Eleonora Moratto’s research while affiliated with Imperial College London and other places


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Publications (10)


Visualizing liquid distribution across hyphal networks with cellular resolution
  • Article

October 2024

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18 Reads

Biomicrofluidics

Amelia J. Clark

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Emily Masters-Clark

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Eleonora Moratto

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[...]

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Claire E. Stanley

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 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 movement of liquid along hyphae 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.


Schematic of the 3D-printed V-box set-up for root infection assays with P. palmivora zoospores in the presence of external electric fields. The negative and positive electrodes are connected to an external power supply. (A) Global electric field set-up. The roots are enveloped in a constant ionic current. (B) Local electric field set-up. The electrodes are mounted in a mock root located on one side of the V-box, so roots are not enveloped in the ionic current. (C) Mock root used to generate the local electric field, with slots used to insert the positive and negative electrodes.
Zoospore attachment to Arabidopsis roots is reduced by exposure to the global electric field. Distributions of the number of zoospores on the surface of roots exposed to 0.5 V/cm, 0.7 V/cm, and 1.0 V/cm in the global configuration. Each point represents snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} of one root; each color represents one technical replicate. * = p-value < 0.05, **** = p-value < 0.0001 ; Wilcoxon test (n = 6 replicates, each with 5 roots).
Zoospore attachment to Arabidopsis roots is reduced by exposure to the local electric field. (A) Distributions of snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for each Arabidopsis root exposed to ~ 250 µA generated by the local electric field. Each point represents snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for one root; each color represents one technical replicate. **** = p-value < 0.0001; Wilcoxon rank sum exact test (n = 6 replicates, each with 6 roots). (B) Distributions of snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for each Arabidopsis root exposed to ~ 550 µA generated by the local electric field. Each point represents snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for one root; each color represents one technical replicate. ** = p-value < 0.01; Wilcoxon rank sum exact test (n = 4 replicates, each with 6 roots). (C) Distributions of snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for Arabidopsis roots exposed to 550 µA generated by the local electric field. The x-axis describes the distance of each plant root from the local electric field (mock root) (n = 4 roots).
The global EF configuration is more effective than the local (mock root) one in reducing zoospore attachment to Arabidopsis roots. (A) Representative confocal images of roots after 2h infection in control conditions or exposed to ~ 250 µA in the local or global configurations. (B) A global electric field is more effective at reducing zoospore attachment to Arabidopsis roots regardless of current intensity. Distributions of snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for Arabidopsis roots exposed to ~ 250 µA (left panel) and ~ 530 µA (right panel) in the global and local electric field configurations. Each point represents the snorm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{norm}$$\end{document} for one root; each color represents one technical replicate. *** = p-value < 0.001, **** = p-value < 0.0001; Wilcoxon test (n = 4 replicates, each with 5 roots). (C) Pathogen biomass after 24h infection measured as relative expression of a pathogen housekeeping gene (ppWS21) compared to a plant housekeeping gene (atUBC21); each color represents one technical replicate. ns = p-value > 0.05; Tukey test (n = 3 replicates, each with 5 roots).
24h exposure of Arabidopsis roots to ~ 250 µA mildly reduces root growth but does not affect its gravitropic response. (A) Distribution of root growth of seedlings exposed to ~ 250 µA for 24h. Each point represents the growth in mm of one primary root, each color represents one technical replicate. ns = p-value > 0.05, * = p-value < 0.05; Wilcoxon rank sum exact test (n = 3 replicates, each with 5 roots). (B) Distribution of root gravitropism response angle of seedling exposed to ~ 250 µA and turned 90° relative to gravity for 24h. Each point represents the angle between one primary root and the gravity vector; each color represents one technical replicate. ns = p-value > 0.05, * = p-value < 0.05; Wilcoxon rank sum exact test (n = 3 replicates, each with 5 roots).

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Reduction of Phytophthora palmivora plant root infection in weak electric fields
  • Article
  • Full-text available

August 2024

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18 Reads

Scientific Reports

The global food security crisis is partly caused by significant crop losses due to pests and pathogens, leading to economic burdens. Phytophthora palmivora, an oomycete pathogen, affects many plantation crops and costs over USD 1 billion each year. Unfortunately, there is currently no prevention plan in place, highlighting the urgent need for an effective solution. P. palmivora produces motile zoospores that respond to weak electric fields. Here, we show that external electric fields can be used to reduce root infection in two plant species. We developed two original essays to study the effects of weak electric fields on the interaction between P. palmivora’s zoospores and roots of Arabidopsis thaliana and Medicago truncatula. In the first configuration, a global artificial electric field is set up to induce ionic currents engulfing the plant roots while, in the second configuration, ionic currents are induced only locally and at a distance from the roots. In both cases, we found that weak ionic currents (250–550 μA) are sufficient to reduce zoospore attachment to Arabidopsis and Medicago roots, without affecting plant health. Moreover, we show that the same configurations decrease P. palmivora mycelial growth in Medicago roots after 24 h. We conclude that ionic currents can reduce more than one stage of P. palmivora root infection in hydroponics. Overall, our findings suggest that weak external electric fields can be used as a sustainable strategy for preventing P. palmivora infection, providing innovative prospects for agricultural crop protection.

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Figure 3: A) Phase contrast (PC) and fluorescence (fluo) microscopy image of P. ultimum and
Visualizing Liquid Distribution Across Hyphal Networks with Cellular Resolution

August 2024

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15 Reads

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.


Figure 1. Assay for zoospore electrotaxis. (A) Diagram of early P. palmivora zoospore life cycle stages. Swimming P. palmivora spores are characterised by two flagella used for propulsion; encysted spores lose their flagella and undergo organelle rearrangement, causing a change of shape from bean-like to almost spherical; germination is characterised by the emergence of the germ tube; vesicles fuse with the plasma membrane at the tip of the germ tube allowing for directional growth. (B) Schematic of the 3D-printed V-slide set-up for P. palmivora electrotaxis experiments. The negative and positive electrodes are connected to an external power supply; the three horizontal regions are marked −, M, +; the three vertical layers are marked in yellow (top), green (central) and blue (bottom); V, voltmeter; A, ammeter. (C) Time-course of the distribution of spores exposed to a strong sucrose gradient. At each time-point, each bar represents the proportion of swimming spores in the slide's regions with high (H, dark grey), mid (M, grey) and low (L, light grey) sucrose concentration; error bars, s.e.p. After only 10 min, the proportion of spores in the high-sucrose concentration region becomes significantly higher than the one in the low-concentration region ( * * = p-value < 0.01 between H and L; t-test, n = 5).
Figure 2. Zoospores are electrotropic towards the positive electrode. Time-course of the distribution of spores detected in the top, central and bottom layers of the medium in the V-slide. At each timepoint, each bar represents the proportion of spores close to the positive electrode or the right side (dark grey), the negative electrode or the left side (light grey), and in the middle (medium grey); error bars, s.e.p. The grey line is the proportion of all spores detected in that layer, at each timepoint. The spores in the central layer accumulated at the positive pole when exposed to 0.7 and 1.0 V cm −1 approximately 30-70 min after the start of the field (H & K; * = p-value < 0.05 between positive and negative regions; t-test, n = 9).
Figure 3. Ionic currents in the medium are necessary for zoospore electrotaxis. Time-course of the distribution of spores detected in the top, central and bottom layers of the medium in the V-slide containing 0.5 mM Sodium Phosphate. At each timepoint, each bar represents the proportion of spores close to the positive electrode or the left side (dark grey), the negative electrode or the right side (light grey), and in the middle (medium grey); error bars, s.e.p. The grey line is the proportion of all spores detected in that layer, at each timepoint. At 1.0 V cm −1 a significant difference has been observed between the zoospore population at the positive and negative poles is observed at 0, 10 and 20 min in the middle layer, from 10 to 100 min in the bottom layer (A, B, C, D, E & F; * = p-value between positive and negative <0.05; * * = p-value between positive and negative <0.01; t-test, n = 8).
Figure 4. Electric fields do not affect zoospore encystment but affect other aspects of zoospore germination. (A) Proportional encystment for spores pipetted near the positive (green line) or negative (blue line) electrode; error bars, s.e.m. (n = 6). (B) Distribution of the proportion of spore germination when exposed to various electric fields; * * = p-value < 0.01 between the applied field and 0 V cm −1 ; Wilcoxon rank sum exact test (n = 3) (C) Distribution of germ tube length when exposed to various electric fields; * = p-value < 0.05 between the applied field and 0 V cm −1 ; Wilcoxon rank sum test (>23 000 germ tubes in n = 3 biological reps) (D) distribution of germ tube orientation observed within ±45 • from 0 • ( i.e. towards the positive pole) and within ±45 • from 180 • (i.e. towards the negative pole), when exposed to various electric fields. We compared the two distributions in each electric field: * * p-value < 0.01 between the orientations 0 • ± 45 • and 180 • ± 45 • ; Wilcoxon rank sum test.
Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields

July 2023

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58 Reads

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2 Citations

Physical Biology

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 tube’s 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.



Figure 1: Assay for zoospore electrotaxis. A) Diagram of early P. palmivora zoospore life cycle stages. Swimming P. palmivora spores are characterised by two flagella used for propulsion; encysted spores lose their flagella and undergo organelle rearrangement, causing a change of shape from bean-like to almost spherical; germination is characterised by the emergence of the germ tube; vesicles fuse with the plasma membrane at the tip of the germ tube allowing for directional growth B) Schematic of the 3D-printed V-slide set-up for P. palmivora electrotaxis experiments. The black and red electrodes are connected to an external power supply; the three imaging regions are marked -, M, +; V, voltmeter; A, ammeter. C) Time-course of the distribution of spores exposed to a strong sucrose gradient. At each time-point, each bar represents the proportion of swimming spores in the slide's regions with high (H, dark grey), middle (M, grey) and low (L, light grey) sucrose concentration; error bars, s.e.p. After only 10 minutes, the proportion of spores in the high-sucrose
Figure 2: Zoospores are electrotropic towards the positive electrode. Time-course of the distribution of spores detected in the top, central and bottom layers of the medium in the V-slide. At each timepoint, each bar represents the proportion of spores close to the positive electrode or the right side (dark grey), the negative electrode or the left side (light grey), and in the middle (medium grey); error bars, s.e.p. The grey line is the proportion of all spores detected in that layer, at each timepoint. The spores in the central layer accumulated at the positive pole when exposed to 0.7 and 1.0 V/cm approximately 30 to 70 minutes after the start of the field (H & K; *= p-value < 0.05 between positive and negative regions; t-test, n = 9).
Figure 3: Ionic currents in the medium are necessary for zoospore electrotaxis. Time-course of the distribution of spores detected in the top, central and bottom layers of the medium in the V-slide containing only deionised water. At each timepoint, each bar represents the proportion of spores close to the positive electrode or the right side (dark grey), the negative electrode or the left side (light grey), and in the middle (medium grey); error bars, s.e.p. The grey line is the proportion of all spores detected in that layer, at each timepoint. No significant difference has been observed between the zoospore population at the positive and negative poles, in any of the three layers (p-value between positive and negative > 0.05; t-test, n = 6).
Figure 4: Electric fields do not affect zoospore encystment but affect other aspects of zoospore germination. A) Proportional encystment for spores pipetted near the positive (green line) or negative (blue line) electrode; error bars, s.e.m. (n =6). B) Distribution of the proportion of spore germination when exposed to various electric fields; ** = p-value < 0.01 between applied field and 0V/cm; Wilcoxon rank sum exact test (n = 3) C) Distribution of germ tube length when exposed to various electric fields; ** = p-value < 0.01 between applied field and 0V/cm; Tukey multiple comparisons of means test (n = 3) D) Distribution of germ tube orientation observed within ±45° from 0° (i.e. towards the positive pole)
Figure 5: Model of zoospore movement in an electric field in vitro. When exposed to an external electric field in vitro, P. palmivora swimming zoospores are mostly concentrated in the very top layers of the liquid media. The swimming is biased towards the positive pole (electrotaxis), where the zoospores begin encysting. As zoospores encyst, they precipitate to the bottom of the V-slide.
Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields

January 2023

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89 Reads

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 tube’s 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.


Fig. 2. The first alpha helix of ZAR1 is functional in NRC4 and does not alter its localization. (A) Single-plane confocal image showing ZAR1 NRC4α1 -GFP does not focally accumulate at EHM but is cytoplasmically localized. (B) Single-plane confocal image showing NRC4 ZAR1α1 -GFP focally accumulates at EHM with RFPRem1.3. (C) NRC4 ZAR1α1 -GFP and NRC4-GFP but not EV-GFP genetically complements Rpi-blb2 nrc4a/b background by triggering HR when coexpressed with AVRblb2 and resistance when expressed alone and infected 1 dpai. The HR assay was repeated with the same results in 28 plants over two independent experiments. Images were taken at 8 dpai. Scattered points in scatter violin plot represent the area (mm 2 ) occupied by infection spots measured on white light images and averaged per leaf, where each construct had n > 29 plants/leaves. The mean and SE are shown as points and bars, respectively. Three independent biological replicates were conducted as indicated by color of dots. Ultraviolet (shown) and white light imaging was taken at 8 dpi. Unpaired Wilcoxon tests gave P values of 3.0 × 10 −11 for NRC4 ZAR1α1 -GFP to GUS-GFP, 1.6 × 10 −11 for NRC4-GFP to GUS-GFP, and 6.3 × 10 −6 for NRC4-GFP to NRC4 ZAR1α1 -GFP. (D-F) Homology model of NRC4 based on ZAR1 in the (D) inactive ADP-bound state (possibly monomeric), (E) newly active ATP-bound state (possibly monomeric), and (F) where five copies of NRC4 oligomerize into a pentameric resistosome and the five α1-helices insert into the membrane. CC domain (pink) consists of region 1 to 140 aa, 141 to 157 is a disordered linker region, NB-ARC domain (green) 158 to 495 aa, and LRR (blue) 496 to 843 aa. (G) Model showing location of swap for NRC4 ZAR1α1 -GFP chimera from A.
Fig. 3. NRC4 requires a CC domain to accumulate to the EHM. NRC4, NRC2, and ZAR1 C-terminally fused GFP chimeras or truncates were coexpressed with PM and EHM marker RFP-Rem1.3, cytoplasmic marker BFP-EV, and silencing suppressor P19 to boost expression (with the exception of 4CC-GFP, which showed very strong expression). Leaves were infected and after 3 d were imaged with a Leica SP8 microscope. Single-plane micrographs were captured with the names of the constructs blinded to reduce acquisition bias; the image names were also randomized to reduce bias during quantification. A custom ImageJ/FIJI macro was made (see Materials and Methods), which allowed us to quantify the GFP signal at the peak RFP position (EHM) and divide it by the GFP signal at the peak BFP position. This is the EHM enrichment, and a number of 1 is no enrichment, and a number of more than 1 is enrichment at the EHM. BFP and RFP channels are available in SI Appendix, Fig. S5. Cartoon of chimeric swaps uses green to indicate NRC4, orange to indicate NRC2, and yellow to indicate ZAR1. Each dot on the scatter boxplot corresponds to a single measurement from one haustorium. Significance groupings on the Right were determined by first averaging the within-plant values (technical replicates) and then performing an unpaired t test.
Fig. 6. NRC4 is activated by Rpi-blb2 during infection and forms puncta that associate with the EHM and PM. (A-D) Single-plane confocal micrographs showing the localization of active and inactive variants of NRC4, with PM marker RFP-Rem1.3 during infection with P. infestans. (Scale bars, 10 μm.) (A) NRC4 L9E -GFP forms puncta associated with the EHM and PM in Rpi-blb2 nrc4a/b plants and partially but not fully accumulates at the EHM. (B) NRC4 L9E -GFP focally accumulates to the EHM in nrc4a/b plants. (C) NRC4 L9E/D478V -GFP does not focally accumulate at the EHM but instead forms puncta on the PM and EHM in nrc4a/b plants. (D) EV-GFP does not focally accumulate or form puncta in nrc4a/b plants. (E) Model depicting possible modes of action of NRC4 where NRC4 gets activated via sensor NLRs (not shown) at the EHM by detecting perihaustorial effectors. NRC4 then oligomerizes into a resistosome and targets the EHM, but a population of resistosomes dissociates from the EHM to target the PM. These resistosomes insert into the membrane to cause programmed cell death.
Dynamic localization of a helper NLR at the plant–pathogen interface underpins pathogen recognition

August 2021

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328 Reads

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50 Citations

Proceedings of the National Academy of Sciences

Significance Plant NLRs function as intracellular immune sensors of pathogen virulence factors known as effectors. In the 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 reorganizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary receptors but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.


Figure 1. NRC4, but not NRC2 or ZAR1, accumulates at the EHM
Figure 2: NRC4 localises to an EHM microdomain. (A) Peak NRC4-mKOk perihaustorial fluorescence does not overlap with the cytoplasmic marker EV-GFP. (B) NRC4-GFP shows a similar but not identical localisation to the PM and EHM marker Solanum tuberosum StRFPRemorin1.3 (RFP-Rem1.3). (C) NRC4-mKOk shows a perihaustorial localisation partially overlapping that of EHM markers RPW8.2-BFP and RFP-Rem1.3. Line profile shown below merge image. (D) Some vesicles of RPW8.2-YFP are not labelled by NRC4, including one at the EHM (arrowhead). (E) NRC4-GFP perihaustorial signal is mutually exclusive to that of the ER marker SP-RFP-HDEL. (F) NRC4-mKOk shows a perihaustorial localisation mostly exclusive to that of GFP-SYT1. C was taken with Leica SP8, others SP5. Scale bars = 10 μm.
Figure 3 The first alpha helix of ZAR1 is functional in NRC4 and does not alter its localisation. (A) Single plane confocal micrograph showing ZAR1 NRC4a1 -GFP does not focally accumulate at EHM but is cytoplasmically localised. (B) Single plane confocal micrograph showing NRC4 ZAR1a1 -GFP focally accumulates at EHM with RFP-Rem1.3. Model depicts the chimeras of NRC4 ZAR1a1 . (C) NRC4 chimera with the N-terminal 17 amino acids from ZAR1, and NRC4-GFP but not EV-GFP genetically complements Rpi-blb2 nrc4a/b background by triggering HR when coexpressed with AVRblb2 and resistance when expressed alone and infected 1 dpai. The HR assay was repeated with the same results in 28 leaves, over two independent biological reps where N > 11 leaves. Images were taken at 8 dpai. Scattered points in scatter violin plot represent the area (mm 2 ) occupied by infection spots measured on white light images, where each construct had N > 88 infection spots. The mean and standard error are shown as point and bars respectively. Three independent biological reps were conducted as indicated by colour of dots. UV (shown) and white light imaging was taken at 8 dpi. Unpaired Wilcoxon tests gave p-values of 1.33 x10 -10 for NRC4-GFP to NRC4 ZAR1a1 -GFP, 1.84 x 10 -29 for NRC4 ZAR1a1 -GFP to GUS-GFP and 6.99 x 10 -27 for NRC4-GFP to GUS-GFP. (D-F) Homology model of NRC4 based on ZAR1 in the (D) inactive ADP-bound state (possibly monomeric), (E) newly active ATP-bound state (possibly monomeric) and (E) where five copies of NRC4 oligomerise into a pentameric resistosome and the five a1 helices insert into the membrane. CC domain (pink) consists of region 1-140 amino acids (aa), 141-157 is a disordered linker region, NB-ARC domain (green) 158-495 aa, LRR (blue) 496-843 aa.
Figure 7: NRC4 is activated by Rpi-blb2 during infection and forms puncta that associate with the EHM and plasma membrane.
Fig. S3. RFP-Rem1.3, BFP-EV and Merge channels for Figure 4, western blots showing chimeras are expressed. Single-plane confocal micrographs of C-terminally tagged GFP constructs of NRC4 truncates, NRC4-NRC2 chimeras or NRC4-ZAR1 chimeras, co-expressed in nrc4a/b plants with RFP-Rem1.3 and BFP-EV, with P19 for all but 4CC-GFP due to its high expression. Leaves were infected 3 hours post infiltration and imaged 3 days later. Arrowheads indicate haustoria. (A) NRC4∆CC-GFP localises to the cytoplasm throughout the cell, including around the haustorium, but does not accumulate to the EHM; it also forms some (motile) puncta in the cytoplasm. (B) 4CC-GFP localises to the cytoplasm throughout the cell, including around the haustorium, but does not accumulate to the EHM. (C) NRC4 2CC -GFP accumulates at the EHM. (D) NRC4 Z1CC -GFP accumulates at the EHM, particularly around the haustorium neck. (E) ZAR1 4CC -GFP localises to the cytoplasm throughout the cell, including around the haustorium, but does not accumulate to the EHM. (F) NRC2 4CC -GFP accumulates at the EHM. (G) NRC2 4CCa4 -GFP accumulates at the EHM and forms filaments like NRC2, but less frequently. (H) NRC2 4CCa1-3 -GFP localises to the cytoplasm throughout the cell, including around the haustorium, shows some EHM labelling, but does not accumulate to the EHM. (I) NRC2-GFP localises to filaments dispersed throughout the cytoplasm, some of which associate with EHM, but NRC2 does not focally accumulate at the EHM. (J) NRC4-GFP accumulates at the EHM. (K) Western blotting shows expression of chimeras. Chimeras were expressed in nrc4a/b plants for 3 days with P19 to boost expression and 4 leaf disks were collected using #4 size cork borer RLU is rubisco large subunit stained by Coomassie Brilliant Blue (CBB) or Ponceau Stain (PS). Chimeras are all expected to be approximately 130 kilodaltons (kDa).
Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

March 2021

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541 Reads

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1 Citation

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 statement Plant 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.


Figure 1-figure supplement 1. Images of N. benthamiana leaves expressing truncated NRC4 DV ::Mu-STOP variants.
Figure 5-figure supplement 1. Bar graph of MADA/MADAL-CC-NLRs according to HMM score.
An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species

November 2019

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347 Reads

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175 Citations

eLife

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 a1 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.


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

July 2019

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1,001 Reads

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2 Citations

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 α1 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.

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Citations (5)


... To test the main hypothesis that an external EF can affect early-stage root infection, we first implemented the global configuration (Fig. 1A) to expose roots to nominal EFs known to cause P. palmivora zoospore electrotaxis 22 : 0.5 V/cm, 0.7 V/cm, and 1.0 V/cm, with the corresponding current and current density measured in the system (Table 1). After 2h exposure, when zoospore attachment and germination had occurred 10 , we observed the roots under a confocal microscope and calculated a zoospore-attachment index normalized to take into account variation in total zoospore numbers within each replicate: ...

Reference:

Reduction of Phytophthora palmivora plant root infection in weak electric fields
Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields

Physical Biology

... In the first mechanism, operating in both local and global configurations (Fig. 7B,C), the applied EF competes with the root endogenous bioelectric field 25 to attract the zoospores via their natural electrotropic response. Since the endogenous EFs measured in roots are generally quite weak, generating current densities ranging between 0.002 µA/mm 2 in Ryegrass (Lolium perenne) and 0.027 µA/mm 2 in Peanut (Arachis hypogaea) 25 , the stronger current densities generated by the artificial EFs in this work ( Table 2) seem likely to win such competition and to drive the zoospores away from the natural roots. In the second mechanism, only www.nature.com/scientificreports/ ...

The Bioelectricity of Plant–Biotic Interactions

Bioelectricity

... Following resistosome assembly, CC-NLR oligomers insert themselves into the plasma membrane and presumably act as calcium channels, initiating immune signalling and cell death, as shown for AtZAR1 and the wheat CC-NLR TmSr35 [7,14]. In previous studies from our group, we showed that NbNRC2 and NbNRC4 form plasma membrane associated oligomers upon activation [15,16]. More recently, cryo-EM based structural studies showed that NbNRC2 and SlNRC4 form inflammasome-like resistosomes upon activation, although unlike AtZAR1 and TmSr35, these NRC assemblies are hexameric [17,18]. ...

Dynamic localization of a helper NLR at the plant–pathogen interface underpins pathogen recognition

Proceedings of the National Academy of Sciences

... Predicting the location and function of putative gene-promoter cis-acting elements is helpful for a deeper understanding of gene transcriptional regulation processes, revealing details and mechanisms of gene expression regulation [23]. In this study, three HbZAR1 genes promoter sequences contain different types and numbers of putative cis-acting elements, with the HbZAR1.1 promoter clearly having the most diverse types and numbers of putative cis-acting elements, followed by HbZAR1.3, and HbZAR1.2 ...

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

eLife

... All of the mutant proteins accumulated to similar levels, except for L10E, when expressed in N. benthamiana leaves indicating that the observed loss-of-function phenotypes were not due to protein destabilization (Fig. 3I). Together, our results suggest that hydrophobic amino acids at the It has been reported that only 29 amino acids of NRC4 is sufficient to induce cell death in N. benthamiana (47). To define the minimal region of G10-CC to be sufficient for autoactivity, we found that G10-CC domain has five α helices on their N-terminus through secondary structure analysis and generated the N-terminal or C-terminal deletion mutants of CC309 (Fig. 4A). ...

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