Biology subjects

Sattely, E.

Publications and source records attributed to Sattely, E..

12 recordsLinked to original sources

bioRxiv2026

Microbiota- and diet-specific T cells become Tregs by default

CD4+ T cells recognize antigens from microbiota, diet, and pathogens via T cell receptors (TCRs) and orchestrate immunity by differentiating into tolerogenic regulatory (Treg) or pro-inflammatory effector (Teff) lineages (e.g. TH1 or TH17) (1). Dysregulation of these responses underlies numerous gastrointestinal inflammatory and infectious diseases (2-6). The prevailing paradigm suggests that individual microbes and dietary antigens drive distinct cell fates (e.g., segmented filamentous bacteria [SFB] induce TH17 cells (7) whereas Helicobacter hepaticus (8) and diet (9) induce Tregs). However, the generality of this model is uncertain: several key organisms are atypical, and foundational studies often omitted a complex microbiome or a diverse polyclonal TCR repertoire. Here we develop a high-throughput pipeline to screen hundreds of TCRs from mice colonized from birth with a 116-strain human microbiota (hCom2v), demonstrating that TCRs recognizing microbiota or dietary antigens are overwhelmingly enriched in the induced Treg (iTreg) lineage. Endogenous CD4+ T cells specific for these antigens adopt a uniform iTreg phenotype in vivo, both in hCom2v-colonized and conventional mice. This baseline tolerance is robust to acute inflammation but breaks down following a 'two-hit' combination of inflammation and genetic susceptibility, allowing Teff to emerge against otherwise Treg-restricted antigens. These data support a revised paradigm in which antigen-specific Treg induction is the default response to foreign antigens in the healthy gut, and effector responses are an exception reflecting a perceived threat. Reframing gastrointestinal immunity as a tolerance-first system provides a framework for understanding inflammatory disease pathogenesis and suggests that therapeutic strategies should aim to restore a Treg-predominant baseline.

immunology↗

bioRxiv2026

FoTO1 orchestrates Taxol biosynthesis through catalytic and non-catalytic mechanisms

Taxol is a blockbuster chemotherapeutic derived from the Pacific Yew tree. Recent work in our group has identified a complete pathway to baccatin III, a key intermediate, that hinges on a novel accessory protein, Facilitator of Taxane Oxidation (FoTO1). This protein dramatically improves yield and alters enzyme product profiles when reconstituting the Taxol pathway in N. benthamiana. FoTO1 has been shown to act early in the biosynthetic pathway improving the yields of product generated by the combination of a plastidial diterpene synthase (taxadiene synthase) and an endoplasmic reticulum (ER) localized cytochrome P450 (T5-alpha-hydroxylase). Here, we show that FoTO1 is an enzyme capable of converting taxadiene-(4),5-epoxide, the likely product of T5H oxidation, into taxadien-5-ol. FoTO1 is also functional in yeast, resolving a key bottleneck for development of a bioproduction route to Taxol in this host. Targeted mutagenesis of key catalytic residues in FoTO1 abrogates function in vitro but not in planta, suggesting non-catalytic contributions of FoTO1 to the taxane pathway. A combination of proximity labelling, bimolecular fluorescence complementation assays, and co-immunoprecipitation studies revealed that FoTO1 interacts with and organizes various P450s in the Taxol pathway. These approaches highlight the importance of both FoTO1s catalytic and non-catalytic functions in improving yields in the early Taxol pathway. Beyond Taxol biosynthesis, FoTO1 boosts yields for diverse diterpene pathways from across phylogeny, suggesting a general role of this protein class in mediating metabolism across the plastid and ER in plants.

biochemistry↗

bioRxiv2024

Zinc-starved Brassicaceae Plants Secrete Peptides thatInduce Root Expansion

Zinc (Zn) deficiency is recognized as a global crisis as it is observed in half of all agricultural soils. However, the molecular mechanisms that drive plant physiological responses to soil Zn deficiency are not well understood. We used an untargeted metabolomics approach to search for metabolites exuded from roots during Zn deficiency stress, which led to the discovery of a collection of secreted small defensin-like peptides in Arabidopsis thaliana (named Zinc-Deficiency Responsive Peptides (ZDRPs)). Phylogenetic analysis and untargeted metabolomics revealed ZDRPs in at least eleven accessions of A. thaliana and nine members of the Brassicaceae family. Analysis of Arabidopsis gene mutants and overexpressing lines, in combination with chemical complementation experiments, unveiled a critical role of these peptides in plant root growth. We hypothesize that Brassicaceae secreted peptides enable plants to expand their root mass to reach Zn-rich soil layers and optimize Zn uptake. These data reveal a critical relationship between plant survival, Zn status, root morphology and peptide production. Taken together, our results expand our knowledge regarding micronutrient deficiency responses in plants and could enable in engineering approaches to make plants more resilient to low Zn conditions. SignificanceZinc deficiency is the most abundant micronutrient deficiency affecting about 50% of arable lands thus presenting a high burden for plant health and agriculture globally. In this study, we reveal a metabolic strategy by Brassicaceae to deal with low Zn concentrations. We characterize the role of peptides expressed upon zinc deficiency in a variety of important crop plants. The discovery of a cryptic class of peptides that are made by plant roots specifically suffering from Zn deficiency provides critical insight into the molecular mechanisms by which plants dynamically acclimate to nutrient-limited soils. The identification of peptides actively secreted by zinc-deprived plants has translational value for sustainable agriculture, human health, and bioengineering approaches to enable tolerance to low zinc.

plant biology↗

bioRxiv2024

Transcriptome analysis reveals role for WRKY70 in early N-hydroxy-pipecolic acid signaling

N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and amplifying systemic acquired resistance (SAR) following pathogen attack. Early phases of NHP signaling leading to immunity have remained elusive. Here we report the early transcriptional changes mediated by NHP and the role salicylic acid (SA) plays during this response. We show that distinct waves of expression within minutes to hours of NHP treatment include increased expression of WRKY transcription factors as the primary transcriptional response, followed by the induction of WRKY-regulated defense genes as the secondary response. The majority of genes induced by NHP within minutes were SA-dependent, whereas those induced within hours were SA-independent. These data suggest that NHP induces the primary transcriptional response in a low SA environment and new SA biosynthesis is dispensable for induction of the secondary transcriptional response. We demonstrate that WRKY70 is required for the induced expression of a set of genes defining some of the secondary transcriptional response, SAR protection, and NHP-dependent enhancement of ROS production in response to flagellin treatment. Taken together, our study highlights the key genes and pathways defining early NHP responses and a role for WRKY70 in the regulation of NHP-dependent transcription.

plant biology↗

bioRxiv2023

A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A lead example is the >150 Amaryllidaceae alkaloids (AmAs) including galantamine, an FDA-approved treatment for Alzheimers disease. We show that while AmAs accumulate to high levels in many tissues in daffodils, biosynthesis is localized to nascent, growing tissue at the base of leaves. A similar trend is found for the production of steroidal alkaloids (e.g. cyclopamine) in corn lily. This model of active biosynthesis enabled elucidation of a complete set of biosynthetic genes for the production of AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodil. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as aboveground tissue develops.

biochemistry↗

bioRxiv2023

Multiplicity of Agrobacterium infection of Nicotiana benthamiana for transient DNA delivery

Biological DNA transfer into plant cells mediated by Agrobacterium represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has recently been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single vs. multiple transfection events per cell. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Establishing MOI for Agrobacterium T-DNA delivery at the level of single plant cells would allow users to design genomic library delivery conditions (at most 1 event/cell), or maximize co-delivery of T-DNA loads from separate Agrobacterium (>1 event/cell). Here, we analyze the Poisson probability distribution of T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/Nicotiana benthamiana. These data delineate the relationship between an individual Agrobacterium strain infiltration OD600, plant cell perimeter and leaf age, as well as plant cell co-infection rates. Our analysis also establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and the reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.

plant biology↗

bioRxiv2022

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

bioengineering↗

bioRxiv2020

Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in the response have not yet been elucidated. Here we report that Arabidopsis thaliana UGT76B1 can generate glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants do not accumulate NHP-Glc and accumulate less glycosylated salicylic acid (SA-Glc) than wild type plants. The metabolic changes in ugt76b1 mutant plants are accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of SAR molecules NHP and SA by UGT76B1 plays an important role in defense modulation. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato increases NHP-Glc production and reduces NHP accumulation in local tissue, and abolishes the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

plant biology↗

bioRxiv2019

Conservation of N-hydroxy-pipecolic acid-mediated systemic acquired resistance in crop plants

Signal propagation and the coordination of whole-organism responses in plants rely heavily on small molecules. Systemic acquired resistance (SAR) is one such process in which long-distance signaling activates immune responses in uninfected tissue as a way to limit the spread of a primary, localized infection. Recently, N-hydroxy pipecolic acid (NHP) was discovered and shown to coordinate SAR in Arabidopsis. Here, we provide metabolic and biochemical evidence that NHP is conserved across the plant kingdom and demonstrate a role for NHP in mediating SAR responses in tomato and pepper. We reconstituted the NHP biosynthetic pathway in planta and show that transient expression of two NHP biosynthetic genes in tomato induces enhanced resistance to a bacterial pathogen in distal tissue. Our results suggest engineering strategies to induce NHP-mediated SAR are a promising route to improve broad-spectrum pathogen resistance in crops. IN BRIEFEngineering NHP production is a promising strategy to enhance disease resistance in crops. HIGHLIGHTSO_LIArabidopsis N-hydroxy-pipecolic acid (NHP) pathway is conserved across the plant kingdom C_LIO_LIApplication of NHP to tomato and pepper plants induces a robust SAR response C_LIO_LIMetabolic engineering of the Arabidopsis NHP pathway in Solanum lycopersicum leads to enhanced NHP production and defense priming C_LIO_LIGenetic engineering for enhanced NHP production is a promising strategy to protect crop plants from multiple pathogens C_LI

plant biology↗

bioRxiv2018

A pathogen-responsive gene cluster for the production of highly modified fatty acids in tomato

In response to biotic stress, plants reshape their complement of lipids to produce suites of highly modified fatty acids that bear unusual chemical functionality. Despite their chemical complexity, proposed roles in pathogen defense and presence in crop plants, little is known about the biosynthesis of these decorated fatty acids. Falcarindiol is a prototypical member of a suite of acetylenic lipids from carrot, tomato, and celery that inhibits growth of several fungal strains and human cancer cell lines. Here we report a set of clustered genes in tomato (Solanum lycopersicum) that are required for the production of falcarindiol in leaves in response to treatment with an adapted fungal pathogen, Cladosporium fulvum. Our approach is based on correlation of untargeted transcriptomic and metabolomic data sets in order to rapidly identify a candidate biosynthetic pathway. By reconstituting the initial biosynthetic steps in a heterologous host (Nicotiana benthamiana) and generating stable transgenic pathway mutants in tomato, we demonstrate a direct role for three genes in the cluster in falcarindiol biosynthesis. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens, and provides critical insight into the biochemistry of alkynyl lipid production.\n\nOne Sentence SummaryA biosynthetic gene cluster for the production of falcarindiol, a highly modified antifungal oxylipin found in edible plants.

plant biology↗

bioRxiv2018

N-hydroxy-pipecolic acid is a mobile signal that induces systemic disease resistance in Arabidopsis

Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity of the mobile defense signal that moves systemically throughout plants to initiate SAR has remained elusive. In this paper, we describe a new metabolite, N-hydroxy-pipecolic acid (N-OH-Pip), and provide evidence that this molecule is a mobile signal that plays a central role in initiating SAR signal transduction in Arabidopsis thaliana. We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-PIP moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant, and enhances resistance to a bacterial pathogen. This work provides new insight into the chemical nature of a mobile signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.

plant biology↗

bioRxiv2018

HEx: a heterologous expression platform for the discovery of fungal natural products

For decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.\n\nSummaryHere we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform.

synthetic biology↗