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1.
Physiol Plant ; 176(3): e14351, 2024.
Article in English | MEDLINE | ID: mdl-38779764

ABSTRACT

Fluorescent labelling of proteins enables the determination of their spatiotemporal localization but, sometimes, it can perturb their activity, native localization, and functionality. Spot-tag is a12-amino acid peptide recognized by a single-domain nanobody and could potentially resolve the issues associated with large fluorescence tags due to its small size. Here, using as an example the microtubule motor CENTROMERIC PROTEIN E-RELATED KINESIN 7.3 (KIN7.3), we introduce the spot-tag for protein labelling in fixed and living plant cells. Spot-tagging and detection by an anti-spot nanobody of ectopically expressed KIN7.3 did not interfere with its native localization. Most importantly, our spot-tagging pipeline facilitated the localization of KIN7.3 much more rapidly and likely accurately than labelling with large fluorescent proteins or even immunolocalization approaches. We should, though, note some limitations we have not resolved yet. Spot-tagging is functional only in fixed cells; it is available only as two fluorophores and may create a noisy background during imaging. However, we foresee that, besides the limitations of this method, spot-tagging will apply to many proteins, offsetting activity perturbations and low photon quantum yields of other protein-tagging approaches.


Subject(s)
Plant Cells , Plant Cells/metabolism , Kinesins/metabolism , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/genetics
2.
Plant Cell ; 36(3): 559-584, 2024 Feb 26.
Article in English | MEDLINE | ID: mdl-37971938

ABSTRACT

Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because these subcellular structures lack a delineating membrane, determining their compositions is difficult. Here we describe a proximity-biotinylation approach for capturing the RNAs of the condensates known as processing bodies (PBs) in Arabidopsis (Arabidopsis thaliana). By combining this approach with RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal conditions and heat stress. PBs showed a much more dynamic RNA composition than the total transcriptome. RNAs involved in cell wall development and regeneration, plant hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA-binding proteins and the liquidity of PBs modulated RNA recruitment, while RNAs were frequently recruited together with their encoded proteins. In PBs, RNAs follow distinct fates: in small liquid-like PBs, RNAs get degraded while in more solid-like larger ones, they are stored. PB properties can be regulated by the actin-polymerizing SCAR (suppressor of the cyclic AMP)-WAVE (WASP family verprolin homologous) complex. SCAR/WAVE modulates the shuttling of RNAs between PBs and the translational machinery, thereby adjusting ethylene signaling. In summary, we provide an approach to identify RNAs in condensates that allowed us to reveal a mechanism for regulating RNA fate.


Subject(s)
Arabidopsis , RNA , Processing Bodies , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Heat-Shock Response , Arabidopsis/genetics , Arabidopsis/metabolism
3.
PLoS Biol ; 21(9): e3002305, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37721949

ABSTRACT

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

4.
Trends Plant Sci ; 28(10): 1101-1112, 2023 10.
Article in English | MEDLINE | ID: mdl-37183142

ABSTRACT

Cellular condensation is a reinvigorated area of study in biology, with scientific discussions focusing mainly on the forces that drive condensate formation, properties, and functions. Usually, condensates are called 'membrane-less' to highlight the absence of a surrounding membrane and the lack of associated contacts. In this opinion article we take a different direction, focusing on condensates that may be interfacing with membranes and their possible functions. We also highlight changes in condensate material properties brought about by condensate-membrane interactions, proposing how condensates-membrane interfaces could potentially affect interorganellar communication, development, and growth, but also adaptation in an evolutionary context. We would thus like to stimulate research in this area, which is much less understood in plants compared with the animal field.


Subject(s)
Cell Membrane , Plant Cells , Plants , Plant Cells/physiology
5.
Plant Cell ; 35(9): 3187-3204, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37162152

ABSTRACT

Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, 2 of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.


Subject(s)
Biomolecular Condensates , Proteome
6.
EMBO J ; 42(9): e111885, 2023 05 02.
Article in English | MEDLINE | ID: mdl-36741000

ABSTRACT

Cellular condensates can comprise membrane-less ribonucleoprotein assemblies with liquid-like properties. These cellular condensates influence various biological outcomes, but their liquidity hampers their isolation and characterization. Here, we investigated the composition of the condensates known as processing bodies (PBs) in the model plant Arabidopsis thaliana through a proximity-biotinylation proteomics approach. Using in situ protein-protein interaction approaches, genetics and high-resolution dynamic imaging, we show that processing bodies comprise networks that interface with membranes. Surprisingly, the conserved component of PBs, DECAPPING PROTEIN 1 (DCP1), can localize to unique plasma membrane subdomains including cell edges and vertices. We characterized these plasma membrane interfaces and discovered a developmental module that can control cell shape. This module is regulated by DCP1, independently from its role in decapping, and the actin-nucleating SCAR-WAVE complex, whereby the DCP1-SCAR-WAVE interaction confines and enhances actin nucleation. This study reveals an unexpected function for a conserved condensate at unique membrane interfaces.


Subject(s)
Actins , Arabidopsis Proteins , Arabidopsis , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Processing Bodies
8.
Trends Plant Sci ; 28(2): 142-153, 2023 02.
Article in English | MEDLINE | ID: mdl-36404175

ABSTRACT

Recent evidence sheds light on the peculiar type of plant intelligence. Plants have developed complex molecular networks that allow them to remember, choose, and make decisions depending on the stress stimulus, although they lack a nervous system. Being sessile, plants can exploit these networks to optimize their resources cost-effectively and maximize their fitness in response to multiple environmental stresses. Even more interesting is the capability to transmit this experience to the next generation(s) through epigenetic modifications that add to the classical genetic inheritance. In this opinion article, we present concepts and perspectives regarding the capabilities of plants to sense, perceive, remember, re-elaborate, respond, and to some extent transmit to their progeny information to adapt more efficiently to climate change.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Epigenesis, Genetic/genetics , Plants/genetics , Epigenetic Memory , Stress, Physiological/genetics
9.
Plant Cell ; 34(9): 3400-3424, 2022 08 25.
Article in English | MEDLINE | ID: mdl-35640532

ABSTRACT

For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host's pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.


Subject(s)
Arabidopsis , Xanthomonas campestris , Bacterial Proteins , Plant Diseases , Plant Immunity , Virulence
10.
Front Genet ; 13: 818727, 2022.
Article in English | MEDLINE | ID: mdl-35251130

ABSTRACT

Crop adaptation to climate change is in a part attributed to epigenetic mechanisms which are related to response to abiotic and biotic stresses. Although recent studies increased our knowledge on the nature of these mechanisms, epigenetics remains under-investigated and still poorly understood in many, especially non-model, plants, Epigenetic modifications are traditionally divided into two main groups, DNA methylation and histone modifications that lead to chromatin remodeling and the regulation of genome functioning. In this review, we outline the most recent and interesting findings on crop epigenetic responses to the environmental cues that are most relevant to climate change. In addition, we discuss a speculative point of view, in which we try to decipher the "epigenetic alphabet" that underlies crop adaptation mechanisms to climate change. The understanding of these mechanisms will pave the way to new strategies to design and implement the next generation of cultivars with a broad range of tolerance/resistance to stresses as well as balanced agronomic traits, with a limited loss of (epi)genetic variability.

12.
Plant Physiol ; 188(2): 1043-1060, 2022 02 04.
Article in English | MEDLINE | ID: mdl-34633458

ABSTRACT

In plants, auxin transport and development are tightly coupled, just as hormone and growth responses are intimately linked in multicellular systems. Here we provide insights into uncoupling this tight control by specifically targeting the expression of TINY ROOT HAIR 1 (TRH1), a member of plant high-affinity potassium (K+)/K+ uptake/K+ transporter (HAK/KUP/KT) transporters that facilitate K+ uptake by co-transporting protons, in Arabidopsis root cell files. Use of this system pinpointed specific root developmental responses to acropetal versus basipetal auxin transport. Loss of TRH1 function shows TRHs and defective root gravitropism, associated with auxin imbalance in the root apex. Cell file-specific expression of TRH1 in the central cylinder rescued trh1 root agravitropism, whereas positional TRH1 expression in peripheral cell layers, including epidermis and cortex, restored trh1 defects. Applying a system-level approach, the role of RAP2.11 and ROOT HAIR DEFECTIVE-LIKE 5 transcription factors (TFs) in root hair development was verified. Furthermore, ERF53 and WRKY51 TFs were overrepresented upon restoration of root gravitropism supporting involvement in gravitropic control. Auxin has a central role in shaping root system architecture by regulating multiple developmental processes. We reveal that TRH1 jointly modulates intracellular ionic gradients and cell-to-cell polar auxin transport to drive root epidermal cell differentiation and gravitropic response. Our results indicate the developmental importance of HAK/KUP/KT proton-coupled K+ transporters.


Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Indoleacetic Acids/metabolism , Plant Roots/anatomy & histology , Plant Roots/growth & development , Plant Roots/genetics , Plant Roots/metabolism , Potassium/metabolism , Biological Transport/drug effects , Biological Transport/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Gene Expression Regulation, Plant , Genes, Plant , Ion Transport/genetics
13.
Methods Mol Biol ; 2382: 233-243, 2022.
Article in English | MEDLINE | ID: mdl-34705243

ABSTRACT

Membrane trafficking is central to cell plate construction during plant cytokinesis. Studies on cell plate formation can provide answers to basic biology questions including molecular mechanisms of membrane trafficking, tissue patterning, and cytoskeletal dynamics. Consequently, a detailed understanding of cytokinesis depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles and delivery of proteins to the developing and maturing plate. This chapter describes a pipeline based on fluorescence recovery after photobleaching (FRAP) to measure and analyze turnover of peripheral or transmembrane proteins on the cell plate. The approach described here can also be applied in other biological contexts.


Subject(s)
Cytokinesis , Cytoplasm , Fluorescence Recovery After Photobleaching
14.
EMBO J ; 40(17): e105043, 2021 09 01.
Article in English | MEDLINE | ID: mdl-34287990

ABSTRACT

Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling.


Subject(s)
Cytoplasmic Granules/metabolism , Intrinsically Disordered Proteins/metabolism , Protein Interaction Maps , Arabidopsis , Arabidopsis Proteins/metabolism , Binding Sites , Heat-Shock Response , Intrinsically Disordered Proteins/chemistry , Protein Binding , Protein Serine-Threonine Kinases/metabolism
15.
Materials (Basel) ; 14(9)2021 Apr 30.
Article in English | MEDLINE | ID: mdl-33946231

ABSTRACT

The outburst of plastic pollution in terrestrial ecosystems poses a potential threat to agriculture and food safety. Studies have already provided evidence for the uptake of plastic microparticles by several plant species, accompanied by numerous developmental effects, using fluorescence labelling techniques. Here, we introduce the implementation of confocal Raman spectroscopy, a label-free method, for the effective detection of microplastics (MPs) accumulation in the roots of a common edible root vegetable plant, Raphanus sativus, after treatment with acrylonitrile butadiene styrene (ABS) powder. We also demonstrate the concomitant occurrence of phenotypic defects in the polymer-treated plants. We anticipate that this work can provide new insights not only into the extent of the impact this widespread phenomenon has on crop plants but also on the methodological requirements to address it.

16.
Int J Mol Sci ; 21(21)2020 Oct 29.
Article in English | MEDLINE | ID: mdl-33138028

ABSTRACT

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.


Subject(s)
Beta vulgaris/immunology , Lipoprotein(a)/pharmacology , Plant Diseases/immunology , Plant Proteins/pharmacology , Protease Inhibitors/pharmacology , Rhizoctonia/physiology , Virulence , Beta vulgaris/drug effects , Beta vulgaris/growth & development , Beta vulgaris/microbiology , Plant Diseases/microbiology , Soil Microbiology
17.
Plant Cell ; 32(11): 3388-3407, 2020 11.
Article in English | MEDLINE | ID: mdl-32843435

ABSTRACT

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.


Subject(s)
Biotin/chemistry , Plant Proteins/metabolism , Protein Interaction Maps , Arabidopsis/cytology , Arabidopsis/metabolism , Biotin/metabolism , Biotinylation , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , Cell Membrane/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lotus/genetics , Lotus/metabolism , Solanum lycopersicum/chemistry , Solanum lycopersicum/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plants, Genetically Modified , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Temperature , Nicotiana/genetics , Nicotiana/growth & development , Nicotiana/metabolism
18.
Proc Natl Acad Sci U S A ; 117(31): 18832-18839, 2020 08 04.
Article in English | MEDLINE | ID: mdl-32709746

ABSTRACT

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.


Subject(s)
NLR Proteins , Plant Immunity , Plant Proteins , Recombinant Fusion Proteins , Signal Transduction , Animals , Inflammasomes/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Mammals , NLR Proteins/chemistry , NLR Proteins/genetics , NLR Proteins/immunology , NLR Proteins/metabolism , Plant Immunity/genetics , Plant Immunity/immunology , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Protein Domains/genetics , Protein Domains/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Signal Transduction/immunology
19.
Mol Cell ; 77(5): 927-929, 2020 03 05.
Article in English | MEDLINE | ID: mdl-32142688
20.
Trends Plant Sci ; 25(4): 325-328, 2020 04.
Article in English | MEDLINE | ID: mdl-32191869

ABSTRACT

Hormonal pathways often converge on transcriptional repressors that can be degraded by the proteasome to initiate a response. We wish to draw attention to developments in a less-explored proteolytic branch called 'limited proteolysis' that, in addition to the classical proteolytic pathways, seems to regulate auxin and ethylene signaling.


Subject(s)
Indoleacetic Acids , Proteasome Endopeptidase Complex , Proteolysis , Transcription Factors
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