Biology subjects

Lahaye, T.

Publications and source records attributed to Lahaye, T..

7 recordsLinked to original sources

bioRxiv2026

TALE-independent transcriptional activation of the rice executor gene Xa23 is regulated via histone acetylation during zygote development

Transcription activator-like effectors (TALEs) from Xanthomonas activate transcription of executor (E) genes in host plants, leading to cell death and thereby restricting proliferation of biotrophic pathogens. Because E gene transcripts had only been detected upon activation by cognate Xanthomonas TALEs, E genes were thought to function exclusively in plant immunity. Here, we detect TALE-independent transcription of the rice E gene Xa23 in zygotes 4-6 hours after gamete fusion. Histone deacetylase inhibition induces Xa23 transcription in unfertilized egg cells, implicating histone acetylation in Xa23 regulation. We identified potential cis-regulatory elements and transcription start sites associated with native Xa23 transcription during zygote development. Together, our findings suggest that Xa23 is a developmentally regulated gene with a native role during early zygote development. This supports a previously proposed model in which E genes have native functions in development, while fortuitous upstream polymorphisms can create TALE-binding sites that convert them into immune executors.

plant biology↗

bioRxiv2026

An ancient cis-element regulates translation of arginine decarboxylase to control downstream polyamine biosynthesis and stress responses

Polyamines (PAs) are ubiquitous metabolites that, despite their simple structure, profoundly influence plant growth, development, and stress adaptation. Their cellular levels are largely determined by arginine decarboxylase (ADC), a key rate-limiting enzyme in their biosynthesis. We previously identified a [~]50 bp GC-rich sequence in the 5' untranslated region (UTR) of plant ADC genes, termed the ADC-box, that is conserved across land plants. Transient reporter assays in tomato, in which ADC upstream regions were decoupled from their native coding sequences and fused to reporter genes, suggested that this element represses translation. However, its function in the native genomic context and its impact on PA homeostasis remain unclear. Here, we combined CRISPR-Cas9 genome editing, metabolite profiling, enzymatic assays, and RNA structure probing to define ADC-box function in tomato and in the seedless land plant Marchantia polymorpha, which retains a conserved [~]20 bp core region. Mutation of the M. polymorpha ADC-box increased ADC activity and altered PA levels, indicating that the ADC-box functions as a conserved translational repressor. In tomato, disruption of the ADC-boxes in SlADC1 and SlADC2 increased ADC activity, demonstrating that the ADC-box acts as a translational repressor in its native context. These ehects were most pronounced under cold stress, when ADC transcript levels increase, suggesting that the ADC-box buhers stress-induced translation. Metabolically, ADC-box disruption led to agmatine accumulation and alterations in upstream intermediates, while downstream PA pools remained largely unchanged. SHAPE analysis revealed that the tomato ADC-box folds into a three-stem RNA structure, with a central stem representing the major inhibitory module. ADC-box mutants displayed altered plant-microbe interactions, with enhanced resistance to Pseudomonas syringae and Tobacco rattle virus, but increased susceptibility to Ralstonia solanacearum and Tomato yellow leaf curl virus. Together, these findings establish the ADC-box as an evolutionarily conserved cis-regulatory element that stabilizes PA homeostasis and modulates plant-microbe interactions.

plant biology↗

bioRxiv2025

Optimized LC-MS method for simultaneous polyamine profiling and ADC/ODC activity quantification and evidence that ADCs are indispensable for flower development in tomato

Polyamines (PAs) are essential for plant development and stress responses, requiring tight homeostatic regulation. Many PA enzymes are regulated post-transcriptionally, making traditional transcript-based methods ineffective in determining their abundance, highlighting the need for alternative approaches to study PA homeostasis. Here, we refined a liquid chromatography-mass spectrometry (LC-MS) based method to simultaneously quantify activities of two key PA synthesizing enzymes - arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) - from plant tissues using stable isotope substrates. By optimizing substrate concentrations, we increased assay sensitivity >10-fold in tomato leaf tissue. We further adapted this protocol for Nicotiana benthamiana, a model plant widely used for transient recombinant protein expression. Expression of epitope-tagged ADCs in this system revealed a direct correlation between protein abundance and enzymatic activity, demonstrating that ADC activity can infer its protein abundance in native tissues. Proof-of-principle experiments with the N. benthamiana expression system, confirm substrate specificity of tomato ADC and ODC enzymes and essential catalytic residues of tomato ADCs. Beyond enzymatic activities, our LCMS-based method also permits quantification of 11 PA network metabolite concentrations from the same LCMS sample. Visualizing this data as a heatmap pathway diagram, alongside ADC/ODC activities provides a comprehensive overview of PA metabolism in plant tissues. We also studied tomato CRISPR-Cas9-induced mutants deficient in ADC or ODC, complemented by phenotypic analysis. LC-MS analysis of an adc1/adc2 double mutant - an embryo lethal genotype in Arabidopsis - had no detectable agmatine, the product of ADCs. Additionally, despite a reduction in putrescine, no impact on the downstream PAs, spermidine and spermine, was found. The adc1/adc2 double mutant showed severe developmental abnormalities, including complete flower loss, demonstrating the indispensable role of ADCs in flower development. In summary, our optimized LC-MS approach for simultaneous quantification of ADC/ODC enzyme activity and PA-pathway metabolites, the ability to transiently express and functionally analyze recombinant ADC/ODC proteins in planta, and a collection of tomato CRISPR mutants deficient in these enzymes collectively establish a versatile new experimental toolkit to dissect PA homeostasis and PA-dependent developmental processes in plants.

plant biology↗

bioRxiv2024

NUDIX Hydrolases Target Specific Inositol Pyrophosphates and Regulate Phosphate and Iron Homeostasis, and the Expression of Defense Genes in Arabidopsis

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wildtype (WT) and subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

plant biology↗

bioRxiv2021

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas. Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H2O2, a hallmark metabolite in plant immune reactions. Histochemical staining of infected pepper leaves and a translational fusion of Bs3 to the redox reporter roGFP2 both indicated that the Bs3-dependent HR induces a local increase in H2O2 levels in planta. Moreover, our in vitro studies with recombinant Bs3 protein confirmed its NADPH oxidase activity. To test if the NADPH oxidation of Bs3 induces HR, we adapted previous studies which have uncovered mutations in fungal FMOs that result in higher NADPH oxidase activity. We replicated one of these mutations and demonstrated that the generated recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3 in vitro. Translational fusions to roGFP2 showed that Bs3S211A also had increased NADPH oxidase activity in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta. Ultimately, this reveals that Bs3 produces H2O2 in planta, but that the H2O2 produced by Bs3 on its own is not sufficient to trigger HR. We also demonstrated that expression of Bs3 not only triggered HR in plants, but also inhibited proliferation of yeast, which lends this model system to be utilized for the genetic dissection of Bs3 function in future studies. One sentence summaryThe executor-type resistance protein Bs3 from pepper (Capsicum annuum) acts as an NADPH oxidase but reactive oxygen species produced by Bs3 are not sufficient to trigger plant cell death

plant biology↗

bioRxiv2020

A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

ABSTRACTForward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between the mutant and a wildtype individual. Ultimately, two additional generations are needed to pinpoint causal DNA changes upon mutant identification. We postulated that genome-wide allele frequency distributions within the mutants of M2 families facilitate discrimination of causal versus non-causal mutations, essentially eliminating the need for F2 populations. In a proof-of-principle experiment, we aimed to identify signalling components employed by the executor-type resistance (R) protein, Bs4C, from pepper (Capsicum pubescens). In a native setting, Bs4C is transcriptionally activated by and mediates recognition of the transcription activator-like effector AvrBs4 from the bacterial pathogen Xanthomonas. Arabidopsis containing an estradiol-inducible Bs4C transgene was used in a conditionally lethal screen to identify second-site suppressor mutations. Whole genome sequencing was used for M2 mutant allele-frequency distribution (MAD) mapping in three independent M2 families. MAD-mapping uncovered that all three families harboured mutations in XRN4, a novel component of executor R protein pathways. Our work demonstrates that causal mutations observed in forward genetic screens can be identified immediately in M2 families instead of derived F2 families. Notably, the timesaving concept of MAD mapping should be applicable to most crop species and will advance the appeal of forward genetics beyond applications in fundamental research.SIGNIFICANCE Forward genetics has uncovered numerous genes that govern plant immune reactions. This procedure relies on mutant plants with modified immune reactions followed by identification of causal DNA changes in derived F2 progeny. We developed a novel forward genetics concept where causal DNA changes are identified in the initial M2 mutants, making time consuming establishment of F2 populations obsolete. To confirm the feasibility of the concept, we mutagenized transgenic Arabidopsis seeds containing the cell death executing resistance gene Bs4C from pepper. Whole-genome sequencing of identified mutant families that lack a Bs4C-dependent cell death revealed the XRN4 gene as a novel component of Bs4C-dependent cell death. This confirms our hypothesis that causal mutations can be identified directly within phenotypically selected mutant families.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Measuring both microbial load and diversity with a single amplicon sequencing library

The ratio of microbial population size relative to the amount of host tissue, or "microbial load", is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because conventional methods to determine load, such as serial dilution plating or quantitative PCR, add substantial experimental burden, they are only rarely paired with amplicon sequencing. Alternatively, whole metagenome sequencing of DNA contributed by host and microbes both reveals microbial community composition and enables determination of microbial load, but host DNA typically greatly outweighs microbial DNA, severely limiting the cost-effectiveness and scalability of this approach. We introduce host-associated microbe PCR (hamPCR), a robust amplicon sequencing strategy to quantify microbial load and describe interkingdom microbial community composition in a single, cost-effective library. We demonstrate its accuracy and flexibility across multiple host and microbe systems, including nematodes and major crops. We further present a technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without loss of information. Because of its simplicity, and the fact that it provides an experimental solution to the well-known statistical challenges provided by compositional data, hamPCR will become a transformative approach throughout culture-independent microbiology.

bioengineering↗