Biology subjects

Meile, L.

Publications and source records attributed to Meile, L..

7 recordsLinked to original sources

bioRxiv2025

A high-throughput heterologous expression platform for plant synthetic biology based on Arabidopsis suspension cells

Efficient heterologous expression platforms are essential for plant synthetic biology, particularly for engineering complex multigene pathways. Here, we establish a high-throughput system for both transient and stable transformation of Arabidopsis thaliana suspension cells using plant cell pack infiltration. This method requires no specialized equipment or consumables and is compatible with several cell lines. It enables rapid generation of 100 g of transgenic cells within two weeks and allows expression of at least 6 stacked genes from a single construct. We characterized constitutive promoters for gene expression in Arabidopsis cells and validated plastid targeting peptides. A library of NifB homologs was screened for expression and solubility and several archaeal variants suitable for plant expression were identified. We further engineered stable cell lines expressing up to six genes, encoding the NifB module components NifU, NifS, FdxN, and NifB, demonstrating that the newly developed platform integrates into an established workflow for nitrogenase engineering. The platform accelerates design-build-test cycles and facilitates the production of delicate proteins that require large amounts of transgenic biomass. It thus represents a versatile and scalable tool for advancing synthetic biology and for tackling major biotechnological challenges, such as biological nitrogen fixation. HighlightWe developed a fast and scalable expression platform in Arabidopsis suspension cells, enabling transient and stable multigene expression for applications in plant synthetic biology such as nitrogenase engineering.

plant biology↗

bioRxiv2024

Zymoseptoria tritici stealth infection is facilitated by stage-specific down-regulation of a β-glucanase

Plant cell walls constitute a major defence barrier against pathogens, although it is unclear how specific cell wall components impact pathogen colonisation. Pathogens secrete cell wall degrading enzymes (CWDEs) to facilitate plant colonisation, but damaged, infected cells are often a source of cell wall-derived oligosaccharides that trigger host immunity. The mechanisms by which pathogens minimize the release of cell wall-derived oligosaccharides while colonizing the host remain to be elucidated. We combined biochemical, molecular genetics and transcriptomic analyses to functionally characterize a glycoside hydrolase (ZtGH45) from the wheat pathogen Zymoseptoria tritici. ZtGH45 gene is expressed during the necrotrophic phase of the fungus, coinciding with an accumulation of wheat {beta}-1,3/1,4-mixed-linked glucan (MLG)-derived oligosaccharides. We show that overexpression of ZtGH45 enhances {beta}-1,3/1,4-glucan hydrolysis and the derived oligosaccharides trigger an immune response in wheat, which hinders Z. tritici virulence. The results demonstrate that tight regulation of ZtGH45 is critical for the infection process to prevent early accumulation of MLG oligosaccharides that would prematurely induce host immunity counterbalancing fungal virulence. We suggest that the balance between plant cell wall degradation by fungal CWDE and the release of immunogenic wall-derived oligosaccharides governs the outcome of host invasion by pathogens.

plant biology↗

bioRxiv2024

The Zymoseptoria tritici avirulence factor AvrStb6 accumulates in hyphae close to stomata and triggers a wheat defense response hindering fungal penetration

Zymoseptoria tritici, the causal agent of septoria tritici blotch, is one of Europes most damaging wheat pathogens, causing significant economic losses. Genetic resistance is a common strategy to control the disease, Stb6 being a resistance gene used for over 100 years in Europe. This study investigates the molecular mechanisms underlying Stb6-mediated resistance. Utilizing confocal microscopy imaging, we identified that Z. tritici epiphytic hyphae mainly accumulates the corresponding avirulence factor AvrStb6 in close proximity to stomata. Consequently, the progression of AvrStb6-expressing avirulent strains is hampered during penetration. The fungal growth inhibition co-occurs with a transcriptional reprogramming in wheat characterized by an induction of immune responses, genes involved in stomata regulation, and cell wall-related genes. Overall, we shed light on the gene-for-gene resistance mechanisms in the wheat - Z. tritici pathosystem at the cytological and transcriptomic level, and our results highlight that stomata penetration is a critical process for pathogenicity and resistance.

plant biology↗

bioRxiv2023

Sas3-mediated histone acetylation regulates effector gene activation in a fungal plant pathogen

Effector proteins are secreted by plant pathogens to enable host colonization. Typically, effector genes are tightly regulated, have very low expression levels in axenic conditions, and are strongly induced during host colonization. Chromatin remodeling contributes to the activation of effector genes in planta by still poorly known mechanisms. In this work we investigated the role of histone acetylation in effector gene derepression in plant pathogens. We used Zymoseptoria tritici, a major pathogen of wheat, as a model to determine the role of lysine acetyltransferases (KATs) in plant infection. We showed that effector gene activation is associated with chromatin remodeling, featuring increased acetylation levels of histone H3 lysine 9 (H3K9) and 14 (H3K14) in effector loci. We functionally characterized the role of Z. tritici KATs and demonstrated their distinct contributions to growth, development, and infection. Sas3 is required for host colonization and pycnidia production, while Gcn5 has a major role in pycnidia production. Furthermore, we demonstrated that Sas3 is involved in acetylation of H3K9 and H3K14 in effector loci and in effector gene activation during plant infection. We propose that Sas3-mediated histone acetylation is required for spatiotemporal activation of effector genes and virulence of Z. tritici. IMPORTANCEPathogen infections require the production of effectors that enable host colonization. Effectors have diverse functions and are only expressed at certain stages of the infection cycle. Thus, effector genes are tightly regulated by several mechanisms, including chromatin remodeling. Here, we investigate the role of histone acetylation in effector gene activation in the fungal wheat pathogen Zymoseptoria tritici. We demonstrated that lysine acetyltransferases (KATs) are essential for the spatiotemporal regulation of effector genes. We show that two KATs, Sas3 and Gcn5, are involved in leaf symptom development and pycnidia formation. Importantly, our results indicated that Sas3 controls histone acetylation of effector loci and is a regulator of effector gene activation during stomatal penetration. Overall, our work demonstrates the key role of histone acetylation in regulating gene expression associated with plant infection.

plant biology↗

bioRxiv2018

Precision phenotyping reveals novel loci for quantitative resistance to septoria tritici blotch in European winter wheat

O_LIAccurate, high-throughput phenotyping for quantitative traits is the limiting factor for progress in plant breeding. We developed automated image analysis to measure quantitative resistance to septoria tritici blotch (STB), a globally important wheat disease, enabling identification of small chromosome intervals containing plausible candidate genes for STB resistance.\nC_LIO_LI335 winter wheat cultivars were included in a replicated field experiment that experienced natural epidemic development by a highly diverse but fungicide-resistant pathogen population. More than 5.4 million automatically generated phenotypes were associated with 13,648 SNP markers to perform a GWAS.\nC_LIO_LIWe identified 26 chromosome intervals explaining 1.9-10.6% of the variance associated with four resistance traits. Seventeen of the intervals were less than 5 Mbp in size and encoded only 173 genes, including many genes associated with disease resistance. Five intervals contained four or fewer genes, providing high priority targets for functional validation. Ten chromosome intervals were not previously associated with STB resistance.\nC_LIO_LIOur experiment illustrates how high-throughput automated phenotyping can accelerate breeding for quantitative disease resistance. The SNP markers associated with these chromosome intervals can be used to recombine different forms of quantitative STB resistance that are likely to be more durable than pyramids of major resistance genes.\nC_LI

plant biology↗

bioRxiv2018

Transposable element insertions shape gene regulation and melanin production in a fungal pathogen

Background\n\nVariation in gene expression contributes to phenotypic diversity within species and adaptation. However, very few cases of adaptive regulatory changes have been reported and the mechanisms underlying variation in gene expression remain largely unexplored. Fungal pathogen genomes are highly plastic and harbour numerous insertions of transposable elements, which can potentially contribute to gene expression regulation. In this work we elucidated how transposable elements contribute to variation of melanin accumulation, a quantitative adaptive trait of fungal pathogens that is involved in survival under stress conditions.\n\nResults\n\nWe demonstrated that differential transcriptional regulation of the gene encoding the transcription factor Zmr1, which controls expression of the genes in the melanin biosynthetic gene cluster, is responsible for variation in melanin accumulation in the fungal plant pathogen Zymoseptoria tritici. We show that differences in melanin levels between two strains of Z. tritici are due to two levels of transcriptional regulation: 1) variation in the promoter sequence of Zmr1, and 2) an insertion of transposable elements upstream of the Zmr1 promoter. Remarkably, independent insertions of transposable elements upstream of Zmr1 occurred in 9% of Z. tritici strains from around the world and negatively regulated Zmr1 expression, contributing to melanin accumulation variation.\n\nConclusions\n\nOur studies demonstrate that different layers of transcriptional control fine-tune the synthesis of melanin. These regulatory mechanisms potentially evolved to balance the fitness costs associated with melanin production against its positive contribution to survival in stressful environments.

molecular biology↗

bioRxiv2018

A fungal avirulence factor encoded in a highly plastic genomic region triggers partial resistance to septoria tritici blotch

O_LICultivar-strain specificity in the wheat-Zymoseptoria tritici pathosystem determines the infection outcome and is controlled by resistance genes on the host side, of which many have been identified. However, on the pathogen side, the molecular determinants of specificity are largely unknown.\nC_LIO_LIWe used genetic mapping, targeted gene disruption and allele swapping to characterize the recognition of the new avirulence factor Avr3D1. We then combined population genetic and comparative genomic analyses to estimate the evolutionary trajectory of Avr3D1.\nC_LIO_LIAvr3D1 is specifically recognized by cultivars harboring the resistance gene Stb7 and triggers a strong defence response without preventing pathogen infection and reproduction. Avr3D1 resides in a cluster of putative effector genes located in a region populated by independent transposable element insertions. The gene is present in all 132 investigated strains and is highly polymorphic, with a total of 30 different protein variants. We demonstrated that certain amino acid mutations in Avr3D1 led to evasion of recognition.\nC_LIO_LIThese results demonstrate that quantitative resistance and gene-for-gene interactions are not mutually exclusive per se. Location of avirulence genes in highly plastic genomic regions likely facilitates accelerated evolution that enables escape from recognition by resistance proteins.\nC_LI

plant biology↗