Biology subjects

Evangelisti, E.

Publications and source records attributed to Evangelisti, E..

15 recordsLinked to original sources

bioRxiv2026

Data-driven inference of local behavioural rules predicts emergent properties of Phytophthora zoospore dispersal

Motile microorganisms explore complex environments in search of nutrients, hosts and favourable ecological niches. Plant-pathogenic oomycetes, for instance, undergo such an exploratory phase through biflagellate zoospores that actively swim through water-filled soil pores before infecting host tissues. Linking individual zoospore swimming behaviour to emergent dispersal remains challenging. Here, we present an end-to-end, data-driven framework that transforms time-lapse microscopy image sequences into generative agent-based simulations of zoospore dispersal by inferring local behavioural rules directly from experimental trajectories. Using Phytophthora nicotianae as a model system, we isolated nearly 60,000 zoospore trajectories and quantified both local behavioural descriptors and emergent trajectory properties. Local behavioural measurements were first used to infer an empirical two-state model distinguishing SLOW and FAST swimming regimes while capturing temporal memory and the coupling between speed and turning. Implemented within an agent-based cellular automaton, this model reproduced the principal emergent properties of experimental dispersal. We then independently inferred the behavioural organisation of zoospore swimming using hidden Markov models. The most parsimonious two-state HMM recovered a closely related behavioural organisation, while revealing that the inferred states jointly reflected swimming speed, turning dynamics and directional persistence rather than speed alone. Finally, we challenged the inferred behavioural rules in an independent obstacle-filled environment. Combined with simple collision hypotheses, the model reproduced emergent dispersal without recalibrating the swimming rules and identified transient post-collision slowdown as a key response required to account for the experimental trajectories. Together, these results demonstrate that experimentally inferred local behavioural rules possess predictive power beyond the conditions used for their calibration. More broadly, this work establishes a predictive framework linking quantitative microscopy, behavioural-rule inference and generative modelling of microbial dispersal.

microbiology↗

bioRxiv2026

Live imaging reveals polarized calcium transients during plant pathogen development and host colonization

Ca2+ signaling mediates rapid cellular responses across eukaryotes, but its spatiotemporal dynamics remain largely inaccessible in many genetically less tractable microbial lineages. Oomycete plant pathogens, including Phytophthora species, undergo rapid transitions between motile, encysted, germinating, and invasive stages, yet the organization of Ca2+ dynamics during these transitions is poorly understood. Here, we adapt the genetically encoded ratiometric biosensor MatryoshCaMP8s for in vivo calcium imaging in Phytophthora palmivora. The reporter is stably expressed without major detectable effects on sporulation or virulence and reports rapid ratiometric responses to cold shock and calcimycin/A23187. Using live-cell imaging, we uncover stage-specific Ca2+ dynamics across the pre-infective and early infection cycle. Sporangia approaching zoospore release display spatially heterogeneous Ca2+ transients, newly formed cysts occasionally exhibit Ca2+ transients, and germinating cysts show recurrent Ca2+ transients at the germ tube tip. Similar sharp ratiometric pulses occur during early plant infection, indicating that polarized Ca2+ transients are not restricted to in vitro germination but recur at the host surface. Together, our work establishes live ratiometric calcium imaging in oomycetes, reveals polarized Ca2+ transients as recurrent signatures of developmental transitions and early host colonization, and opens the way to mechanistic dissection of signaling, polarity, and infection in a major group of plant pathogens.

microbiology↗

bioRxiv2026

Engineering quantitative root disease resistance in barley by targeting conserved SCAR susceptibility genes without compromising seed yield or mycorrhizal symbiosis

Durable resistance to soil-borne pathogens remains elusive in cereals, partly because susceptibility (S) genes that facilitate root infection have not been identified in monocots. In the model legume Medicago truncatula, the SCAR/WAVE complex member MtAPI functions as a root S-gene for microbial invasion. Whether SCAR gene associated susceptibility function is conserved in monocots, and whether SCAR gene inactivation can enhance root resistance in cereals, remains unknown. Here, we identify and characterize three SCAR genes in barley: HvSCAR-A, HvSCAR-B, and HvSCAR-C. Cross-species complementation assays indicate that HvSCAR-B and HvSCAR-C are functionally similar to MtAPI. While hscar-b and hvscar-c single mutants exhibited no major growth defects, hvscar-a mutants showed strongly reduced seed production, and a hvscar-b/c double mutant displayed shorter root hairs. Notably, the hvscar-b/c double mutant exhibited increased resistance to the hemibiotrophic pathogen Phytophthora palmivora but greater colonization by the symbiotic arbuscular mycorrhizal fungus Funneliformis mosseae, underscoring a complex role in plant root - microbe interactions. Our findings reveal a conserved susceptibility function of SCAR genes in monocots and identify api monocot homologs as promising targets for engineering disease resistance in cereals. This study offers new insights into SCAR protein functional diversification and its potential for improving root health in crop plants.

plant biology↗

bioRxiv2026

MycorrhizaFinder: an efficient machine learning tool to quantify endomycorrhizal colonisation of real-world roots

AbstractO_ST_ABSBackground and aimsC_ST_ABSRoot colonisation by endomycorrhizal fungi has long been one of the most widely used metrics in mycorrhizal studies. However, due to the significant time required to assess colonisation using traditional microscope techniques, studies of colonisation at large scales are impractical. AI-powered approaches may increase output and facilitate ecosystem assessments. MethodsWe trained an AI-powered tool (MycorrhizaFinder) on field roots from diverse grasslands and heathlands hosting common Northern European plants with a range of arbuscular (AM) and ericoid mycorrhizal (ErM) fungal structures, and dark septate endophytes (DSE), also common in field-sourced roots. We incorporated a user-customized confidence threshold to encourage the user to engage with inevitable morphological ambiguities, in e.g. ErM and DSE. A Macro F1 statistic was used to assess the tools development. ResultsWe provide a sample workflow from root processing and microscope slide scanning to semi-automated model training and performance evaluation. Without human supervision, our automated baseline Macro F1 is 66% for arbuscular and at 57% for ericoid mycorrhizal colonisation assessment. ConclusionMycorrhizaFinder is user friendly, requires no programming skills and offers flexibility for advanced agronomists or ecologists who wish to train the tool using their own labelled mycorrhizal root datasets, including images acquired from different instruments or staining protocols. This adaptability allows users to customize the model for specific ecosystems or experimental designs. Leveraged with molecular identification and/or functional assessment of fungi, MycorrhizaFinder could support scalable and repeatable monitoring across ecosystems to assess mycorrhizal status and track land-use changes over time.

ecology↗

bioRxiv2026

Deep learning enables quantitative subcellular analysis of plant-microbe interfaces

Specialized host-microbe interfaces are central to cellular interactions in plants. Intracellular structures such as haustoria formed by filamentous pathogens mediate nutrient exchange and effector delivery to host cells. Despite their biological importance, the lack of quantitative frameworks has largely confined the study of these interfaces to qualitative observations, limiting our ability to compare infection strategies, cellular responses, and spatial organization across cells and tissues. Here, we present HFinder, a deep learning-based framework for automated detection, segmentation, and quantitative analysis of plant-microbe interfaces in confocal images. Using an object-centric deep learning approach, HFinder enables robust identification of haustoria, microbial hyphae, and host organelles across diverse imaging conditions and pathosystems. We demonstrate that this framework supports quantitative analyses of subcellular processes at host-microbe interfaces, including effector secretion, perturbation of host cellular processes, and immune receptor accumulation at haustoria. HFinder provides a practical and scalable solution for the systematic digitalization of plant infection imaging data and establishes a general framework for quantitative studies of cellular dynamics at host-microbe contact zones.

cell biology↗

bioRxiv2025

Microtubules in the coenocyte Phytophthora function in nuclear positioning and sustaining tip growth

The microtubule cytoskeleton consists of dynamic intracellular filaments and is involved in numerous processes, ranging from nuclear division to intracellular transport. Some of these microtubule-mediated processes are conserved in all eukaryotic lineages while others are specific for certain groups of organisms. Here, we focus on the microtubule cytoskeleton of oomycetes in the genus Phytophthora, a group of harmful plant pathogens. For visualizing microtubule organization and dynamics, we generated transgenic Phytophthora palmivora lines expressing GFP-tagged -tubulin. Besides a conserved localization in the mitotic spindle, we observed cytoplasmic microtubules originating from microtubule-organizing centers associated with nuclei in the coenocytic hyphae. These dynamic microtubules initiated long-lasting, antiparallel connections with microtubules originating from adjacent nuclei. After mitosis, these microtubules rapidly increased in length while maintaining their antiparallel interaction. Frequent buckling events suggest that microtubule-based force generation plays a role in nuclear spacing. This idea was strengthened by erratic nuclear motility and positioning in hyphae exposed to the microtubule depolymerizing drug oryzalin. Besides aberrant nuclear positioning we also observed defects in hyphal growth; in oryzalin-treated hyphae lacking microtubules, tip growth was less sustained than in non-treated hyphae. This suggests that microtubules radiating into the hyphal tip from the apical nucleus have a function in sustaining tip growth. Altogether, this study provides novel insights in the localization, dynamics and functions of the microtubule cytoskeleton in the coenocytic Phytophthora hyphae.

cell biology↗

bioRxiv2025

An approach to enhance symbiotic nitrogen fixation

Through symbiosis with nitrogen-fixing bacteria, cultivated legumes provide themselves and subsequent crops with nitrogen, making genetic improvements of symbiotic efficiency particularly attractive. Here, we identify the symbiosis-specific GBP1 gene which negatively regulates nitrogen fixation by attenuating bacterial nitrogenase activity in Medicago truncatula nodules. GBP1 inactivation increases nitrogen fixation without affecting nodule development and numbers, providing inroads for engineering legumes with increased productivity for sustainable nitrogen provision.

plant biology↗

bioRxiv2025

Membrane Proteome of Phytophthora parasitica Zoospores: How Does Sensing Occur?

Phytophthora plant pathogens rely on motile biflagellated zoospores to efficiently locate and colonise host tissues. While rhizospheric signals guiding zoospore movement toward roots are known, the protein composition of membranes mediating these responses remains unclear. Here, we used liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic data mining to analyse membrane fractions from the flagella and cell bodies of Phytophthora parasitica zoospores. Major classes of membrane proteins (receptors, transporters and enzymes) were identified and their subcellular distribution between flagella and cell bodies quantified. Immunolocalization revealed that while most membrane proteins are evenly distributed, a subset localizes to the flagella, suggestive of specialized roles in sensing and movement regulation, particularly for sterol recruitment and ion flux variations. These findings advance our understanding of protein-mediated dispersal and host targeting by zoospores and support the hypothesis that zoospores use polarized signal perception mechanisms for environmental sensing and movement.

microbiology↗

bioRxiv2024

LbCas12-mediated multiplex gene editing and 2-fluoroadenine counter-selection in Phytophthora palmivora

CRISPR-Cas systems have moved forward genetic engineering in virtually any organism amenable to genetic modification. In particular, these systems have unlocked unprecedented possibilities to generate mutants in oomycetes, a group of filamentous microbes comprising over two hundred Phytophthora species, including the cacao killer Phytophthora palmivora. Here, we showcase multiplex gene editing in P. palmivora using LbCas12. We have developed a straightforward protocol to simultaneously knock out two genes encoding adenine phosphoribosyltransferase (APT), an essential enzyme of the purine salvage pathway. We show that APT knockouts ({Delta}PpATP1/2) are insensitive to 2-fluoroadenine (2-FA) and retain full virulence on Nicotiana benthamiana. We rely on zoospore electroporation using an all-in-one construct to facilitate the rapid editing of multiple genes. This work enhances the genetic toolbox for Phytophthora species and simplifies the exploration of gene function, laying the groundwork for future innovations aiming to tackle oomycete plant diseases.

microbiology↗

bioRxiv2024

An oomycete effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant- derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TBC1D15L, to remodel the host-pathogen interface. The effector, PiE354, hijacks TBC1D15L as a susceptibility factor to usurp its GAP activity on Rab8a--a key Rab GTPase crucial for defense-related secretion. By hijacking TBC1D15L, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co- opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

plant biology↗

bioRxiv2022

A pathogen effector FOLD diversified in symbiotic fungi

Pathogenic fungi use secreted effector proteins to suppress immunity and support their infection, but effectors have also been reported from fungi that engage in nutritional symbioses with plants. Sequence based effector comparisons between pathogens and symbiotic arbuscular mycorrhizal (AM) fungi are hampered by the huge diversity of effector sequences even within closely related microbes. Here we used a systematic protein structure modelling approach to classify the secretome of the AM fungus Rhizophagus irregularis. We identified secreted proteins with high structural similarity to Fusarium oxysporum f. sp. lycopersici dual domain (FOLD) effectors, which occur in low numbers in fungal pathogen genomes. Contrastingly, genes encoding FOLD proteins from AM fungi (MycFOLDs) are found in enlarged and diversified gene families. Our structure-model comparison suggests that MycFOLDs are similar to carbohydrate binding motifs. Different MycFOLD genes are expressed during colonisation of different hosts and MycFOLD-17 transcripts accumulate in plant intracellular arbuscules. The exclusive presence of MycFOLDs across unrelated plant-colonising fungi, their inducible expression, lineage specific sequence diversification, and transcripts in arbuscules support the hypothesis that FOLD proteins act as effectors during plant colonisation by symbiotic and pathogenic fungi.

microbiology↗

bioRxiv2021

Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species and have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements (TEs) for generating genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Characterising the epigenomic status of R. irregularis provides evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a potential role for TEs in shaping the genome, and roles for DNA methylation and small RNA-mediated silencing in regulating TEs. A well-controlled balance between TE activity and repression may therefore contribute to genome evolution in AM fungi.

genomics↗

bioRxiv2021

Artificial intelligence enables the identification and quantification of arbuscular mycorrhizal fungi in plant roots

Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the abundance of hyphal structures in mutant roots rely on staining and human scoring involving simple, yet repetitive tasks prone to variations between experimenters. We developed AMFinder which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks. AMFinder delivered high-confidence predictions on image datasets of roots of multiple plant hosts (Nicotiana benthamiana, Medicago truncatula, Lotus japonicus, Oryza sativa) and captured the altered colonisation in ram1-1, str and smax1 mutants. A streamlined protocol for sample preparation and imaging allowed us to quantify mycobionts from the genera Rhizophagus, Claroideoglomus, Rhizoglomus and Funneliformis via flatbed scanning or digital microscopy including dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed here: https://github.com/SchornacklabSLCU/amfinder.git

plant biology↗

bioRxiv2019

Centrin-anchored hydrodynamic shape changes underpin active nuclear rerouting in branched hyphae of an oomycete pathogen

Polyenergid fungi and oomycetes are phylogenetically distant but structurally similar. To address whether they share similar nuclear dynamics we carried out time-lapse imaging of fluorescently labelled Phytophthora palmivora nuclei. Nuclei underwent coordinated bi-directional movements during plant infection. Within hyphal networks growing in planta or in axenic culture nuclei are either dragged passively with the cytoplasm or actively become rerouted toward nuclei-depleted hyphal sections and often display a very stretched shape. Benomyl-induced depolymerization of microtubules reduced active movements and the occurrence of stretched nuclei. A centrosome protein localized at the leading end of stretched nuclei, suggesting that like fungi astral microtubule-guided movements contribute to nuclear distribution within oomycete hyphae. The remarkable hydrodynamic shape adaptations of Phytophthora nuclei contrast those in fungi and likely enable them to migrate over longer distances. Therefore, our work summarises mechanisms which enable a near equal nuclear distribution in an oomycete. We provide a basis for computational modelling of hydrodynamic nuclear deformation within branched tubular networks.

cell biology↗

bioRxiv2017

Time-resolved dual root-microbe transcriptomics reveals early induced Nicotiana benthamiana genes and conserved infection-promoting Phytophthora palmivora effectors

BackgroundPlant-pathogenic oomycetes are responsible for economically important losses on crops worldwide. Phytophthora palmivora, a broad-host-range tropical relative of the potato late blight pathogen, causes rotting diseases in many important tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus.\n\nTranscriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped understanding of how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce.\n\nResultsWe employed the model plant N. benthamiana to study the P. palmivora root infections at the cellular and molecular level. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features.\n\nConclusionsThese results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

plant biology↗