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A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora

Abstract

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a ‘brute-force’ approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation ‘naifu’ invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

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Fig. 1: Phytophthora invades real and artificial hosts at an oblique angle without forming an appressorium.
Fig. 2: Visualizing the surface mechanics of artificial host entry by Phytophthora.
Fig. 3: Quantitative surface mechanics reveal naifu mechanism.
Fig. 4: Polarized growth, substrate adherence and pressure generation are necessary for naifu-based host invasion.

Data availability

Raw data associated with the figures in this manuscript have been archived at https://doi.org/10.4121/14115461 and are publicly available at https://data.4tu.nl/articles/dataset/Data_underlying_the_publication_Phytophthora_pathogens_exploit_slicing_action_for_host_invasion/14115461. Source data are provided with this paper.

Code availability

All data analysis algorithms used to perform the work presented in this manuscript were written by the authors using MatLab (v.2018b) and have been made publicly available at https://github.com/jorissprakel/Phytopthora_invasion.

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Acknowledgements

This research is supported by research programme ECHO with project number 712.016.001, financed by the Dutch Research Council (NWO) (to J. Bronkhorst and J.S.); NWO Science domain (NWO-ENW) project GSGT.GSGT.2018.024 (to M.K.); and the European Research Council (ERC) project CoG-SOFTBREAK (to J.v.d.G). We thank V. Vleeshouwers (WUR Plant Breeding) for supplying seedlings.

Author information

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Authors

Contributions

J. Bronkhorst, T.K., F.G. and J.S. conceived and designed the project. J. Bronkhorst, M.K., S.v.V., J.M.C. and K.K. performed experimental work. J. Bronkhorst, S.v.V., J. Buijs and J.S. conceived image analysis routines and performed the analysis. J. Bronkhorst, J.v.d.G. and J.S. performed mathematical modelling. J. Bronkhorst, T.K., F.G. and J.S. wrote the paper with assistance and input of all coauthors.

Corresponding author

Correspondence to Joris Sprakel.

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The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Nicholas Money, Richard Wilson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Spiropyran fracture sensor reveals locus of surface invasion by the pathogen.

Additional examples of time series that reveal the time and locus of crack nucleation by invasion of P. infestans - 14:3 eGFP into a force-sensor elastomer substrate. We define t = 0 min as the moment of crack nucleation. All scale bars represent 5 μm.

Extended Data Fig. 2 Additional time series of surface deformations during invasion of elastomer substrates by P. infestans – 14:3 eGFP.

Colors indicate surface deformations in micrometers. Red: positive deformations due to adhesion; blue: negative deformations due to indentation. Arrows in b point to the location of the cyst, showing weak indentation but no adhesion. Scale bars: 5 μm.

Extended Data Fig. 3 Additional time series of surface deformations during invasion by P. palmivora – wt.

Colors indicate surface deformations in micrometers. Red: positive deformations due to adhesion, blue: negative deformations due to indentation. Scale bars: 5 μm.

Extended Data Fig. 4 Time series of surface deformations during invasion on elastomer substrates by P. infestans – wt.

Colors indicate surface deformations in micrometers. Red: positive deformations due to adhesion, Blue: negative deformations due to indentation. Scale bars: 5 μm.

Extended Data Fig. 5 Time series of surface deformations pathogenic invasion on elastomer substrates by P. capsici – wt.

Colors indicate surface deformations in micrometers. Red: positive deformations due to adhesion, blue: negative deformations due to indentation. Scale bars: 5 μm.

Extended Data Fig. 6 Mechanical invasion exhibits three stages.

Two additional examples of total indentation force Fi as a function of time (t) for the mechanical invasion of P. infestans - 14:3 eGFP into 0.58 MPa elastomer substrates, exhibiting three temporal stages (see Fig. 1a): I) germ tube growth is observed but no detectable surface deformations occur, II) pressure application increases until a fracture nucleates, III) pathogen invades into the crack opening, during which the crack propagates at relatively constant force.

Source data

Extended Data Fig. 7 Leaf infection assays.

a, Detached leaf assay to test the effect of cytoskeletal disruption with LatB on P.inf-wt infectivity on Nb-sobir1 leaves. b, Detached leaf assay to test the effect of a non-adhesion coating on P.inf-wt infectivity on Nb-sobir1 leaves. Leaf images are deep-red images that reveal lesions as bright orange-white zones (a-b). Schematic illustrations created with BioRender.com. c, Infectivity, determined as the percentage of inoculated spots that show a clear lesion 7dpi, for various controls and treatments, showing clear reduction in pathogenicity for high LatB concentrations and the non-adhesion coating. Numbers N indicate the total number of leaves tested for each sample.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–10, Methods, Discussion, Tables 1 and 2 and table of experimental details.

Reporting Summary

Supplementary Video 1

Three-dimensional reconstruction of P. infestans invasion into a potato stem: Several fluorescent pathogens (P. infestans 14-3-GFP, green) invade the stem of an etiolated potato cultivar (Bintje), with plant plastids shown in pink, at distinct oblique angles. Scale: the 3D volume has a size of 130 × 60 × 50 μm (l × w × h). Stem surface is located at approximately 35 μm from the base of the volume.

Supplementary Video 2

Side view of P. infestans invasion into an elastomer substrate: Time series of maximum-intensity projections in the xz-plane showing the invasion of P. infestans 14-3-GFP (green) into a fluorescent elastomer substrate (red).

Supplementary Video 3

Top view of P. infestans invasion into a fracture-reporting substrate: Time series of maximum-intensity projections of P. infestans 14-3-GFP (green) invasion in a fracture-reporting elastomer surface, which is covalently modified to carry the molecular mechanosensor spiropyran (magenta). This video reveals the initial stage of surface-fracture nucleation.

Supplementary Video 4

Surface-deformation maps for P. infestans 14-3-GFP invasion: Right: time series of reconstructred surface-deformation maps for P. infestans 14-3-GFP into a fluorescent elastomer substrate. Colour bar indicates the surface-deformation amplitude in μm. Left: corresponding GFP channel.

Supplementary Video 5

Surface-deformation maps for P. palmivora WT invasion: Right: time series of reconstructred surface-deformation maps for P. palmivora WT into a fluorescent elastomer substrate. Colour bar indicates the surface-deformation amplitude in μm.

Supplementary Video 6

Surface-deformation maps for P. infestans WT invasion: Right: time series of reconstructred surface-deformation maps for P. infestans WT into a fluorescent elastomer substrate. Colour bar indicates the surface-deformation amplitude in μm. Left: corresponding bright-field channel (black rectangle indicates the corresponding area for surface-deformation analysis).

Supplementary Video 7

Surface-deformation maps for P. capsici WT invasion: Right: time series of reconstructred surface-deformation maps for P. capsici WT into a fluorescent elastomer substrate. Colour bar indicates the surface-deformation amplitude in μm.

Supplementary Video 8

Fitting of surface-deformation profiles with model: Time series of surface-deformation profiles (symbols) and fits to the mathematical model (lines) for P. infestans 14-3-GFP invasion into a fluorescent elastomer substrate.

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Source data

Source Data Fig. 1

Statistical source data for Fig. 1d–i.

Source Data Fig. 2

Statistical source data for Fig. 2d,e.

Source Data Fig. 3

Statistical source data for Fig. 3b,d,e.

Source Data Fig. 4

Statistical source data for Fig. 4k,l.

Source Data Extended Data Fig. 6

Statistical source data for Extended Data Fig. 6a,b.

Source Data Extended Data Fig. 7

Statistical source data for Extended Data Fig. 7c.

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Bronkhorst, J., Kasteel, M., van Veen, S. et al. A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora. Nat Microbiol 6, 1000–1006 (2021). https://doi.org/10.1038/s41564-021-00919-7

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