucleosome-enriched chromatin at the nuclear periphery. Loss of all three PNET2s leads to severely disrupted growth and development, concomitant activation of abiotic and biotic stress responses, and ultimate lethality in Arabidopsis. The pent2 triple mutant also displays drastic transcriptome changes accompanied by a globally altered chromatin architecture revealed by HiC analysis. Our study identified PNET2 as an inner nuclear membrane (INM) component of the NL, which associates with chromatin and play a critical role in orchestrating gene expression and chromatin organization in plants.

Regulation of Plant Immunity by Nuclear Membrane-Associated Mechanisms.

Unlike animals, plants do not have specialized immune cells and lack an adaptive immune system. Instead, plant cells rely on their unique innate immune system to defend against pathogens and coordinate beneficial interactions with commensal and symbiotic microbes. One of the major convergent points for plant immune signaling is the nucleus, where transcriptome reprogramming is initiated to orchestrate defense responses. Mechanisms that regulate selective transport of nuclear signaling cargo and chromatin activity at the nuclear boundary play a pivotal role in immune activation. This review summarizes the current knowledge of how nuclear membrane-associated core protein and protein complexes, including the nuclear pore complex, nuclear transport receptors, and the nucleoskeleton participate in plant innate immune activation and pathogen resistance. We also discuss the role of their functional counterparts in regulating innate immunity in animals and highlight potential common mechanisms that contribute to nuclear membrane-centered immune regulation in higher eukaryotes.

The BORDER family of negative transcription elongation factors regulates flowering time in Arabidopsis.

Transcription initiation has long been considered a primary regulatory step in gene expression. Recent work, however, shows that downstream events, such as transcription elongation, can also play important roles.1-3 A well-characterized example from animals is promoter-proximal pausing, where transcriptionally engaged Pol II accumulates 30-50 bp downstream of the transcription start site (TSS) and is thought to enable rapid gene activation.2 Plants do not make widespread use of promoter-proximal pausing; however, in a phenomenon known as 3' pausing, a significant increase in Pol II is observed near the transcript end site (TES) of many genes.4-6 Previous work has shown that 3' pausing is promoted by the BORDER (BDR) family of negative transcription elongation factors. Here we show that BDR proteins play key roles in gene repression. Consistent with BDR proteins acting to slow or pause elongating Pol II, BDR-repressed genes are characterized by high levels of Pol II occupancy, yet low levels of mRNA. The BDR proteins physically interact with FPA,7 one of approximately two dozen genes collectively referred to as the autonomous floral-promotion pathway,8 which are necessary for the repression of the flowering time gene FLOWERING LOCUS C (FLC).9-11 In early-flowering strains, FLC expression is repressed by repressive histone modifications, such as histone H3 lysine 27 trimethylation (H3K27me3), thereby allowing the plants to flower early. These results suggest that the repression of transcription elongation by BDR proteins may allow for the temporary pausing of transcription or facilitate the long-term repression of genes by repressive histone modifications.

A karyopherin constrains nuclear activity of the NLR protein SNC1 and is essential to prevent autoimmunity in Arabidopsis.

The nucleotide-binding and leucine-rich repeat (NLR) proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants. The activity of some NLRs, particularly the Toll-like/interleukin-1 receptor (TIR) type, is highly correlated with their nucleocytoplasmic distribution. However, whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear. Here, we identified a nuclear transport receptor, KA120, which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation. We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features. Through a targeted genetic screen using an artificial NLR microRNA library, we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120. Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120-induced autoimmune activation, and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level. Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants. Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed, supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity. Consistently, compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120-induced autoimmunity. Collectively, our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1, possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism.

Exportin-4 coordinates nuclear shuttling of TOPLESS family transcription corepressors to regulate plant immunity.

The regulated nucleocytoplasmic exchange of macromolecules is essential for the eukaryotic cell. However, nuclear transport pathways defined by different nuclear transport receptors (NTRs), including importins and exportins, and their significance in activating distinct stress responses are poorly understood in plants. Here, we exploited a CRISPR/Cas9-based genetic screen to search for modifiers of CONSTITUTIVE EXPRESSION OF PATHOGENESIS-RELATED GENE 5 (cpr5), an Arabidopsis thaliana nucleoporin mutant that activates autoimmune responses that partially mimic effector-triggered immunity (ETI). We identified an NTR gene, Exportin-4 (XPO4), as a genetic interactor of CPR5. The xpo4 cpr5 double mutant activates catastrophic immune responses, which leads to seedling lethality. By leveraging the newly developed proximity-labeling proteomics, we profiled XPO4 substrates and identified TOPLESS (TPL) and TPL-related (TPR) transcription corepressors as XPO4-specific cargo. TPL/TPRs target negative regulators of immunity and are redundantly required for ETI induction. We found that loss-of-XPO4 promotes the nuclear accumulation of TPL/TPRs in the presence of elevated salicylic acid (SA), which contributes to the SA-mediated defense amplification and potentiates immune induction in the cpr5 mutant. We showed that TPL and TPRs are required for the enhanced immune activation observed in xpo4 cpr5 but not for the cpr5 single-mutant phenotype, underscoring the functional interplay between XPO4 and TPL/TPRs and its importance in cpr5-dependent immune induction. We propose that XPO4 coordinates the nuclear accumulation of TPL/TPRs, which plays a role in regulating SA-mediated defense feedback to modulate immune strength downstream of CPR5 during ETI induction.

Structural and functional insight into the nuclear pore complex and nuclear transport receptors in plant stress signaling.

Nuclear pore complexes (NPC) are highly conserved mega protein complexes that penetrate the double-layered nuclear membrane and form channels to allow bi-directional transport of macromolecules between the nucleus and the cytosol. Non-passive nucleocytoplasmic transport also requires nuclear transport receptors (NTR), which bind cargo molecules and shuttle them across the NPC. The NPC and NTRs constitute two fundamental layers of regulatory mechanisms that together determine the selective nuclear translocation of signal molecules and play essential roles in activating the precise response of a cell to environmental stimuli. Here we discuss recent findings in the NPC made by advanced structural biology approaches, and dissect distinct functions of different NPC components and NTRs in plants' responses to various biotic and abiotic stresses.

Proximity labeling proteomics reveals critical regulators for inner nuclear membrane protein degradation in plants.

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.

Global profiling of plant nuclear membrane proteome in Arabidopsis.

The nuclear envelope (NE) is structurally and functionally vital for eukaryotic cells, yet its protein constituents and their functions are poorly understood in plants. Here, we combined subtractive proteomics and proximity-labelling technology coupled with quantitative mass spectrometry to understand the landscape of NE membrane proteins in Arabidopsis. We identified ~200 potential candidates for plant NE transmembrane (PNET) proteins, which unravelled the compositional diversity and uniqueness of the plant NE. One of the candidates, named PNET1, is a homologue of human TMEM209, a critical driver for lung cancer. A functional investigation revealed that PNET1 is a bona fide nucleoporin in plants. It displays both physical and genetic interactions with the nuclear pore complex (NPC) and is essential for embryo development and reproduction in different NPC contexts. Our study substantially enlarges the plant NE proteome and sheds new light on the membrane composition and function of the NPC.