The EMBO Journal

Coronaviruses hijack host membranes to assemble ER-derived double-membrane vesicles (DMVs) that shield viral RNA replication from the cell intrinsic surveillance. Although DMVs morphologically resemble autophagosomes, whether and how autophagy factors actively support their biogenesis has remained elusive. Here, we identify a non-canonical requirement for the autophagy protein LC3C in {beta}-coronavirus replication. Loss of LC3s impaired viral RNA replication, whereas genetic ablation of ATG7 did not, indicating that canonical ATG7-dependent lipidation is dispensable in this context. Reconstitution experiments showed that only LC3C substantially restored replication in LC3-deficient cells and that LC3C phospho-mutants, differing in accessibility to ATG4-mediated processing, displayed distinct proviral activities. Additionally, ATG4D, the main protease responsible for maintaining the LC3 non-lipidated pool, is selectively required for viral replication. Both ATG4D and LC3s depletion triggers formation of aberrant DMV-like structures and potently suppresses SARS-CoV-2 replication. Ultrastructural analysis of nsp3-nsp4-induced membranes showed that depletion of LC3s or ATG4 proteases altered DMV abundance and morphology, supporting a role for the LC3C-ATG4D axis in replication organelle biogenesis. These data establish that {beta}-coronaviruses repurpose ATG4D-driven LC3C de-lipidation for non-canonical LC3 recruitment to replication organelles, identifying the lipidation state of LC3 as a molecular determinant of replication organelle biogenesis and efficient viral replication. HighlightsThe manuscript shows that {beta}-coronavirus replication depends on LC3 proteins and particularly on LC3C in reconstitution experiments, that this dependency is independent of ATG7-mediated lipidation, and that ATG4D promotes efficient replication and replication organelle morphology. Together, the data support a model in which a non-canonical LC3C-ATG4D pathway contributes to DMV biogenesis and viral RNA replication.

MTCH2 (mitochondrial carrier homolog 2) is a noncanonical member of the solute carrier family with five transmembrane (TM) helices, localized to the mitochondrial outer membrane. MTCH2's atypical topology creates a membrane-accessible hydrophilic groove, predicted to be necessary for its protein insertase and lipid scramblase activities. MTCH2 is linked to lipid metabolism and obesity and is required for starvation-induced mitochondrial hyperfusion. Here, we show that MTCH2 is a stable component of a complex containing the Armadillo (ARM) repeat-containing protein, ARMC1, and the DnaJ/Hsp40 chaperone protein, DNAJC11. Protein crosslinking, protein structural modeling, and molecular dynamics simulations demonstrate that the ARMC1 alpha-helical C-terminal domain (CTD) inserts into and stably interacts with the MTCH2 hydrophilic groove and blocks its lipid scramblase activity. We observe that starvation-induced mitochondrial fatty acid oxidation (FAO) is negatively regulated by the ARMC1-MTCH2 interaction. In ARMC1-deficient cells, FAO is stimulated compared to wild-type cells and lipid droplet abundance is significantly reduced. The altered lipid phenotype of ARMC1-/- cells is strictly dependent on MTCH2 and is reversed by ARMC1 expression in a manner dependent on its CTD. Beyond this metabolic axis, we also identify a function for ARMC1 in regulating lysosomal distribution and autophagic flux that is independent of its CTD and interaction with MTCH2. Thus, our data support a model in which the MTCH2-ARMC1 interaction functions as a metabolic switch during starvation to regulate the balance between fat storage and fat burning.

The recruitment and activation of abscission machinery following mitosis is tightly regulated in time and space, yet the mechanisms controlling this process are poorly understood. We find that RNA localization and local translation at the midbody regulates when and where abscission-regulating proteins are expressed. The 3'UTR of NET1 mRNA contains an element that is necessary and sufficient for RNA targeting to the midbody. Mislocalization of NET1 mRNA results in a loss of NET1 protein, a Rho family GEF, throughout the intercellular bridge as well as slower cell proliferation and delayed abscission. This leads to a loss of Arp2/3 at the abscission site and is dependent upon NET1 binding to Rho family GTPases. These findings establish midbody RNA localization and local translation as a key layer of regulation over abscission timing and identify a role for NET1 as a regulator of Arp2/3-mediated branched actin accumulation at the abscission site.

Eukaryotic cells contain multiple biochemically distinct actin filament networks, which enable versatile functions of actin in a range of cellular processes. Yet, the mechanisms by which specific actin filament networks are assembled in a common cytoplasm remain elusive. Here, we investigated how functionally distinct actin nanoscale layers, specified by -actininand tropomyosin isoforms, Tpm1.6 and Tpm3.2, are assembled at focal adhesions. By combining genetic perturbations with mitochondrial-targeting of actin polymerases, we discovered that DAAM1 formin assembles Tpm3.2-actin filaments, whereas Ena/VASP family proteins polymerize -actinin cross-linked actin filament bundles at focal adhesions. Consequently, loss of DAAM1 dampened Tpm3.2 protein levels and impaired focal adhesion disassembly, thus phenocopying Tpm3.2-deficient cells. In contrast, Ena/VASP depletion led to defective focal adhesion maturation and loss of -actinin from focal adhesions. More broadly, our study highlights specific roles for formin and Ena/VASP family proteins in assembling biochemically and functionally distinct linear actin filament arrays in cells.

mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3'-5' helicase activity and pulling on the mRNA from its 3' end.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S- palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

Set promotes cohesion removal in mitosis by evicting phosphorylated Histone H1 and counteracting Sgo1. In addition, Set promotes chromosome alignment by counteracting Aurora B activation. The underlying molecular mechanisms through which Set performs these activities remain insufficiently characterized, but roles of Set as a Histone chaperone and PP2A inhibitor have been proposed. Building on our previous observations that Set promotes pericentromeric Cohesin removal in oocyte meiosis II, we generated an oocyte-specific conditional knock-out of Set to address its functions in meiosis. Similar to mitosis, Set depletion caused chromosome alignment and cohesion defects. We found that Set is required for accurate error correction by localizing Aurora B/C, and for efficient cleavage of the meiosis-specific Cohesin subunit Rec8 by Separase. Paired chromosomes and sister chromatids were often incompletely separated, likely a primary cause of missegregation. Set performed both its roles in a Sgo2-dependent manner, but, unexpectedly, independently of interaction with PP2A-B56. In line with a role of Set as a Histone chaperone, accumulation of phosphorylated Histone H1 in Set knock-out oocytes occurs concomitantly with reduction of oocyte-specific H1foo on chromosome arms, indicating that Set is required to create the optimal chromatin environment for efficient Rec8 cleavage by Separase in meiosis.

Circadian rhythms in mammals are generated by negative feedback loops, in which CLOCK and BMAL1 bind to E-box to activate transcription of Period (Per) and Cryptochrome (Cry) and the E-box-dependent transactivation is inhibited by PER and CRY proteins. Although the core transcriptional feedback loop of the circadian clock has been well defined, how this machinery interfaces with broader nuclear regulatory systems remains incompletely understood. Here, we identify nardilysin (NRDC), a metalloendopeptidase previously implicated in nuclear transcriptional regulation and metabolic homeostasis, as an unexpected modulator of the circadian clock. NRDC deficiency led to elevated PER2 protein levels in the liver and enhanced PER2 dynamics in cell-autonomous circadian oscillators, and was accompanied by a significant shortening of behavioral rhythms in mice. Biochemical analyses demonstrated that NRDC selectively associates with PER2 and CRY2 and antagonizes PER2-mediated repression of CLOCK-BMAL1-dependent transcription. Genome-wide chromatin immunoprecipitation analyses reveal that NRDC is enriched at promoter-proximal E-box-containing regions, frequently co-localizing with CLOCK binding sites. Together, these findings uncover a previously unrecognized link between circadian timing and protease-based nuclear regulation, positioning NRDC as a critical modulator of PER2 function and circadian period determination.

During spermiogenesis, early round spermatids differentiate into specialized spermatozoa through an extensive reshaping of the nucleus driven by coordinated cytoskeletal and chromatin-based mechanisms. In mammals, this process critically relies on the transient manchette, a microtubule-based structure that remodels the spermatid nucleus and serves a track to transport material required for flagellum assembly. However, the existence, organization, and molecular composition of such a structure in other vertebrates have remained poorly investigated. Here, we establish that an organized microtubule network is present in Xenopus spermatids and shares key architectural and molecular hallmarks of the mammalian manchette. We further uncover a large structural heterogeneity of spermatid microtubules, with variable protofilament numbers, skew angles, and lattice organizations. We reveal the presence of a Spaca9-Saxo2 internal scaffold in spermatid microtubules suggesting an internal reinforcement mechanism necessary for extensive nuclear reshaping and cytoplasm remodeling.

Telomeres shield chromosome ends from DNA damage response through T-loops, lariat DNA structures formed and stabilized by the shelterin protein TRF2. During prolonged mitotic arrest, telomeres lose this protection through a process termed mitotic arrest-dependent (MAD) telomere deprotection, which elicits telomere-specific DNA damage signaling in the absence of telomere shortening or chromosome end-to-end fusions. We previously demonstrated that the RecQ helicase BLM promotes MAD telomere deprotection, whereas the related helicase WRN suppresses it independently of its catalytic activities. However, the molecular interplay between BLM and WRN at mitotic telomeres and whether additional recombination-associated enzymes contribute to MAD telomere deprotection have remained unresolved. Here, we identify the Dimerization Helical Bundle in the N-terminal (DHBN) domain of BLM helicase as the critical determinant of MAD telomere deprotection and show that WRN selectively restrains this activity without interfering with BLM's canonical genome-protective functions. We further show that both MUS81 and GEN1 contribute to MAD telomere deprotection. Moreover, the exacerbation of MAD telomere deprotection observed upon TRF2 depletion is strongly attenuated by additional depletion of these enzymes, demonstrating that TRF2 normally protects T-loop junctions from their enzymatic activities. Collectively, our findings reveal how telomeres become selectively vulnerable during prolonged mitotic arrest and uncover a regulated enzymatic mechanism that repurposes recombination machinery at chromosome ends.

Malaria infections are initiated by mosquito bites, during which Plasmodium sporozoites are injected into the host skin. Sporozoites migrate rapidly to find and enter blood capillaries and ultimately invade hepatocytes. Sporozoite migration and invasion is mediated by the transmembrane protein thrombospondin-related anonymous protein (TRAP), which links the extracellular substrate to the actomyosin complex powering gliding motility, while the abundant, GPI-anchored circumsporozoite protein (CSP) covers most of the parasite membrane and modulates adhesion. Both proteins are secreted onto the parasite surface and deposited in a membranous trail originating at the parasite rear. The surface dynamics of these essential sporozoite proteins and the mechanism of deposition, however, are not understood. Here, using orbital total internal reflection fluorescence microscopy (TIRF), we reveal the dynamics of TRAP adhesion site formation and disassembly as well as CSP and TRAP deposition rates. We find that TRAP assembles into distinct adhesion sites, which then undergo retrograde translocation as the sporozoite moves forward. Around half of the TRAP adhesins, together with CSP, remain associated in small membrane droplets on the substrate after the sporozoite has disengaged from the adhesion site. These droplets seem to originate from nanotubes, that presumably decay under high tension. Strikingly, we observe a change in actin filament accumulation if proteolytic cleavage of TRAP is inhibited, providing the first visual evidence for outside-in signaling in sporozoites. Our study reveals a relation between adhesion dynamics and trail formation in Plasmodium sporozoites that might also be relevant for other cell types.

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an alpha-helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an -helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

A missense variant in TM6SF2 (transmembrane 6 superfamily member 2, TM6SF2E167K) is a major risk factor for steatotic liver disease1, while protecting against coronary artery disease2. TM6SF2 is a polytopic resident protein of the smooth endoplasmic reticulum (ER) and ER-Golgi intermediate compartment that promotes lipidation of hepatic ApoB-containing lipoproteins before secretion into the circulation. Here, we used cryo-electron microscopy (cryo-EM) to determine the structures of TM6SF2 and TM6SF2E167K at 3.64 [A] and 3.58 [A] resolution, respectively. TM6SF2 comprises 10 transmembrane helices that bind a single cholesterol molecule within a transmembrane cavity. The protein assembles into homodimers and homotetramers that interact with ApoB. Structural and biochemical analyses show that the E167K substitution reduces cholesterol binding and ApoB interaction without disrupting overall protein structure. Expression of wild-type, but not mutant, TM6SF2 restores hepatic triglyceride secretion in TM6SF2-deficient hepatocytes. Together, these findings establish the first structural framework for the bulk lipidation step in hepatic lipoprotein biogenesis, the principal pathway for hepatic triglyceride and cholesterol export into the circulation.

Heat shock factor 1 (HSF1) is the master transcriptional regulator of cellular response to disrupted cytosolic protein homeostasis. Temperature change, oxidation, and other stresses drive the trimerization and activation of HSF1 to induce expression of molecular chaperones, such as heat shock proteins Hsp70 and Hsp90, which sit at the center of cellular proteostatic networks. In turn, the HSPs and co-chaperones regulate HSF1, but mechanistic details of this cycle remain largely unknown. We developed a FRET-based approach to simultaneously monitor HSF1 conformational change and oligomeric state throughout activation and inactivation. By reconstituting Hsp-HSF1 interactions in vitro, we find that monomerization of HSF1 resembles fibril disassembly through coordinated Hsp40-Hsp70 activity. We then used site-specific photocrosslinking to track HSF1 loading into Hsp90 complexes, Hsp90 cycling, and stress-induced shifts in Hsp90-HSF1 interactions. Whereas Hsp90 inhibitors force 'loading state' type Hsp90-HSF1 interactions, heat shock promotes faster Hsp90 cycling. In this reconstituted system, HSF1-Hsp90 interactions are unexpectedly strongly dependent on the co-chaperone HOP, in contrast to canonical Hsp90 clients. Combining cryo-EM structures of Hsp90-HSF1 loading state and maturation state complexes with crosslinking mass spectroscopy and biophysical experiments, we show that Hsp90 holds HSF1 in a pre-activated, extended monomer state that is primed for trimerization. We propose this state both enhances responsivity but also promotes cytoplasmic-nuclear shuttling through exposure of the NLS. Notably, Hsp90-bound HSF1 can trimerize and bind DNA, placing it on-pathway for transcriptional activation. Together, our results unify previously contradictory view on Hsp90's role in HSF1 regulation. The integrated combination of in vitro reconstitution, photocrosslinking, cryoEM and MS is an exciting new paradigm for the study of many dynamic systems including other complex proteostasis components.

Streptomyces sp. AgN23 is an epiphytic rhizobacterium that establishes in the Arabidopsis rhizosphere by activating plant immune responses. This activity depends on the secretion of polyketide galbonolides, which inhibit host inositol phosphoceramide synthase (IPCS) and thereby perturb sphingolipid homeostasis. However, the downstream signalling events linking IPCS inhibition to AgN23 enrichment in the rhizosphere remain unclear. Here, we show that AgN23 activates ethylene- and salicylic acid-dependent immune signalling, leading to coordinated stimulation of phenylalanine- and tryptophan-derived secondary metabolism. Using Arabidopsis mutants defective in these pathways, we show that these metabolites mitigate AgN23-induced root growth inhibition. We further show that the npr1 mutant is strongly compromised in AgN23-triggered secondary metabolic responses, resulting in reduced rhizosphere colonization by AgN23. By comparing rhizosphere microbiota from wild-type and npr1 plants, we distinguished direct AgN23 effects linked to intermicrobial competition from indirect effects mediated by host metabolic activation. In particular, AgN23 colonization occurred at the expense of several Streptomycetaceae ASVs and coincided with changes in bacterial and fungal taxa belonging to Flavobacteriaceae and Mucoromycota. Together, these findings define a mechanistic framework in which Streptomyces AgN23 interacts with NPR1-dependent signalling to reprogram root metabolism and rhizosphere community structure, notably through the production of specialized metabolites such as galbonolides.