Nature Plants

Fluctuating light strongly limits photosynthetic efficiency in plants, yet the mechanisms coordinating stomatal dynamics with rapid photosynthetic induction remain incompletely understood. Here, through a forward physiological screen in Arabidopsis, we identify the abscisic acid (ABA) transporters NPF2.4 and NPF2.5 as regulators of photosynthetic responses to fluctuating light. Loss-of-function npf2.4 and npf2.5 mutants exhibited accelerated stomatal opening and photosynthetic induction during light transitions, resulting in enhanced biomass accumulation and seed yield without compromising long-term drought tolerance. NPF2.4 and NPF2.5 mediated ABA uptake in yeast and were predominantly expressed in the root pericycle and cortex/epidermal tissues, respectively. Reciprocal grafting, xylem sap ABA measurements, and ABA treatment to the roots demonstrated that both transporters regulate root-to-shoot ABA transport. Our findings reveal that root-derived ABA modulates stomatal dynamics and photosynthetic efficiency under fluctuating light, establishing long-distance hormone transport as a key regulator of plant carbon gain in dynamic environments.

Precise quantitative and spatial control of plant gene expression is typically attributed to cis-regulatory elements in promoters and other non-coding sequences. The extent to which exon-encoded regulatory information shapes plant gene expression and how remains unclear. Here we used BRASSINOSTEROID INSENSITIVE 1 (BRI1) as a model locus and found that it contains an exonic enhancer (EE), which is essential to establish the spatial BRI1 expression pattern. Its disruption restricts BRI1 expression and impairs BRI1-dependent root growth, linking exon-encoded regulatory information and development. The BRI1 EE acts in concert with its promoter, and related receptor kinase genes likewise harbor regulatory information within their coding sequences. Genome-wide surveys show that candidate EEs are associated with highly connected chromatin interaction networks and transcriptionally active loci. Moreover, species comparisons suggest that common features of exon-encoded regulatory information extend across diverse plant lineages. Thus, exon-encoded regulatory information is a vital component of plant gene regulation.

Cellulose synthase complexes build the load-bearing cellulose microfibrils of plant cell walls, yet how the abundance of their catalytic CELLULOSE SYNTHASE A (CESA) subunits is maintained remains unclear. Here, we used multi-bait TurboID proximity labelling with ten cellulose-synthesis-associated baits and six subcellular controls to define a high-confidence cellulose synthase neighbourhood. Stringent spatial and recurrence-based filtering yielded a core network of 119 interactions among 44 proteins and identified three members of the conserved Bcl-2-associated athanogene (BAG) family as previously unrecognised regulators of cellulose synthase homeostasis. BAG1-3 associated with primary-wall CESAs in reciprocal proximity-labelling experiments. Arabidopsis bag mutants showed reduced cellulose accumulation, hypersensitivity to cellulose-synthesis inhibitors, and markedly decreased CESA protein abundance without corresponding changes in CESA transcript levels. Loss of BAG function also increased the accumulation of CESA6 in vacuolar compartments. These findings identify BAG proteins as previously unrecognised regulators of cellulose synthase homeostasis and link a conserved proteostasis-associated protein family to plant cell wall biosynthesis. More broadly, the study establishes multi-bait proximity labelling, combined with cell location-specific controls, as a strategy for resolving dynamic protein networks whose components traffic through multiple subcellular compartments.

Coordinated organogenesis and rhythmic stem development determine shoot morphogenesis, which governs distinct plant architecture and crop yield. The shoot apical meristem (SAM) drives organ initiation and stem growth, with its activity sustained by the spatially defined CLAVATA3 (CLV3)-WUSCHEL (WUS) feedback loop. However, how the temporal CLV3-WUS module modulates SAM activity remains elusive. Here, we demonstrate that both CLV3 and WUS are required for circadian stem elongation. The clock component TIMING OF CAB EXPRESSION1 (TOC1) directly activates and sustains the circadian expression of CLV3, and this regulatory process is controlled by the APETALA1-CIRCADIAN CLOCK-ASSOCIATED1 module. Notably, the rhythmic expression pattern of CLV3, rather than its transcript abundance, plays a more important role in regulating WUS expression and sustaining SAM activity, and CLV3 confers circadian rhythmicity to WUS protein accumulation. Transcriptome and ChIP seq analyses reveal that WUS modulates multiple developmental programs, including meristem homeostasis, phytohormone responses, and cell wall remodeling. Mechanistically, WUS directly activates PECTIN METHYLESTERASE5 expression to promote stem elongation. Collectively, our findings uncover a spatiotemporal regulatory cascade that fine-tunes SAM activity to coordinate rhythmic organogenesis and stem development, thereby establishing optimal phyllotactic patterning in plants.

Although mitochondrial DNA (mtDNA) is subject to damage and high mutation rates in many eukaryotes, plants have a variety of mtDNA repair pathways that somehow result in exceptionally low mutation rates. However, the stoichiometry of biomolecules in plant mitochondria raises challenges for the DNA repair process. The protein complexes responsible for repair pathways contain subunits present at low copy numbers per mitochondrion. Mitochondria on average contain less than a full genome, limiting the capacity for templated repair, and raising the risk that repair machinery is localised to a DNA-empty mitochondrion where it cannot be used. Here we build on recent experimental characterisation of the copy numbers and physical processes involved to model the capacity for DNA repair in plant mitochondria. We find that targeted import of protein machinery, biased fusion of mitochondria bearing DNA, and exchange of biomolecules upon fusion can improve repair capacity through different mechanisms. Specifically, our model indicates that base excision repair can keep ~95% of mtDNA intact even without targetting or exchange rules and that targetting protein import to DNA-bearing mitochondria or exchanging protein content drives this to effectively 100%. In contrast, template repair cannot keep a majority of mtDNA intact unless mtDNA is shared across mitochondria through fusion, in which case its efficacy increases. Exchanging of metabolons (repair protein complexes) increases repair efficiency across all circumstances. We discuss the implications of our model for plant development and organelle inheritance.

Aerial roots of certain maize (Zea mays) and sorghum (Sorghum bicolor) genotypes secrete a carbohydrate-rich mucilage that sustains nitrogen-fixing bacteria, and the underground roots of most plants secrete mucilage at their tips. In both settings, border cells released from the root cap produce this mucilage, yet the genes controlling their formation remain largely unknown. We sequenced the RNA of about 23,000 nuclei from mucilage-secreting roots of maize, sorghum, and Medicago truncatula, sampled before and after secretion. Each species yielded 10 to 15 clusters, which we annotated with published markers and with new markers that we validated by in situ hybridization in the two cereals and by promoter-reporter fusions in M. truncatula. We then inferred modular and cell type-specific gene regulatory networks. Transcriptional programs diverged across the three species except in the quiescent center and the meristem, and in the clusters containing border cells, where a shared set of regulators emerged. Silencing three of five candidate regulators reduced border cell number, while silencing a partially overlapping set of three regulators reduced root diameter in M. truncatula. Silencing the C2H2 zinc finger protein MtrunA17_Chr1g0148421 also reduced border cell viability. A maize insertion mutant of bzip89 released fewer border cells than the wild type. These networks offer entry points for tuning mucilage production in cereal and legume crops.

Zea mays (maize) flowering time is genetically determined and a critical yield determinant. Yet mechanistic understanding of maize flowering remains poor. Indeterminate1 (Id1), a zinc-finger transcription factor (TF), is a monocot-conserved master regulator of maize flowering. Epistasis between Id1 and the ZeaCentroradialis-Delayed Flowering1 (Zcn-Dlf1) inductive pathway partly explains ID1 floral control; however, the strong mutant id1- floral delay is not explained by this pathway alone. To better characterize Id1 actions, we performed single-cell assay for transposase-accessible chromatin and single nucleus RNA sequencing (scATAC-seq and snRNA-seq) comparing Id1+ and id1- developing leaves. These analyses reveal id1- chromatin remodeling via TEOSINTE BRANCHED1 CYCLOIDEA PROLIFERATING CELL FACTOR (TCP) and APETALA2/ETHYLENE RESPONSEFACTOR (AP2/ERF) transcription factors and provide candidate direct targets that include AP2/ERF genes. These candidate direct targets include the family of {beta}-glucosidase genes that lose expression in id1-. Unexpectedly, CRISPR/Cas9 {beta}-glucosidase edits produced plants that phenocopied terminal ear1- (te1-) mutants. This phenocopy prompted an investigation into the genetic relationship between id1-, te1- and flowering. Surprisingly, id1- te1- plants exhibited a synergistic floral delay, producing ~90 leaves before inflorescence production. Beyond highlighting hitherto unappreciated Te1 autonomous flowering roles, this genetic synergy raises the hypothesis that meristem leaf primordia cessation underpins maize flowering.

Plastids form stroma-filled tubular extensions, termed stromules, that are implicated in inter-organellar communication. In pathogen-challenged Nicotiana benthamiana, stromule formation is driven by kinesins acting along microtubules. However, whether this mechanism also operates in unstressed plants or in other species remains unknown. Using systematic genetic analysis of all class XI myosin genes and quantitative time-lapse imaging in Arabidopsis (Arabidopsis thaliana), we show that basal stromule formation is predominantly myosin dependent through two distinct mechanisms. Myosin XI-I generates most stromules by driving oscillatory nuclear movement that mechanically pulls stromules from stationary plastids within the approx. 8m wide stromule-promoting zone. Myosins XI-K, XI-1, and XI-2 additionally drive a movement-independent, nucleus-associated stromule subpopulation within the same zone, establishing two mechanistically distinct pathways. We propose that movement-coupled stromules function as dynamic tethers maintaining plastid-nucleus proximity during nuclear oscillations, while movement-independent stromules expand the plastid membrane area within the perinuclear space, increasing the potential contact surface with the nucleus and surrounding environment. Together, both populations are proposed to enhance signal and metabolite exchange at the plastid-nucleus interface. These findings indicate that stromule biogenesis is both mechanistically and functionally context dependent, challenging the universality of the kinesin-microtubule model across plant species and physiological states.

Ethylene signaling orchestrates plant development and stress adaptation, yet how post-translational modifications of its receptors coordinate growth regulation remains unresolved. Here, we uncover a proteolytic processing mechanism of the soybean ethylene receptor GmERS1-1 that directly gates cell cycle progression. The GmERS1-1 mutants exhibit elevated yield under field conditions, whereas overexpression suppresses growth and hypersensitizes plants to ethylene. Ethylene triggers nuclear translocation of C-terminal domain (GmERS1-1-CEND), which is indispensable for receptor function. Mechanistically, GmERS1-1-CEND scaffolds a trimeric complex with GmACR1 and GmCDK7, enforcing G2/M phase arrest, thereby decoupling cell division from canonical ethylene transcriptional responses. Strikingly, ethylene-induced cell cycle blockade during apple fruit maturation mirrors this pathway, suggesting deep conservation across angiosperms. Our work defines a receptor processing-dependent signaling axis that directly interlinks phytohormone perception with cell cycle control, proposing tunable targets (e.g., GmERS1-1 cleavage efficiency) for engineering crop architecture and productivity.

Protein import across the chloroplast outer membrane is mediated by the TOC complex and regulated by GTP hydrolysis at two receptor GTPases, large and small. The former are the {beta}-barrel-containing Toc159 family of proteins (Sun and Jarvis 2023; H. Li and Chiu 2010; Shi and Theg 2013a; Kessler and Schnell 2009). The smallest member of this family in higher plants is Toc90, which has long been regarded as functionally unimportant due to the absence of obvious phenotypes in early mutant studies (Kubis et al. 2004; Infanger et al. 2011a). It was hypothesised that Toc90 is a relic of an ancestral Toc159 protein from early green lineage organisms (Hofmann and Theg 2003; Reumann et al. 2005). Here, we challenge this view by combining genetic, biochemical, phylogenetic, and in silico structural analyses to show that Toc90 is not an ancient remnant, but rather a neofunctionalised derivative of Toc159, and likely the most recently evolved member of the family. We show that Toc90 physically associates with Toc33 as a part of a photosynthetic TOC complex. Furthermore, we identify Toc90 as a regulator of chloroplast ageing. Our findings redefine Toc90 as an active and specialised component of the chloroplast protein import machinery, playing a pivotal role in senescence, especially in reducing photosynthetic protein abundance. Together, these results highlight Toc90 as a promising target for manipulating chloroplast longevity and plant senescence, with potential to improve crop productivity and yield.

Edible waxy maize is a unique domesticated cereal valued for its superior sensory and nutritional properties, yet high quality gap free genomes remain lacking, hindering the exploration of structural variations (SVs) and domestication related divergence. Here, we assembled two chromosome level, gap free genomes of the elite waxy maize inbred lines Tongxi 5 and Hengbai 522, the parents of the widely cultivated hybrid Suyunuo 1. Comparative genomic analysis revealed substantial genome size variation, prominent megabase scale SVs, and extensive knob repeat expansion in Tongxi 5. We identified widespread gene presence absence variation and hyperdivergent regions enriched in transposable elements, which largely underpin genomic differentiation between waxy and field maize. Population genomic analyses further demonstrated asymmetric introgression from field maize into the two parents, whereas the conserved waxy haplotype supports a shared ancestral origin. Integrative GWAS based on SNPs, InDels and SVs revealed that SVs substantially contribute to phenotypic diversity and mediate the co-regulation of yield traits and flavor-related metabolites. Our results uncover key genomic events underlying post-domestication divergence and highlight the essential roles of SVs in heterosis and flavor-yield balance, providing valuable genomic resources for waxy maize improvement.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

Castilleja (Orobanchaceae) is a diverse, facultatively hemiparasitic plant genus characterized by a complex evolutionary history of reticulate evolution and significant taxonomic ambiguity. High-quality genomic resources have historically been limited, hindering robust macro-evolutionary and macro-ecological inquiries. Here, we present the first high-quality, phased, chromosome-level reference genome for the diploid species Castilleja foliolosa. Utilizing a hybrid assembly strategy, we integrated long-read PacBio HiFi sequencing with Omni-C proximity ligation data to generate a highly contiguous nuclear assembly, complemented by reconstructed mitochondrial and chloroplastic genomes. The primary nuclear haplotype spans approximately 510 Mbp, containing 39,417 predicted genes and a substantial repetitive landscape covering ~66% of the genome. Our organellar assemblies reveal notable structural complexity: the chloroplast maintains heteroplasmy via two primary structural haplotypes, while the 611 kbp mitogenome adopts a circular master topology existing in two isomeric forms. Comparative genomic analyses demonstrate a largely conserved chromosomal architecture relative to the closely related Pedicularis cranolopha, punctuated by the presence of lineage-specific genes. Functional enrichment of Castilleja-specific orthogroups identifies a consistent association with telomere maintenance, DNA integration, and zinc ion binding, likely stemming from the substantial repeat content. This reference genome provides a robust framework for dissecting the genomic drivers of taxonomic diversification and adaptation within the Orobanchaceae family. This resource significantly enhances our capacity to untangle reticulate evolutionary patterns, offering a definitive foundation for future comparative studies of genome evolution in the Orobanchaceae lineage of parasitic plants.

Ordinal scoring of plant disease severity by human raters compresses variation in lesion color, size, and number. Inter-rater variability further complicates comparisons and integrated analyses across environments. We developed a low-cost portable imaging chamber to rapidly image large numbers of leaves under standardized lighting, orientation, and backdrops in the field and employed this system to image more than 11,000 leaves across three states. Embeddings from vision encoders predicted human-assigned disease severity scores. No significant GWAS hits were identified using human-assigned or vegetation-index-based disease severity scores, but GWAS using embeddings identified twelve genomic hotspots controlling leaf appearance. Nine were linked to variation in disease symptom severity. Five hotspots corresponded to previously characterized sorghum genes: all three hotspots not linked to disease and two of the nine that were. Roughly one-third of tested embedding--hotspot associations replicated across at least two states, and twenty replicated across all three. eQTL, PheWAS, and large-effect variant analyses identified single candidate genes with plausible mechanistic links to disease symptom severity for six of the seven hotspots not mapping to characterized genes. These results demonstrate the power of combining scalable, standardized leaf imaging with pretrained image encoders to capture genetically controlled variation in diverse disease symptoms that human ordinal scoring misses.

Ayahuasca, used religiously for centuries by Amazonian Indigenous peoples and increasingly studied for its therapeutic potential, owes its psychoactivity to two plants: Psychotria viridis supplies N,N-dimethyltryptamine (DMT), whereas Banisteriopsis caapi supplies beta-carbolines that render DMT orally active. Here we show that the two plants have distinct genome histories: P. viridis is hexaploid (2n = 6x = 66), consistent with autopolyploidy, and B. caapi is diploid (2n = 2x = 20). CENH3-bound regions of both species are nonetheless enriched in CRM-type Ty3/Gypsy retrotransposons, with additional LINE elements and CRM-derived satellites in B. caapi. In P. viridis, a single multifunctional methyltransferase catalyzes both N-methylations of tryptamine to DMT. In B. caapi, beta-carboline biosynthesis combines dedicated enzymes with an acetaldehyde-dependent Pictet-Spengler cyclization that can proceed non-enzymatically, followed by enantioselective oxidation to harmaline. These resources link the ethnobotanical history of ayahuasca to its molecular basis, providing genomes of both source plants, the genetic structure of traditionally recognized cultivated varieties, and the enzymes behind the alkaloids long combined in this decoction.

Protein stability, the capacity to maintain a folded structure and function, matters as much for transporters and regulators as for enzymes, and heat erodes it. Wheat, rice, maize and soybean supply two-thirds of human caloric intake, and warming reduces yields. Rubisco activase variants selected for thermostability increased photosynthesis, biomass and yield under heat. They came from gene shuffling; no method proposes residues at proteome scale. Genome editing has made such changes routine, yet predictors estimate melting temperature, name no residue, and have not been reported for a plant proteome. Here we present CropStabDB: 1,425,716 proteins from 30 plant species scored on six interpretable components, with an Engineering Atlas of 2,097,576 candidate substitutions in 51,440 proteins, 785,622 at Engineering Confidence Score [≥] 0.70. Every candidate is a residue a more stable orthologue carries at that column. Substitution-type scores match the direction of measured folding free energies in 842 of 926 domains (90.9%). On three proteomes from families absent from training, the model reaches an area under the curve of 0.919. Applied blind to wheat, the score reproduces the measured thermostability ranking of the Rubisco activase isoforms in all three subgenomes. CropStabDB is freely available without registration at https://cropstab.org.

Potassium (K) deficiency is a widespread constraint on maize production, yet how it influences the suberized apoplastic barriers that control radial K transport in roots, and whether the endodermis and the constitutively suberized exodermis respond differently, has remained unclear. We grew maize under K deficiency in soil and across a hydroponic K gradient, with abscisic acid (ABA) and fluridone treatments, and analysed seminal roots by Fluorol Yellow 088 staining, tissue-resolved suberin chemistry of endodermis and exodermis, rubidium (Rb) flux, and RNA-sequencing with weighted gene co-expression network analysis (WGCNA). K deficiency selectively increased endodermal aliphatic suberin and eliminated endodermal passage cells in soil-grown roots, whereas exodermal suberin was unchanged. Although K influx remained high, root-to-shoot Rb translocation fell sharply, consistent with K retention. A single co-expression module linked the suberin-biosynthetic genes with several K transport genes, and the suberin programme responded to exogenous ABA, indicating ABA-dependent co-regulation. Soil and hydroponic systems converged on the same endodermis-specific anatomical response. Endodermal, but not exodermal, barrier reinforcement limits K leakage from the stele in maize roots. This tissue-specific plasticity identifies the endodermal suberin biosynthesis programme, and its coordination with K uptake, as a target for improving K use efficiency.

Plants explore soil environments through their roots, which branch repeatedly to access water and soil-borne nutrients. In many plants, root branches are generated by repeated developmental programs and molecular analyses have not identified markers that distinguish primary from later root branches. Consequently, efforts to engineer plant roots for improved water or nutrient acquisition are limited to changes that affect the entire root system. Here, we developed synthetic genetic circuits to count repeating root development cues and enable differential gene expression in successive root branches of the model plant Arabidopsis thaliana. The circuits use a root primordium-specific promoter to express large serine recombinases, which irreversibly invert segments of DNA each time a new lateral root is initiated. This converts repeating rounds of root formation into distinct, heritable DNA states. Because root branch specification and outgrowth occur over a ~2 day period, we tested circuit designs for their ability to count long-duration input pulses and developed an architecture that produces distinct fluorescence outputs in primary, secondary, and tertiary roots. By linking repeated developmental cues to distinct genetic outputs, synthetic genetic circuits that count enable root system engineering at the resolution of individual branches and offer a general strategy for dissecting developmental processes that rely on repeated signals.

With a sub-3-micrometer cell and a 27-Mbp diploid genome, the small but mighty Picochlorum celeri doubles within three hours and exceeds 30 g m-2 d-1 outdoors with CO2 supplied. In climate-simulating photobioreactors, productivity declined from 39 g m-2 d-1 under 2.25% CO2 to near zero in air and resumed within hours of CO2 restoration with photosystem II photochemistry largely retained. We profiled the transcriptome, proteome, phosphoproteome, ubiquitinome, acetylome, metabolome and lipidome, including the first site-resolved green-algal ubiquitinome. In air, the Rubisco large subunit tripled to 7.6% of protein while its two small-subunit isoforms, 70% identical, exchanged near-reciprocally, a novel CO2-dependent switch that we propose retunes Rubisco to low CO2. Photorespiratory enzymes increased in abundance whereas proteins involved in external nitrogen assimilation decreased, consistent with increased reliance on internal nitrogen recycling as carbon fixation became limiting. Transcript and protein responses were nearly uncoupled, with cytosolic ribosomal proteins declining despite increased transcripts and plastid ribosomal proteins spared. Phosphorylation was extensively remodeled, as were the lysine modifications ubiquitination, acetylation and CO2-dependent carbamylation, where a CO2-competition assay identified 86 bicarbonate-protected lysines, including RbcL K252. CO2 limitation therefore imposes a reversible growth arrest that reduces cytosolic translational investment while preserving photosynthetic capacity for rapid recovery.

Agrobacterium-mediated transformation is a cornerstone of plant genetic engineering, yet many crops remain recalcitrant to transformation and its efficiency is highly species- and genotype-dependent. The limited diversity of disarmed agrobacterial strains available for biotechnology, and the technically challenging workflows typically required for strain modification, constrain efforts to develop improved plant transformation platforms. Here, we describe the development of AGROtools-a versatile, expandable, and user-friendly modular cloning plasmid collection comprising more than 75 individual components, that is designed to simplify genetic engineering of agrobacteria. Constructs assembled with this toolkit couple visual reporters with selectable and counterselectable markers to facilitate identification of recombinant clones, thereby reducing reliance on PCR-based screening. We constructed and tested reporter cassettes that are functional across diverse agrobacterial strains and genomic contexts. By linking a visual reporter combinatorially with various antibiotic resistance markers and orthogonal site-specific recombinase recognition sequences, AGROtools can enable marker removal and recycling for up to four successive genomic modifications. We demonstrate the utility of AGROtools by disarming 10 novel wild-type strains, and accomplishing serial modifications within individual strain backgrounds. In addition to conventional recA-mediated homologous recombination, the collection incorporates components for applying contemporary strain engineering approaches, including CRISPR/dCas9 base editing, INTEGRATE-mediated targeted insertion, and phage-derived recombineering systems. Constructs can be assembled and deployed rapidly, enabling isolation of strains carrying an intended modification in less than two weeks. AGROtools provides a broadly applicable resource for accelerating strain development and genetic studies in agrobacteria, with potential to advance plant biotechnology by aiding improvement of disarmed strains.