Biology subjects

Santiago, J.

Publications and source records attributed to Santiago, J..

14 recordsLinked to original sources

bioRxiv2026

The Receptor Kinase MEE39/ATHE Mediates Cell Wall Integrity Surveillance During Root Vascular Pathogen Infection

Plant cell wall (CW) integrity signaling enables early detection of microbial invasion, yet the receptors involved and their spatial and temporal dynamics during infection remain largely unknown. We identify ATHENA (ATHE)/MEE39, a previously uncharacterized malectin-like leucin-rich repeat receptor kinase (Mal-LRR-RK) that contributes to defense against the root vascular pathogen Fusarium oxysporum (Fo), particularly in outer root layers where colonization begins. ATHE abundance, localization, and endocytic trafficking are rapidly remodeled during infection, and loss of ATHE compromises basal immunity and early pathogen-induced transcriptional reprogramming. ATHE responds to altered cellulose synthesis, cellulose-derived oligosaccharides, mechanically induced CW perturbations, and the fungal secreted peptide Fo-RALF. In most of these contexts, ATHE acts together with the LRR-RK MIK2, forming a pathogen-strengthened RK complex that fine-tunes root responses to Fo. This represents the first example of a receptor complex visualized subcellularly in vivo during a plant-microbe interaction. Although Brassicaceae-specific, heterologous expression of ATHE enhanced tomato resistance to Fo, highlighting its functional relevance across plant lineages and its potential use for crop engineering. Our work reveals a previously unrecognized strategy by which plants decode microbial threats through dynamic CW-integrity surveillance.

plant biology↗

bioRxiv2025

Induction and antiviral activity of human β-defensin 3 against adenovirus and influenza A virus in vitro infection of human airway epithelial cells

BackgroundRespiratory pathogens such as adenoviruses (AdV) and influenza A viruses (IAV) can cause serious upper and lower respiratory infections in infants, the elderly, and immunocompromised individuals. However, options for antiviral drugs targeting respiratory viruses are limited. Human beta-defensins (hBDs) are disulfide-rich peptides that demonstrate broad antimicrobial activity against bacteria, fungi, and some viruses, while also playing immunomodulatory roles. Among defensins, human beta-defensins (hBDs) are the most prevalent, with six peptides identified: hBD-1 through hBD-6. Nonetheless, the production of hBD-3 during respiratory virus infections has not been extensively studied, nor has the effect of hBD-3 on these infections. ObjectiveThis study investigated whether infection with AdV-5 or IAV induces hBD-3 expression in human airway epithelial cells and whether a recombinant form of hBD-3 (rhBD-3) can inhibit in vitro infection by these viruses, one DNA virus and one RNA virus. MethodsIn vitro models of human airway epithelial cells (A549 and HEp-2) infected with AdV-5 and IAV were established. In vitro expression of hBD-3 mRNA was assessed by RT-PCR; hBD-3 protein was examined using immunofluorescence and Western blot. The inhibition of viral infection by hBD-3 was quantified using cytopathic effect and plaque reduction assays. Additionally, epithelial cells of animal origin, MDCK and MDBK, were similarly tested for rHBD-3 antiviral activity against IAV infection. ResultsIAV and AdV infections significantly increased hBD-3 mRNA levels in A549 and HEp-2 cells. Immunofluorescence and Western blotting confirmed the presence of hBD-3 protein on the surface of IAV-infected cells. Cells infected with AdV-5 produced less hBD-3 protein. Additionally, rhBD-3 inhibited both AdV-5 and IAV infections in both cell lines at concentrations of 7.5-50 g/mL, with the strongest effect at the highest concentration. Lastly, rhBD-3 also inhibited IAV infection in MDBK and MDCK cells within the same range. ConclusionsIn vitro infection of human airway epithelial cells with IAV and AdV induces hBD-3 mRNA and protein expression. In turn, rhBD-3 inhibits viral infection in a dose-dependent manner in HEp-2 and A549 cells against these viruses, an RNA virus and a DNA virus. hBD-3 is expressed and localized on the surface of infected cells. rHBD-3 also inhibits IAV infection in bovine and canine epithelial cells. Our findings suggest that hBD-3 plays a broad-spectrum role in defense against respiratory viruses in humans and animals and modulates the innate immune responses. PerspectiveThis work supports the use of hBD-3 as a natural, broad-spectrum antiviral agent, either alone or in combination, for treating viral infections, particularly emerging respiratory agents such as IAV and coronaviruses, for viral pandemic preparedness and response. Data SummaryThere are no supporting external data. Impact StatementThis study demonstrates in vitro induction of human beta defensin type 3 (hBD-3) in human airway epithelial cells (A549 and HEp-2 cells) during viral infection by the influenza A virus (IAV) and the human adenovirus type 5 (AdV-5), and inhibition of the viral replication on these cells by the hBD-3 in a recombinant form; this defensin also showed viral inhibition on bovine and canine epithelial cells; further, these are viruses from two very different families; one is an enveloped RNA virus, and the other is a naked DNA virus. Both results are novel observations in the biomedical literature. Our results suggest that this defensin plays a principal role in innate defense during natural respiratory viral infections in humans, and support the use of hBD-3 alone or in combination as a broad-spectrum antiviral agent for the treatment of these infections, especially for emerging agents such as IAV and coronaviruses, for viral pandemic preparedness and response. In fact, few antiviral drugs are currently approved for treating respiratory virus infections, with only specific inhibitors of influenza and respiratory syncytial viruses.

microbiology↗

bioRxiv2025

Disulfide bond sculpts a peptide fold that mediates phytocytokine recognition

Precise ligand recognition by closely related leucine-rich repeat receptor kinases (LRR-RKs) is essential for plants to coordinate immunity, development, and environmental adaptation. Here, we show how HAESA-LIKE 3 (HSL3) specifically recognizes the folded, disulfide-stabilized CTNIP/SCREW phytocytokines in Arabidopsis. Quantitative binding assays define a minimal CTNIP4 region required for high-affinity HSL3 interaction and signaling activation. A 2.12 [A] crystal structure of the HSL3-CTNIP4 complex reveals a unique C-terminal receptor pocket that accommodates the peptides cyclic architecture through a combination of hydrophobic and polar contacts, a feature absent in the closely related HAE/HSL LRR-RKs. The cyclic CTNIP4 fold further establishes a largely hydrophobic interface that bridges HSL3 to the SERK co-receptor, forming a distinct activation surface. Together, these structural, biochemical and physiological insights uncover a previously unrecognised mechanism of peptide perception and receptor activation, highlighting how subtle architectural variations enable precise ligand selectivity among highly conserved plant receptor kinases.

plant biology↗

bioRxiv2025

Glycan recognition by a plant sentinel immune receptor

Pathogens target and degrade the extracellular matrix surrounding plant cells. A central question is how cell wall-derived damage-associated molecular patterns (DAMPs) are recognized and integrated to trigger immune responses. We address this question by determining the structure of the multidomain receptor IGP1 in both apo form and bound to the cellulose-derived DAMP cellotriose. Structural analyses reveal that constitutive Leucine Rich Repeat-malectin interactions preconfigure IGP1 for ligand recognition and that the receptor features a highly specific sugar-binding pocket in the LRR domain capable of distinguishing fine variations in glycan structures. By directly sensing cello-oligomers, IGP1 acts as a cell wall sentinel that links pathogen-induced wall degradation to immune alerting, equipping plants to mount rapid and robust defense responses.

plant biology↗

bioRxiv2025

Floral innovation through modifications in stem cell peptide signaling.

Understanding how evolution shapes genetic networks to create new developmental forms is a central question in biology. Asteraceae (sunflower family) comprise 10% of flowering plants and have capitula, a novel flowering shoot (inflorescence) that mimics a single flower (1, 2). During capitulum development, shoot stem cells undergo prolonged proliferation relative to other species (3, 4). Here we show that capitulum evolution paralleled decreases in CLAVATA3 peptide (CLV3p) signaling, a conserved repressor of stem cell proliferation. Asteraceae CLV3p displays reduced receptor binding and downstream transcriptional outputs. Reversion of CLV3 to a more active form impairs Asteraceae stem cell regulation and capitulum development. Lastly, we trace CLV3 evolution across the Asterales allowing inferences on capitulum evolution. Our findings reveal novel evolutionary mechanisms in plant reproduction and suggest approaches for engineering crops.

plant biology↗

bioRxiv2025

Alterations in retinal tau phosphorylation in Alzheimer's disease patients identified by mass spectrometry

Most neurodegenerative diseases, including Alzheimers disease (AD) and multiple sclerosis (MS), are associated with abnormal phosphorylation of tau (p-tau) in the brain. Immunostaining studies have revealed an accumulation of p-tau in the AD retina and suggested it may reflect tau pathology in the brain. To validate these findings and further investigate the relationship between retinal and brain tau pathology, we used mass spectrometry to measure p-tau peptides in matched retinal and hippocampal samples from non-demented controls (NC, n=8), AD (n=12), and MS (n=4). We analysed the differences in p-tau levels between diagnoses and explored how retinal p-tau variants correlate with hippocampal p-tau and neuropathological changes. Our results revealed peaks corresponding to tau peptides phosphorylated at T181, S199/S202, T231, S396 + T403/S404, and T403/S404. These p-tau peptides were also detected in the hippocampal samples, along with additional p-tau peptides such as T217 and S262. Total tau phosphorylation and phosphorylation at S199/S202 and T231 were significantly higher in the retina of AD cases compared to NC. These two peptides, along with peptides phosphorylated at S396+T403/S404 and T403/S404, were also higher in cases with high amyloid-beta (A{beta}) Braak stages compared to those with low A{beta} Braak stages. Higher A{beta} Braak stages were also associated with higher mass spectrometry peak intensities of peptides phosphorylated at S199/S202 and S396+T403/S404. Additionally, retinal p-tau peptides at S396+T403/S404 and T403/S404 correlated with neurofibrillary tangle (NFT) Braak stages, and p-tau peptides S396+T403/S404 in the retina were linked to corresponding phosphorylation in the CA1 region. These findings underscore the connection between retinal and brain tau pathology and highlight the potential of retinal tau as a biomarker for AD diagnosis and monitoring while also deepening our understanding of tauopathies in both the retina and brain.

neuroscience↗

bioRxiv2024

Leveraging co-evolutionary insights and AI-based structural modeling to unravel receptor-peptide ligand-binding mechanisms

Secreted signaling peptides are central regulators of growth, development, and stress responses, but specific steps in the evolution of these peptides and their receptors are not well understood. In addition, the molecular mechanisms of peptide-receptor binding are only known for a few examples, primarily owing to the limited availability of structural capabilities to few laboratories worldwide. Plants have evolved a multitude of secreted signaling peptides and corresponding transmembrane receptors. Stress-responsive SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs) were recently identified. Bioactive SCOOPs are proteolytically processed by subtilases and are perceived by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) in the model plant Arabidopsis thaliana. How SCOOPs and MIK2 have (co-)evolved, and how SCOOPs bind to MIK2 are however still unknown. Using in silico analysis of 350 plant genomes and subsequent functional testing, we revealed the conservation of MIK2 as SCOOP receptor within the plant order Brassicales. We then leveraged AlphaFold-Multimer and comparative genomics to identify two conserved putative SCOOP-MIK2 binding pockets across Brassicales MIK2 homologues predicted to interact with the SxS motif of otherwise sequence-divergent SCOOPs. Notably, mutagenesis of both predicted binding pockets compromised SCOOP binding to MIK2, SCOOP-induced complex formation between MIK2 and its co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and SCOOP-induced reactive oxygen species production; thus, confirming our in silico predictions. Collectively, in addition to revealing the elusive SCOOP-MIK2 binding mechanisms, our analytic pipeline combining phylogenomics, AI-based structural predictions, and experimental biochemical and physiological validation provides a blueprint for the elucidation of peptide ligand-receptor perception mechanisms. Significance statementThis study presents a rapid and inexpensive alternative to classical structure-based approaches for resolving ligand-receptor binding mechanisms. It relies on a multilayered bioinformatic approach that leverages genomic data across diverse species in combination with AI-based structural modeling to identify true ligand and receptor homologues, and subsequently predict their binding mechanisms. In silico findings were validated by multiple experimental approaches, which investigated the effect of amino acid changes in the proposed binding pockets on ligand-binding, complex formation with a co-receptor essential for downstream signaling, and activation of downstream signaling. Our analysis combining evolutionary insights, in silico modeling and functional validation provides a framework for structure-function analysis of other peptide-receptor pairs, which could be easily implemented by most laboratories.

biochemistry↗

bioRxiv2023

A pectin-binding peptide with a structural and signaling role in the assembly of the plant cell wall

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides which, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.

plant biology↗

bioRxiv2022

Phytocytokine signaling integrates cell surface immunity and nitrogen limitation

Plant endogenous signaling peptides shape growth, development and adaptations to biotic and abiotic stress. Here, we identified C-TERMINALLY ENCODED PEPTIDEs (CEPs) as novel immune-modulatory peptides (phytocytokines) in Arabidopsis thaliana. Our data reveals that CEPs induce immune outputs and are required to mount resistance against the leaf-infecting bacterial pathogen Pseudomonas syringae pv. tomato. We show that effective immunity requires CEP perception by tissue-specific CEP RECEPTOR 1 (CEPR1) and CEPR2. Moreover, we identified the related RECEPTOR-LIKE KINASE 7 (RLK7) as a novel CEP4-specific CEP receptor contributing to CEP-mediated immunity, suggesting a complex interplay of multiple CEP ligands and receptors in different tissues during biotic stress. CEPs have a known role in the regulation of root growth and systemic nitrogen (N)-demand signaling. We now provide evidence that CEPs and their receptors promote immunity in an N status-dependent manner, suggesting a previously unknown molecular crosstalk between plant nutrition and cell surface immunity. We propose that CEPs and their receptors are central regulators for the adaptation of biotic stress responses to plant-available resources.

plant biology↗

bioRxiv2022

A group of Arabidopsis thaliana Leucine Rich Repeat-Malectin Receptor Kinases are required for the activation of immune responses triggered by cellulose and mixed-linked glucan oligosaccharides

Plant immune system perceives through the extracellular ectodomains (ECDs) of Pattern Recognition Receptors (PRRs) a diversity of carbohydrate ligands from plant and microbial cell walls, which activate Pattern-Triggered Immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked {beta}-1,3/{beta}-1,4-glucans (MLGs, e.g., {beta}-1,4-D-(Glc)2-{beta}-1,3-D-Glc, MLG43) and cellulose (e.g., {beta}-1,4-D-(Glc)3, CEL3). The mechanisms of perception of carbohydrates by plants are poorly characterized, with the exception of that determining recognition of fungal chitin oligosaccharides (e.g., {beta}-1,4-D(GlcNAc)6, CHI6) that involves several PRRs with LysM-ECDs that function as receptor or co-receptors. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp), which are defective in PTI activation mediated by MLG43 and CEL3, but not CHI6. igp1-igp4 are altered in receptor-like kinases [RLKs: AT1G56145 (IGP1), AT1G56130 (IGP2/3), and AT1G56140 (IGP4)] with Leucine-Rich-Repeat (LRR) and Malectin (MAL) domains in their ECDs. igp4 is a T-DNA insertional, loss of function mutant whereas igp1 and the allelic igp2/igp3 harbour point mutations (E906K and G773E, respectively) in their kinase domains, which impact their structure and surface electrostatic potential as revealed by in silico structural analyses. Notably, Isothermal Titration Calorimetry assays with purified ECD-RLKs showed that AT1G56145 binds with high affinity CEL3 (Kd = 1.19 {+/-} 0.03 M) and cellopentaose (Kd = 1.40 {+/-} 0.01 M), but not MLG43, supporting AT1G56145 function as a plant PRR for cellulose oligosaccharides. Our data suggest that these LRR-MAL RLKs are receptor/co-receptors of a novel mechanism of perception of cellulose and MLG-derived oligosaccharides and PTI activation in Arabidopsis thaliana. Significance StatementNew oligosaccharides that trigger plant immunity have been described recently, but the mechanisms of perception of these glycans are unknown. We describe here three Arabidopsis thaliana receptor kinases (AT1G56130, AT1G56140, and AT1G56145) with Leucine Rich Repeat (LRR) and Malectin (MAL) domains in their extracellular ectodomains (ECDs), which function as Pattern Recognition Receptors (PRRs) triggering immune response mediated by oligosaccharides from cellulose ({beta}-1,4-glucan) and mixed-linked {beta}-1,3/1,4-glucans (MLGs) of plant and microbial cell walls. The ECD-AT1G56145 binds cellulose oligosaccharides, but not MLGs, supporting its function as a novel receptor of carbohydrate ligands in plants. Our data indicate that these LRR-MAL-PRRs control a complex mechanism of oligosaccharides perception and immune activation that differs from that of fungal chitin oligosaccharides recognition which involves LysM-PRRs.

plant biology↗

bioRxiv2021

Perception of a conserved family of plant signalling peptides by the receptor kinase HSL3

Plant genomes encode hundreds of secreted peptides; however, relatively few have been characterised. We report here an uncharacterised, stress-induced family of plant signalling peptides, which we call CTNIPs. Based on the role of the common co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) in CTNIP-induced responses, we identified the orphan receptor kinase HAESA-LIKE 3 (HSL3) as the CTNIP receptor via a proteomics approach. CTNIP binding, ligand-triggered complex formation with BAK1, and induced downstream responses all involve HSL3. Notably, the HSL3-CTNIP signalling module is evolutionarily ancient, predating the divergence of extant angiosperms. The identification of this signalling module will help establish its physiological role and provides a resource to understand further receptor-ligand co-evolution.

plant biology↗

bioRxiv2021

A subset of CB002 xanthine analogues bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes including metastasis and therapy resistance. CB002 and structural-analogues restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2-checkpoint. Novel CB002-analogues induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogues target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression and downregulation of essential proteins in DNA-synthesis and -repair. CB002-analogue #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs show activation of p53 pathway signaling in tumors with mutated p53, and target an S-phase checkpoint.

cancer biology↗

bioRxiv2019

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Plant reproduction relies on the highly regulated growth of the pollen tube for proper sperm delivery. This process is controlled by secreted RALF signaling peptides, which have been previously shown to be perceived by CrRLK1Ls membrane receptor-kinases and leucine-rich (LRR) extensin proteins (LRXs). Here we demonstrate that RALF peptides are active as folded, disulfide bond-stabilized proteins, which can bind to the LRR domain of LRX proteins with nanomolar affinity. Crystal structures of the LRX-RALF signaling complexes reveal LRX proteins as constitutive dimers. The N-terminal LRR domain containing the RALF binding site is tightly linked to the extensin domain via a cysteine-rich tail. Our biochemical and structural work reveals a complex signaling network by which RALF ligands may instruct different signaling proteins - here CrRLK1Ls and LRXs - through structurally different binding modes to orchestrate cell wall remodeling in rapidly growing pollen tubes.\n\nSignificancePlant reproduction relies on proper pollen tube growth to reach the female tissue and release the sperm cells. This process is highly regulated by a family of secreted signaling peptides that are recognized by cell-wall monitoring proteins to enable plant fertilization. Here, we report the crystal structure of the LRX-RALF cell-wall complex and we demonstrate that RALF peptides are active as folded proteins. RALFs are autocrine signaling proteins able to instruct LRX cell-wall modules and CrRKL1L receptors, through structurally different binding modes to coordinate pollen tube integrity.

plant biology↗

bioRxiv2018

Crystal structures of two tandem malectin-like receptor kinases involved in plant reproduction

A complex cell-to-cell communication between the male pollen tube and the female reproductive organs is required for plant fertilization. A family of Catharanthus roseus Receptor Kinase-1 ( CrRLK1L) membrane receptors has been genetically implicated in this process. Here we present crystal structures of the CrRLK1Ls ANXUR1 and 2 at 1.48 and 1.1 [A] resolution, respectively. Our structures reveal a novel arrangement of two malectin-like domains connected by a short {beta}hairpin linker and stabilized by calcium ions. The canonical carbohydrate interaction surfaces of related animal and bacterial carbohydrate binding modules are not conserved among plant CrRLK1Ls. In line with this, we failed to detect binding of chemically diverse oligosaccharides to ANXUR1 and HERCULES1. Instead, CrRLK1Ls have evolved a protein-protein interface between their malectin domains, which forms a deep cleft lined by highly conserved aromatic and polar residues. Analysis of the glycosylation pattern of different CrRLK1Ls and their oligomeric states together suggests that this cleft could resemble a binding site for a ligand required for CrRLK1Ls receptor activation.

plant biology↗