A proxitome-RNA-capture approach reveals that processing bodies repress coregulated hub genes.

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.

Stress-related biomolecular condensates in plants.

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.

Plant condensates: no longer membrane-less?

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.

An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome.

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.

Deep inside the epigenetic memories of stressed plants.

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.

The host exocyst complex is targeted by a conserved bacterial type-III effector that promotes virulence.

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.

An Epigenetic Alphabet of Crop Adaptation to Climate Change.

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.

Analyses of Protein Turnover at the Cell Plate by Fluorescence Recovery After Photobleaching During Cytokinesis.

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.

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis.

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.

Do Microplastics Enter Our Food Chain Via Root Vegetables? A Raman Based Spectroscopic Study on Raphanus sativus.

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.

The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response.

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.

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants.

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.

Proteolytic Proteoforms: Elusive Components of Hormonal Pathways?

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.

Proteolysis and nitrogen: emerging insights.

Nitrogen (N) is a core component of fertilizers used in modern agriculture to increase yields and thus to help feed a growing global population. However, this comes at a cost to the environment, through run-off of excess N as a result of poor N-use efficiency (NUE) by crops. An obvious remedy to this problem would therefore be the improvement of NUE, which requires advancing our understanding on N homeostasis, sensing, and uptake. Proteolytic pathways are linked to N homeostasis as they recycle proteins that contain N and carbon; however, emerging data suggest that their functions extend beyond this simple recycling. Here, we highlight roles of proteolytic pathways in non-symbiotic and symbiotic N uptake and in systemic N sensing. We also offer a novel view in which we suggest that proteolytic pathways have roles in N homeostasis that differ from their accepted function in recycling.

Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R-RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector's authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY-domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY-domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a "reversibly closed" conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

Phenotypic novelty by CRISPR in plants.

Genome editing by CRISPR is now routinely used in plant biology for unravelling gene functions and improving agronomical traits. CRISPR opens up the possibility of genome manipulations which would have been unthinkable a few years ago. In this perspective, we discuss and suggest CRISPR-mediated approaches for steering plant development, also highlighting potential challenges.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Cutting in the middleman: hidden substrates at the interface between proteases and plant development.

Contents Summary 916 I. Introduction 916 II. DEK1: towards identification of protease substrates 917 III. Separases: when proteolytic modules attain nonproteolytic functions 918 IV. The peculiar case of a nonredundant subtilisin 919 V. Towards a solution to the protease redundancy problem 920 VI. Matters arising and closing remarks 921 Acknowledgements 921 References 921 SUMMARY: Proteases are integral components of proteome remodelling networks that regulate turnover of proteins and expand their functional diversity. Accumulating evidence highlights the importance of proteases as being central hubs of developmental programs. Yet the molecular pathways that many proteases act on, their natural substrates and their putative nonproteolytic functions remain largely elusive. Here, we discuss recent findings on proteases with functions that converge into plant development regulation, such as DEFECTIVE KERNEL 1 (DEK1), separase and subtilisins, to highlight conspicuous but unexplored aspects of protease biology. We also suggest an exploratory framework for addressing protease functions.