The Plant Cell

Protoplast isolation is widely used for plant functional genomics and single-cell analyses, but its impact on transcriptional and cell state dynamics remains incompletely understood. Here, we generated time-course RNA-seq data from leaf protoplasts of Arabidopsis, maize, and poplar, sampling at multiple time points following isolation, to systematically characterize global transcriptional dynamics across species. We identified two major drivers of transcriptional variation: a persistent protoplast isolation effect and a progressive time-dependent transcriptional program, which can be divided into early, middle, and late stages corresponding to an immediate stress response, metabolic and chromatin regulation dynamics, and sustained metabolic and proteostasis regulation, together with species-specific differences across stages. We observed a rapid loss of cell-type-specific transcriptional signatures within 6 hours in Arabidopsis and maize, whereas poplar showed a slower decline. Single-nucleus RNA-seq at 6 hours in maize confirmed attenuation of cell-type-specific transcriptional structure. Furthermore, leveraging this time-course dataset enables the identification of aberrant cell states in single-cell RNA-seq data, exemplified by clusters showing elevated activity of protoplast isolation-associated, middle-, and late-stage transcriptional programs characteristic of stress-like states. Together, our results provide a cross-species framework for dissecting protoplast-induced transcriptional and cell state dynamics and facilitate the systematic identification of stress-associated cell states in single-cell transcriptomic data.

O_LIChloroplast division is governed by a conserved protein machinery, yet empirical characterization of these regulators remains limited in rice, a primary target for C4 engineering. Increased chloroplast occupancy in bundle sheath cells is a hallmark of the C4 pathway and so manipulating division is a potential strategy to achieve this goal. C_LIO_LIThrough developmental transcript profiling and image analysis, we identified a discrete window of active chloroplast proliferation in rice leaves, coinciding with peak expression of conserved plastid division genes. Functional characterization via overexpression revealed regulatory behaviours distinct from those in Arabidopsis thaliana. Overexpression of OsFtsZ1&2 resulted in fewer, enlarged chloroplasts per bundle sheath cell, whereas OsMCD1&OsMinE restricted plastid expansion without altering division rates. Conversely, overexpressing OsPDV1&2 or OsARC6&OsDRP5B increased plastid size without affecting total count. When OsPDV1&2 were co-expressed with transcriptional regulator ZmG2, we observed modest increases in chloroplast size alongside reduced stomatal aperture, increased stomatal density, and higher intrinsic water-use efficiency. C_LIO_LIThe results define the temporal landscape of plastid biogenesis in rice and demonstrate divergence across lineages. Our findings suggest that manipulating the division apparatus is insufficient to drive C4-like chloroplast biogenesis in the rice bundle sheath, highlighting the complexity of plastid-host cell coordination in cereals. C_LI

Reconstitution of functional nitrogenase in plants requires the coordinated expression of the [Fe-S] cluster assembly proteins NifU and NifS. However, the extent to which these proteins interact with endogenous Fe-S metabolism and affect plant physiology remains unclear. Here, we compared NifU and NifS homologs from diverse diazotrophs to identify variants compatible with the plant chloroplast environment. Selected variants of Azotobacter vinelandii, Fischerella thermalis, and Marinobacter lutimaris were characterized by transient expression in Nicotiana benthamiana and stable transformation in rice. Plant-produced NifU was largely devoid of [Fe-S] clusters when isolated but retained strong capacity for in vitro [Fe-S] cluster reconstitution and apo-NifH activation in a Ft > Av >Ml gradient, indicating correct folding and function but limited cluster loading or stability in vivo. NifU and NifS expression in transgenic rice resulted in variant-dependent proteome and phenotype effects, with A. vinelandii-expressing lines exhibiting severe defects, F. thermalis lines showing intermediate phenotype, and M. lutimaris lines being indistinguishable from wild type. These results reveal a trade-off between the biochemical activity of NifU and NifS and their compatibility with host metabolism, which must be considered for successful nitrogenase engineering in plants. HighlightNifU/NifS homolog selection determines trade-offs between [Fe-S] cluster assembly activity and plant compatibility, identifying variants that minimize physiological disruption while supporting nitrogenase cofactor assembly in chloroplasts.

Understanding stress-induced metabolic reprogramming in crop plants can inform breeding strategies and support the development of stress-resilient varieties. Genome-scale metabolic modelling has shown promise in elucidating network-level responses to changing environments, yet as an optimality-based approach it relies on the definition of an objective function, which is far from trivial for non-optimal conditions. To address this uncertainty, we used a time-resolved, data-informed metabolic model of rice (Oryza sativa L.) cold stress response as a test case, and explored two complementary approaches. We used sampling of the solution space combined with machine learning to identify reactions and pathways best characterizing the stress-induced metabolic shift, and used this information to perform Pareto analysis, placing growth and a stress-related objective in competition. This trade-off analysis identified key branch points in carbohydrate, amino acid, phenylpropanoid, nucleotide, and fatty acid biosynthesis, where resource reallocation towards stress-protection comes at the expense of growth. It further revealed differential flux modes across subcellular compartments and shifts in reducing equivalent provision as distinguishing features of the stress response. Together, these results provide a mechanistic understanding of the metabolic trade-offs and branch points governing cold stress response, and identify potential targets to optimize the cold response-growth trade-off in rice.

Flowering time strongly influences crop adaptation, plant architecture, generation turnover, and breeding efficiency, but the functional organization of florigen genes remains poorly resolved in many polyploid orphan crops. Quinoa (Chenopodium quinoa) is a climate-resilient allotetraploid crop with extensive variation in flowering behavior, and genome analyses have identified multiple FLOWERING LOCUS T (FT)-like homologs. However, genome sequence and expression information alone cannot determine which homologs provide effective florigenic output in planta. Here, we combined apple latent spherical virus-mediated overexpression (VOX) and virus-induced gene silencing (VIGS) in quinoa with heterologous expression in Arabidopsis thaliana, domain-swapping analyses, and cross-germplasm validation to functionally dissect quinoa FT activity. Although several CqFT homologs were transcriptionally induced during the floral transition, their functional outputs were markedly unequal. CqFT1A and CqFT1B-1 acted as the major florigenic activators: overexpression of either gene induced rapid and synchronized flowering, whereas CqFT1-VIGS delayed flowering. In contrast, CqFT2A and CqFT2B retained only weak flowering-promoting activity, whereas CqFT1B-2 showed no detectable promotive effect under the conditions tested, revealing a clear functional hierarchy among transcriptionally induced CqFT homologs. Domain-swapping analyses showed that C-terminal variation contributes to, but does not fully explain, functional divergence among CqFT homologs. In late-flowering highland lines, elevated FT input accelerated flowering, induced coordinated floral transition, and shortened the time to viable seed production. These findings identify CqFT1A and CqFT1B-1 as the major florigenic activators in quinoa and establish a functional genomics framework for resolving and modulating flowering-time control in polyploid orphan crops.

Plastids house the biology of eukaryotic photosynthesis. The majority of a plastids proteome is imported after cytosolic translation, but a few dozen proteins on average remain organelle-encoded, translated by the plastids own ribosomes. While 1000s of plastid genomes have been sequenced, the availability of less than ten proteomes and only two species with full 70S plastid ribosomal structures limit our understanding of land plant evolution. To address this, we optimized a protocol for the rapid isolation of Marchantia polymorpha chloroplasts that provides a highly enriched and intact organelle fraction from gradient volumes as little as 2 mL. Our approach was successfully applied to six other species, including Chlamydomonas reinhardtii and Nicotiana tabacum. Focusing on M. polymorpha, we determined the proteome of the chloroplast fraction, identifying 1337 nuclear-encoded proteins with a high confidence, where 83% belong to orthologs shared with angiosperms. We further isolated large protein complexes by RNA affinity purification using poly-lysine and provide the high-resolution structures of the 50S subunit of the chloroplast ribosome and RuBisCO from this bryophyte using cryogenic EM and image reconstruction to 2.23 and 2.12 [A] resolution, respectively, highlighting the structural conservation of both complexes. For chloroplasts, our data show that the genome reduction event experienced by the common ancestor of bryophytes has had little impact on the organelles complexity and that they underscore a high level of structural conservation of core components of plastid biology. Our data provide novel resources and methods to explore the functional evolution of plastid proteomes and major macromolecular complexes of cyanobacterial origin.

Poplar (Populus spp.) is a model system for tree biology. Specifically, P. tremula x P. alba INRA 717-1B4 (hereafter "poplar 717") has become an important platform for functional genomics and synthetic biology due to its rapid growth and ease of transgenesis. Here, we present a single-cell RNA-seq atlas of the poplar 717 shoot, including apical meristem, primary and secondary stems, and three stages of leaf development. Analysis of ca. 159,000 cells resolved 40 transcriptionally distinct clusters representing 7 major cell types, providing a high-resolution view of shoot development and tissue organization. We focused on the epidermis which constituted >15% of cells in the shoot atlas for in-depth characterization of epidermal heterogeneity. By integrating known marker genes with transcriptomic signatures consistent with established poplar leaf phytochemistry, we annotated epidermal cell subclusters corresponding to developmental stages, spatial location, and specialized cell types, including a distinct population of non-glandular trichomes. Coupling the single-cell RNA-seq atlas with bulk transcriptome data from glabrous mutants enabled the identification of novel trichome markers. Experimental validation of a representative trichome-specific promoter established a tool with potential to support cell type-targeted-metabolic engineering. We provide the poplar 717 atlas to the community through the BioPoplar Atlas Viewer (http://bio-poplar-atlas.com), providing a platform to explore the poplar transcriptome at single-cell resolution and a foundation for data-driven cell type-aware genetic engineering strategies in poplar.

Sugar is both an essential energy source and the major substrate for cell wall biosynthesis during plant growth, yet how growth-promoting hormones regulate sugar synthesis remains unclear. Here, we show that the brassinosteroids (BRs) promote gluconeogenic and photosynthetic sugar synthesis by activating phosphoenolpyruvate carboxykinase (PCK), which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a central step in primary metabolism. Arabidopsis BR-deficient mutants display reduced PCK1 activity and elevated phosphorylation at conserved Ser-62 and Thr-66 residues. BR treatment induces PCK1 dephosphorylation and activation, whereas the GSK3-like kinase BIN2 phosphorylates these sites, altering quaternary structure and inhibiting PCK1. Phospho-blocking mutations of Ser-62/Thr-66 confer BR-independent PCK1 activity and enhance seedling growth, while phosphomimetic mutations reduce PCK1 activity and impair seedling growth and establishment. BR also promotes PCK dephosphorylation and activation in photosynthetic leaves of maize and sorghum. Our study demonstrates that BR regulates primary metabolism via GSK3/BIN2-mediated phosphorylation of PCK, thereby promoting gluconeogenesis and photosynthesis.

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

Ethylene regulates diverse developmental processes, yet the molecular function of its central regulator, ETHYLENE INSENSITIVE 2 (EIN2), has remained unclear. Although EIN2 nuclear import is mediated by the Importin-/{beta} pathway, the molecular events initiated by EIN2 after nuclear entry were unknown. Here we show that EIN2 directly engages the transcription factor EIN3, establishing a mechanistic link between EIN2 nuclear accumulation and transcriptional activity. Microscale thermophoresis, yeast two-hybrid analysis and in planta FLIM-FRET consistently support this interaction. Domain mapping identifies EIN3 residues 86-173 as the core EIN2-binding region, and structural modeling refines the interface to a conserved segment within residues 86-120 that contacts a conserved region near the N-terminus of the EIN2-CEND fragment. In planta, EIN2 residues 1042-1214 are sufficient for EIN3 binding, revealing multiple interaction-competent surfaces with distinct affinities. The chromatin-associated protein ENAP1 also binds EIN2 and competes with EIN3, indicating a dynamic, concentration-dependent regulatory mechanism rather than a stable ternary complex. These findings define the molecular basis of the EIN2-EIN3 interaction and provide a mechanistic framework for EIN2-dependent transcriptional control in ethylene signaling.

Plants detect neighbours through a reduced red-to-far-red ratio (R:FR), triggering elongation growth that reduces crop yield. Although Gibberellin (GA) is required for the neighbour-proximity (NP) elongation response, bioactive GA levels do not increase sufficiently to account for elongation magnitude, suggesting GA sensitivity as an additional regulated variable. Here, we show that GID1C, one of three Arabidopsis GA receptors, is the primary GA receptor involved in NP-induced elongation. GID1C protein accumulates selectively in hypocotyls and root tips under low R:FR without an increase in bioactive GA. The gid1c mutant shows a reduced elongation response that exogenous GA treatment cannot rescue. Transcriptome profiling reveals that GID1C controls 86% of the NP-responsive transcriptome, including genes for cell growth, division, and transcriptional regulation. Hub analysis identifies ICE1 as a GID1C-repressed transcriptional brake. ICE1 transcript is suppressed under low R:FR in a GID1C-dependent manner, and a phosphorylation-resistant ICE1 allele blocks NP-induced elongation. Together, these findings establish GA perception as an additional regulatory layer in NP, with subfunctionalisation among GID1 paralogs shaping the response to neighbouring plants.

Ribosome biogenesis is critical for plant development and environmental responses. A large number of ribosomal proteins (RPs) and ribosomal biogenesis factors (RBFs) are required for ribosome biogenesis, many of which remain uncharacterized in plants. We report here the identification of Arabidopsis RBF FAN and its interacting partner FAN-INTERACTING PROTEIN 1 (FIP1). As their human and yeast orthologues, FAN-FIP1 interact. Both FAN and FIP1 participate in the processing of pre-rRNAs. Functional loss of FAN or FIP1 knock-down results in developmental retardation and hypersensitivity to heat stresses. We demonstrate that FAN-FIP1 positively mediates brassinosteroid (BR) signaling by ensuring the translation efficiency of the BR receptor-coding gene BRASSINOSTEROID INSENSITIVE 1 (BRI1) through the presence of its upstream open reading frame (uORF). Importantly, BR signaling positively mediates the processing of pre-rRNAs, which may be critical not only for development but also for heat tolerance.

Plasmodesmata are intercellular channels that mediate symplastic communication between plant cells. Molecular transport through these channels is critically regulated by dynamic callose deposition and degradation, yet the secretory mechanisms that deliver regulatory components to plasmodesmata remain poorly understood. Here, we identify and characterize a non-canonical plasmodesmata-associated module of the exocyst, an evolutionarily conserved protein complex involved in secretory vesicle tethering and exocytosis. Exocyst subunits EXO70G1, SEC15A, EXO84C, and SEC10A specifically accumulate at plasmodesmata, whereas the canonical exocyst subunits EXO70A1 and SEC8 do not. Genetic and interaction analyses show that EXO70G1 acts as a landmark for recruiting SEC15A and EXO84C to plasmodesmata, revealing a distinct mode of exocyst targeting at these membrane domains. EXO70G1-dependent exocyst targeting to plasmodesmata depends on phosphoinositides and sphingolipids, consistent with the specialized lipid environment of plasmodesmal membranes. Loss of EXO70G1 results in increased callose accumulation and reduced symplastic transport, and strongly enhances developmental defects of a callose-overproducing mutant. In addition, exo70G1 mutants display enhanced resistance to bacterial pathogen Pseudomonas syringae, linking reduced plasmodesmal permeability to anti-bacterial defense. Cross-species analysis further indicates that plasmodesmata association is a derived feature of the EXO70G clade, present in angiosperms but absent from non-angiosperm EXO70 homologs. Together, our findings show that exocyst diversification in plants has generated a specialized trafficking module - plasmodesmata-associated exocyst - that links vesicle delivery to callose homeostasis at plasmodesmata, thereby regulating intercellular communication, development, and immunity. TeaserA specialized secretion module of the exocyst complex regulates plant cell-to-cell connectivity by controlling callose turnover at plasmodesmata

The shift from outcrossing to self-fertilization is a common evolutionary transition in flowering plants. The genus Capsella, comprising the obligate outcrosser C. grandiflora and two self-fertile species, C. rubella and C. orientalis, provides a powerful system to explore genomic consequences of mating system shifts. Despite its utility, existing genomic resources in Capsella are fragmented, incomplete, and particularly deficient in repetitive genomic regions, hindering the study of transposable element (TE) dynamics and gene annotation. Here, we present high-quality, chromosome-scale, near-gapless genome assemblies for C. grandiflora, C. rubella, and C. orientalis. Leveraging these improved genomes, we created high-quality genomic resources for the Capsella genus by performing comprehensive, de novo annotations of protein-coding genes and TEs. Comparative genomic analysis among these species reveals differences in TE abundance, position, and production of small RNAs. These resources provide an unprecedented opportunity to explore how mating system transitions influence genome architecture, TE behavior, and gene evolution. This research also developed a static online platform for Capsella genomic resources, Capsella Database (CapBase, www.capsella.uk), to support community use of these resources. Our findings advance understanding of the genomic impacts of selfing and establish a robust foundation for future research into genomics, epigenomics, and evolutionary biology within Capsella and related plant systems.

Cellulose, a central structural component of plant cell walls, is produced by cellulose synthase complexes (CSCs) at the plasma membrane. Salinity stress is particularly damaging to cellulose biosynthesis, and therefore, plants have developed adaptive mechanisms to cope with these conditions. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are essential for growth under salt stress and show a salt-dependent association with CSCs through an as-yet unknown mechanism. Here, we identify a phosphorylation-dependent regulatory mechanism linking salt stress signaling to cellulose biosynthesis through the coordinated action of TTL3 and the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1). Phosphorylation of Serine 93 in the N-terminal intrinsically disordered region of TTL3 controls its localization, retaining it in the cytosol, while dephosphorylation promotes association with CSCs at the plasma membrane. Biochemical and genetic analysis identified BIK1 as the kinase responsible for TTL3-S93 phosphorylation, with bik1 mutants phenocopying the phosphoablative TTL3S93A in vivo. Transcriptomic analyses reveal a strong overlap of differentially expressed genes between bik1 and a cellulose-deficient mutant, supporting a broader role for BIK1 in cell wall regulation. Notably, TTL proteins do not appear to be involved in the assayed canonical immune responses, suggesting pathway specificity downstream of BIK1. Together, these findings define a signaling module that connects salt stress perception to CSCs regulation and establish BIK1-dependent TTL3 phosphorylation as a molecular switch for maintaining cell wall integrity under abiotic stress.

Shoot regeneration is a powerful model for cell fate reprogramming but how it occurs in nature remains poorly understood because studies in Arabidopsis thaliana conventionally rely on in vitro assays supplemented with exogenous hormones and sugars. In this study, we established the Hormone-autonomY Direct Regeneration Assay (HYDRA) in which removal of the shoot apical meristem (SAM) initiates shoot regeneration from the cotyledon-hypocotyl boundary domain without hormone or sugar supplementation. We show that photosynthesis-derived carbon and the boundary domain are two separable but convergent requirements for shoot regeneration in HYDRA. Carbon availability increases in the boundary domain where it activates cell cycle progression via the RETINOBLASTOMA-RELATED1 (RBR1) pathway. Carbon deprivation blocks regeneration despite induction of SAM marker genes, indicating that carbon-dependent cell cycle activation is a limiting factor for regeneration. In parallel, perturbation of the boundary domain or its regulators reduces regeneration despite sufficient carbon, indicating that boundary domain identity is independently required. Additionally, exogenous carbon supply overcomes the requirement for SAM removal to induce shoot formation, indicating that carbon availability also acts as an initiation cue. Together, this study reveals an inherent capacity for hormone-autonomous shoot regeneration and identifies photosynthesis-derived carbon as a central regulator of this process.

Members of the K+ efflux antiporter (KEA) family fulfill key roles in plastids and the endomembrane system. Plants and green algae possess at least one KEA mediating K+/H+ exchange across the plastid inner envelope (IE) membrane. Recently, IE KEAs were shown to be essential for plastid gene expression (PGE), chloroplast development, and photosynthesis. Plants lacking these antiporters exhibit reduced stromal protein synthesis and accumulation of unprocessed rRNA precursors. KEA proteins comprise a conserved monovalent cation/proton antiporter 2 (CPA2) domain and a regulatory K transport and NAD-binding (KTN) domain. IE KEAs are distinguished by an additional ~500-amino-acid N-terminal extension containing a coiled-coil (CC) domain embedded within a largely intrinsically disordered region (IDR). Intrigued by this unusual architecture, we performed phylogenetic analyses, revealing that this N-terminal fusion arose early and has been conserved throughout the green lineage. We then investigated the oligomeric state, native distribution, and function of the N-terminal domain. Using Arabidopsis thaliana, we found that IE KEAs localize to discrete clusters within the inner envelope membrane and assemble into complexes of approximately 600 kDa. Finally, complementary approaches using a functional KEA1 variant lacking the core N-terminal domains (KEA1{Delta}N) indicate that this extension plays a regulatory rather than an essential role. Our findings uncover an evolutionarily ancient regulatory module that shapes the molecular organization and function of IE KEAs, advancing our understanding of plastid ion and pH homeostasis and plastid ribosome integrity.

Plant regeneration is a dynamic developmental process that spans from cell dedifferentiation to organ reconstruction in response to inductive cues, such as wounding stress and hormonal signals. Although this capacity varies widely both between and within species, a comprehensive understanding of the genetic and epigenetic bases of this variation remains incomplete. To address this issue, we revisited published datasets on natural variation in in vitro regeneration capacity in Arabidopsis thaliana. Using quantitative genetic approaches, including meta-analyses of genome-wide association studies (GWAS) and multi-locus models, we dissected the genetic architecture underlying regeneration traits. Our results showed that shoot regeneration capacity is primarily explained by allelic variation in the cis-regulatory region of WUSCHEL (WUS), a key regulator of shoot meristem formation. Notably, these polymorphisms are also associated with epigenetic variants of the DNA transposon ATDNA2T9C, which is located within the regulatory region. Furthermore, allelic variation in ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a positive regulator of cytokinin signaling, is associated with callus formation and greening traits and may promote shoot formation through genetic interactions with WUS alleles. Although in vitro regeneration is controlled by complex, multilayered gene regulatory networks, our results suggest that, in A. thaliana, natural variation in regeneration capacity is largely shaped by a small number of major-effect modifiers together with epigenetic variation and genetic interactions, despite the substantial heterogeneity observed among natural populations.

In Asteraceae species, the capitulum is a compact inflorescence, featuring a characteristic reproductive structure. Despite the identification of a few key regulatory factors, the transcriptome-level information on the developing capitulum remains limited. Here, we applied single-cell and spatial transcriptome sequencing to investigate the developing Gerbera hybridas capitulum during floret differentiation. We obtained a transcriptomics atlas encompassing different stages of the Gerbera capitulum and analyzed the cellular and spatial dynamics of gene expression. Using marker gene expression and GO enrichment of cluster-specific DEGs, we annotated putative cell types and described changes in gene expression across sampled stages, potentially associated with ongoing developmental processes. We detected activity of previously undescribed MADS-box genes and defined their spatial expression patterns. Notably, the MADS-box gene GAGL12 was found to be enriched in the putative capitulum phloem cells. The GAGL12 protein was shown in yeast two-hybrid assays to interact with several other MADS-domain proteins with hypothesized functions in vasculature development, and further detailed in silico analyses supported a candidate role in the development of capitulum vasculature. Altogether, we provide integrative and dynamic transcriptomic insight into capitulum and floret development and lay a basis for future functional studies of the control and development of this intriguing reproductive structure.

O_LIThe type II secretion (T2S) system is conserved across the Xanthomonas lineage, yet its contributions to pathogenicity and secreted protein repertoires are poorly defined. We demonstrate that T2S systems in Xanthomonas pathovars with divergent hosts and lifestyles are required for disease. C_LIO_LIIn planta quantification of cell wall compositional changes during infection by Xanthomonas euvesicatoria (Xe) revealed that T2S-dependent depletion of galacturonic acid occurs during host colonization, providing experimental evidence for T2 effector (T2E)-mediated cell wall remodeling. C_LIO_LIUsing an in planta label-free proteomics approach, we identified two known and 20 new Xe T2Es from tomato apoplast, many with annotated functions in polysaccharide and protein cleavage. Growth assays on plant cell wall extracts and purified substrates revealed T2S-mediated metabolization of plant cell wall polysaccharides and proteins not only by Xe, but also by Xanthomonas axonopodis pv. glycines (Xag) and Xanthomonas campestris pv. campestris (Xcc). Interestingly, comparative sequence analysis revealed that the T2E repertoires have diversified among these pathogens, with differences in protease repertoire being the most pronounced. C_LIO_LIOur methodology establishes a framework for T2E discovery, enabling future functional dissection of this understudied effector class and its crosstalk with other bacterial virulence factors. C_LI