Biology subjects

Ton, J.

Publications and source records attributed to Ton, J..

11 recordsLinked to original sources

bioRxiv2026

Molding the Rice Methylome for Disease Resistance

In Arabidopsis thaliana, epigenetic changes in the DNA methylome can prime transcriptional responses to biotic and abiotic stress, resulting in enhanced resistance. Epigenetic recombinant inbred lines have further enabled the identification of trans-generationally stable epialleles controlling stress resistance without adverse effects on plant growth, highlighting their potential for crop improvement. Unfortunately, extending these approaches to crops has remained largely unsuccessful due to differences in genome architecture. The rice (Oryza sativa) genome consists for [~]40% of transposable elements and other epigenetically regulated repeat sequences. Therefore, perturbation of DNA methylation typically leads to severe developmental defects, sterility or lethality, which precludes the use of methylome engineering strategies for epiallele mapping and crop improvement. Here, we exploit an inducible system to introduce for the first time widespread epigenetic variation in rice without detrimental phenotypic consequences. We combined the A. thaliana DNA demethylase AtROS1 with the {beta}-estradiol-activated XVE cassette (XVE:AtROS1-YFP) to enable transient DNA demethylation during early development. Induction of the construct in transgenic Nipponbare seedlings yielded genome-wide changes in the DNA methylome which persisted for at least one generation. Strikingly, these methylome changes did not cause developmental defects or reduced seed yield, but instead correlated with enhanced resistance against Xanthomonas oryzae, the causal agent of bacterial leaf blight in rice. Our study demonstrates that controlled, transient DNA demethylation can uncouple epigenetic variation from deleterious phenotypes in rice. This approach provides a practical framework for generating epigenetic mapping populations and opens new avenues for harnessing epigenetic variation in crop improvement.

plant biology↗

bioRxiv2026

Brassinosteroid-regulated transcription factors confer epigenetic changes that repress plant immunity

When organisms encounter pathogens, they rapidly activate complex defense programs to ensure survival. While these immune responses are vital, they often also incur trade-offs, such as reduced growth and development and must therefore be tightly controlled 1,2. In this study, we reveal that the steroid hormones brassinosteroids (BRs) contribute to this control in Arabidopsis thaliana by repressing immunity-related genes. We provide evidence that the BR-regulated basic helix-loop-helix (bHLH) transcription factor CESTA (CES), along with its homologs BR ENHANCED EXPRESSION (BEE)1-3, mediate DNA methylation changes at transposable element (TE)-rich loci containing nucleotide-binding leucine-rich-repeat (NLR)-type receptor genes, including SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1). These CES-induced methylation changes correlate with altered splicing of SNC1 pre-mRNA, a process that requires the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). In support, we show that CES associates with components of the chromatin remodeling and splicing machinery. Together, our findings reveal a previously unrecognized BR-induced mechanism that modulates the epigenetic and post transcriptional regulation of immune genes, enabling plants to prioritize growth over defense. Significance statementSteroid hormones are powerful regulators of growth but also act as potent suppressors of immunity, with well-established clinical applications, for example in treating autoimmune diseases in humans. In plants, the steroid hormones brassinosteroids (BRs) exert similar effects. Here, we uncover that BR-mediated immune suppression involves DNA methylation changes and alternative splicing of a subset of immune receptor genes, that govern resistance responses mediated by the plant hormone salicylic acid. We provide evidence for a function of specific bHLH transcription factors in this process that link BR signaling to chromatin remodelling and RNA processing. Thereby our study reveals a novel mode of steroid hormone activity in immune repression, which involves epigenetic and posttranscriptional adjustment of immune receptor function.

plant biology↗

bioRxiv2025

From recall to reset: the role of DNA (de)methylation in modulating plant immune memory

Immune memory enables plants to respond more effectively to recurrent pathogen infections. Epigenetic changes in DNA methylation and small RNAs have been implicated, yet how their temporal dynamics govern the establishment and erasure of immune memory remains unclear. Using a chemically inducible system in Arabidopsis to activate the DNA demethylase REPRESSOR OF SILENCING 1 (ROS1), we establish immune memory against biotrophic pathogens that persists for up to two weeks. Memory establishment coincides with reduced small RNA accumulation and DNA methylation along chromosome arms, revealing gene targets involved in salicylic acid-dependent immunity and DNA damage repair. These changes are progressively reversed during immune memory erasure. In contrast, transposon-rich pericentromeres retain elevated small RNA and DNA methylation levels, consistent with a CLASSY3-associated redistribution of RNA-directed DNA methylation. Our findings show that transient ROS1 activity drives contrasting epigenetic responses across chromosomal regions, shaping both the establishment and erasure of plant immune memory.

plant biology↗

bioRxiv2025

The mutational dynamics of the Arabidopsis centromeres

Centromeres are specialized chromosomal regions essential for sister chromatid cohesion and spindle attachment during cell division. Although many centromeres consist of highly variable tandem repeat arrays, the mutational processes driving this variability remain poorly understood. Here, using replicated genome assemblies of Arabidopsis thaliana mutation accumulation (MA) lines, we define the centromeric mutation spectrum. We find that point mutations occur at an almost tenfold higher rates than in chromosome arms, largely driven by non-allelic gene conversion between closely linked repeat units. Large kilobase-scale indels are also frequent and consistently preserve the tandem repeat array by adding or removing only complete repeat units. Analysis of MA lines deficient in the helicase RTEL1 supports the involvement of homology-directed DNA repair in these mutational processes. Moreover, simulations of sequence turnover using the centromere-specific mutation spectrum recapitulate the emergence of homogenized repeat blocks characteristic of natural centromeres. Together, our results show that centromere evolution is driven by a distinct mutational spectrum shaped by homology-directed DNA repair, providing a quantitative framework for how local mutational processes generate and maintain the large-scale architecture of centromeric DNA.

genomics↗

bioRxiv2024

The role of carbonic anhydrase in rock weathering and pH regulation by the soil bacterium Burkholderia thailandensis E264

Enhanced rock weathering (ERW) is increasingly recognized as a way to sequester atmospheric carbon dioxide (CO2) to slow global warming, but its effectiveness needs to be optimized. Carbonic anhydrase (CA), an enzyme capable of accelerating rock weathering both in vitro and in soil, offers a valuable target due to its ability to convert CO2 into carbonic acid. This conversion promotes rock dissolution, enabling immediate CO2 absorption through cation release and charge-balance mechanisms. Studies have shown that bacteria grown in axenic, rock-amended media increase CA gene expression, but the influence of bacterial CA on rock dissolution rates remains unclear. To investigate this, we used a reverse genetics approach with the phosphate-solubilizing bacterium Burkholderia thailandensis E264. We examined three CA-inactivated mutants alongside the wildtype, growing them in minimal media with basalt rock dust (0-10% w/v) at an initial pH of 6.0. After 7 days, we measured weathering potential through elemental concentrations, pH, and dissolved inorganic carbon. In 1% basalt medium, inactivation of the CA1 gene (BTH_I1052) significantly reduced base cation weathering by 41% compared to the wildtype, whereas inactivation of CA2 (BTH_I0345) and CA3 (BTH_I1199) had no significant effect. In the highly buffered, 10% basalt medium, CA1 had a minor role in weathering, and both CA2 and CA3 had no effect. These findings suggest that CA genes in B. thailandensis operate differently and that CA1s effect is pH-dependent. Surprisingly, CA1 was localized intracellularly, raising questions about how intracellular CAs might influence mineral dissolution, potentially through acidity export or abiontic enzyme activity after cell lysis. ImportanceWhile purified carbonic anhydrase (CA) protein has been shown to increase mineral dissolution rates in mineral-amended media in vitro, it remains unclear if the bacterial CA gene directly drives this process. This study used CA-inactivated mutants of the soil bacterium Burkholderia thailandensis in basalt-amended liquid media and found that only one of the three CA genes influenced mineral dissolution rates. This finding supports prior evidence that bacterial CAs may contribute to mineral dissolution in soils. Importantly, it also showed that not all CA genes in a bacterium may activate under the same conditions, which could impact how soil bacterial CAs are leveraged to enhance weathering. Furthermore, cellular localisation predictions indicated that all three CA genes in B. thailandensis are cytosolic, challenging the common focus on extracellular CAs and suggesting that CA proteins may influence the external environment without needing to be actively exported from the cell.

microbiology↗

bioRxiv2024

Multi-generational biotic stress increases the rate of spontaneous epimutations in a ROS1-dependent manner

Mistakes in the maintenance of CG methylation are a source of spontaneous epimutations in plants that can be inherited across generations. The extent to which these stochastic events are affected by prolonged exposure to biotic and abiotic stress remains poorly characterized. Here, we grew Arabidopsis Mutation Accumulation (MA) lines for 12 generations in the presence of two biotic stressors: Pseudomonas syringae (Pst) and salicylic acid (SA). We found that multi-generational exposure to Pst and SA led to an 18%-32% and 23%-61% increase in the genome-wide epimutation rate, respectively. These rate increases were mainly targeted to subsets of genes characterized by low steady-state methylation (LM), on average, and a lack of transcriptional responsiveness to stress. We show that these effects are mediated by the DNA demethylase Repressor of Silencing 1 (ROS1). Loss of ROS1 not only buffers transcriptional responses to biotic stress, but also stabilizes epimutation rates, particularly in LM genes, rendering them insensitive to environmental perturbations. Taken together, our data demonstrates that stress can induce heritable epimutations and highlights a ROS1-mediated link between transcriptional plasticity and DNA methylation maintenance fidelity over generations.

evolutionary biology↗

bioRxiv2024

Ancestral reconstruction of polyethylene terephthalate degrading cutinases reveals a rugged and unexplored sequence-fitness landscape

The enzymatic degradation of polyethylene terephthalate (PET) is a promising method of advanced plastic recycling. Traditional protein engineering methods often fall short in exploring protein sequence space for optimal enzymes due to structural and rational design limitations. Our study addresses this by using multiplexed ancestral sequence reconstruction (mASR) to explore the evolutionary sequence space of PET-degrading cutinases. With a dataset of 397 cutinase sequences, we created a diverse library of ancestral sequences. Experimental characterization of 48 ancestral sequences revealed a wide range of PETase activities, highlighting the value of mASR in uncovering functional variants when compared to traditional ASR. Our results showed that PETase activity in cutinases evolved through diverse pathways involving distal mutations to the active site, and is readily accessible within this family. Additionally, our analysis of the PETase fitness landscape using one-hot encoding (OHE) and local ancestral sequence embedding (LASE) highlighted the effectiveness of LASE in capturing sequence features relevant to activity. This work emphasizes the utility of mASR as a protein engineering tool for identifying enhanced PET-degrading enzymes, and the advantages of the LASE embedding scheme in mapping the PETase fitness landscape.

biochemistry↗

bioRxiv2023

NPR1 protects young leaves from systemic salicylic acid-induced damage during bacterial infection

The phytohormone salicylic acid (SA) is an important molecular signal that mediates pathogen defence mechanisms, including triggering Arabidopsis immune responses to the hemi-biotroph Pseudomonas syringae pv. tomato (Pst). SA induces the expression of a myriad of defence genes via its receptor and transcriptional regulator NONEXPRESSER OF PR GENES 1 (NPR1). Here, we used chlorophyll fluorescence imaging of Fv/Fm, to detect damage to photosystem II before Pst-induced disease symptoms were visible. We observed that the pathogen only induced damage, and subsequent cell death, in mature leaves while developing leaves in the center of the rosette appeared to be protected. However, in the npr1-1 mutant, Pst-infected mature leaves were able to systemically transmit a signal that caused damage to the photosynthetic machinery in uninfected young leaves. Reductions in Fv/Fm could also be induced systemically in developing npr1-1 leaves by high levels of SA in mature leaves, and rescued by SA biosynthesis deficiency in npr1-1sid2-2 mutants. Together, these results indicate that, in addition to its well-known role as a positive regulator of SA responses, NPR1 also acts to suppress SA-dependent immune responses and thereby protects developing leaves from autoimmune damage.

plant biology↗

bioRxiv2023

An evolutionary epigenetic clock in plants

Molecular clocks are the basis for dating the divergence between lineages over macro-evolutionary timescales (~105-108 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes possess a clock-like behavior. This epimutation-clock is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation-clocks recapitulate known topologies and branching times of intra-species phylogenetic trees in the selfing plant A. thaliana and the clonal seagrass Z. marina, which represent two major modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.

evolutionary biology↗

bioRxiv2022

Predicting antiviral resistance mutations in SARS-CoV-2 main protease with computational and experimental screening

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold, and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/505060v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@14f0713org.highwire.dtl.DTLVardef@15995feorg.highwire.dtl.DTLVardef@8689a7org.highwire.dtl.DTLVardef@b73a64_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

A single amino acid transporter controls the uptake of priming-inducing beta-amino acids and the associated trade-off between induced resistance and plant growth.

Selected beta-amino acids, such as beta-aminobutyric acid (BABA) and R-beta-homoserine (RBH), can prime plants for resistance against broad-spectrum diseases. Here, we describe a genome-wide screen of fully annotated Arabidopsis T-DNA insertion lines for impaired in RBH-induced immunity (iri) against the downy mildew pathogen Hyaloperonospora arabidopsidis, yielding 104 lines that were partially affected and 4 lines that were completely impaired in RBH-induced resistance. The iri1-1 mutant phenotype could be confirmed by an independent T-DNA insertion in the same gene, encoding the high-affinity amino acid transporter LHT1. Using uptake experiments with IRI1/LHT1-expressing yeast cells and mass spectrometry-based quantification of RBH and BABA in leaves of mutant and over-expression lines of IRI1/LHT1, we demonstrate that IRI1/LHT1 acts as the main transporter for cellular uptake and systemic distribution of RBH and BABA. Subsequent characterisation of mutant and over-expression lines of IRI1/LHT1 for induced resistance and growth responses revealed that the level of IRI1/LHT1 expression determines the trade-off between induced resistance and plant growth by RBH and BABA.

plant biology↗