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.

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.

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.

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.

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.

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.

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.

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.

The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At)SCC4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway, and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show the suspensor overproliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo Our findings define the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.

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.

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.

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.

Tudor staphylococcal nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis.

Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress.

Limited and digestive proteolysis: crosstalk between evolutionary conserved pathways.

Contents 958 I. 958 II. 959 III. 960 IV. 962 V. 962 962 References 963 SUMMARY: Proteases can either digest target proteins or perform the so-called 'limited proteolysis' by cleaving polypeptide chains at specific site(s). Autophagy and the ubiquitin-proteasome system (UPS) are two main mechanisms carrying out digestive proteolysis. While the net outcome of digestive proteolysis is the loss of function of protein substrates, limited proteolysis can additionally lead to gain or switch of function. Recent evidence of crosstalk between autophagy, UPS and limited proteolysis indicates that these pathways are parts of the same proteolytic nexus. Here, we focus on three emerging themes within this area: limited proteolysis as a mechanism modulating autophagy; interplay between autophagy and UPS, including autophagic degradation of proteasomes (proteophagy); and specificity of protein degradation during bulk autophagy.

An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity.

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce.

BACKGROUND: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). RESULTS: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. CONCLUSIONS: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.