Biology subjects

Moratto, E.

Publications and source records attributed to Moratto, E..

5 recordsLinked to original sources

bioRxiv2025

A microfluidic platform for the observation and quantification of fungal highways

Soil is a complex system characterised by intra- and inter-kingdom interactions among microbial communities. While many different types of fungal-bacterial interactions have been described, hyphal-mediated transport of bacteria via the so-called "fungal highway" (FH) has not been mechanistically described. Bacteria require a liquid film for active movement; therefore, liquid saturation is a significant limiting factor for their dispersal in soil. Hyphal networks contribute to the connectivity between discrete soil microbial populations by providing a physical network through the unsaturated soil matrix. This network serves as a scaffold for liquid transport, thus allowing bacteria to migrate further or access previously isolated spaces. Studying these interactions is challenging due to the complex and stochastic nature of the soil environment; this "black box" aspect makes it difficult to visualise interactions at the microbial scale. Microfluidic technology can provide a solution by offering precise imaging at a high resolution in a physically and chemically controlled environment. We designed a microfluidic device, the Fungal Highways Device (FHD), that allows us to culture filamentous organisms in unsaturated environments and visualise and quantify bacterial dispersal along hyphal networks at the single-cell level. We showed that Pythium ultimum is essential for Pseudomonas putida movement across an unsaturated environment, and we identified mycelial biomass and hyphal front length as key factors influencing the bacterias movement towards the outlet. We propose that the liquid transport facilitated by P. ultimum mycelium influences the FH behaviour during its interaction with P. putida.

bioengineering↗

bioRxiv2024

Visualizing Liquid Distribution Across Hyphal Networks with Cellular Resolution

Filamentous fungi and fungal-like organisms contribute to a wide range of important ecosystem functions. Evidence has shown the movement of liquid across mycelial networks in unsaturated environments, such as soil. However, tools to investigate liquid movement along hyphae at the level of the single cell are still lacking. Microfluidic devices permit the study of fungal and fungal-like organisms with cellular resolution as they can confine hyphae to a single optical plane, which is compatible with microscopy imaging over longer timescales and allows for precise control of the microchannel environment. The aim of this study was to develop a method that enables the visualization and quantification of liquid movement on hyphae of fungal and fungal-like microorganisms. For this, the Fungal-Fungal Interaction (FFI) microfluidic device was modified to allow for the maintenance of unsaturated microchannel conditions. Fluorescein-containing growth medium solidified with agar was used to track liquid transported by hyphae via fluorescence microscopy. Our key findings highlight the suitability of this novel methodology for the visualization of liquid movement by hyphae over varying time scales and the ability to quantify the movement of liquid along hyphae. Furthermore, we showed that at the cellular level, extracellular hyphal liquid movement can be bidirectional and highly dynamic, uncovering a possible link between liquid movement and hyphal growth characteristics. We envisage that this method can be applied to facilitate future research probing the parameters contributing to hyphal liquid movement and is an essential step for studying the phenomenon of fungal highways.

microbiology↗

bioRxiv2023

Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields.

Soil-dwelling microorganisms use a variety of chemical and physical signals to navigate their environment. Plant roots produce endogenous electric fields which result in characteristic current profiles. Such electrical signatures are hypothesised to be used by pathogens and symbionts to track and colonise plant roots. The oomycete pathogen Phytophthora palmivora generates motile zoospores which swim towards the positive pole when exposed to an external electric field in vitro. Here, we provide a quantitative characterization of their electrotactic behaviour in 3D. We found that a weak electric field (0.7 - 1.0 V/cm) is sufficient to induce an accumulation of zoospore at the positive pole, without affecting their encystment rate. We also show that the same external electric field increases the zoospore germination rate and orients the germ tubes growth. We conclude that several early stages of the P. palmivora infection cycle are affected by external electric fields. Taken together, our results are compatible with the hypothesis that pathogens use plant endogenous electric fields for host targeting.

biophysics↗

bioRxiv2021

Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly known. Here we show that NRC4, from the NRC solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses. Significance statementPlant NLR immune receptors function as intracellular sensors of pathogen virulence factors known as effectors. In resting state, NLRs localize to subcellular sites where the effectors they sense operate. However, the extent to which NLRs alter their subcellular distribution during infection remains elusive. We describe dynamic changes in spatiotemporal localization of an NLR protein in infected plant cells. Specifically, the NLR protein accumulates at the newly synthesized plant-pathogen interface membrane, where the corresponding effectors are deployed. Following immune recognition, the activated receptor re-organizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary immune receptors, but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.

plant biology↗

bioRxiv2019

An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4--a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal 1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

plant biology↗