Identification of MARVELous Protein Markers for Phytophthora infestans Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are released from cells by unconventional secretion, but little is known about the biogenesis routes, composition or cargoes of EVs from fungal or oomycete plant pathogens. We investigated the proteome of EV-associated proteins secreted by the oomycete Phytophthora infestans, cause of potato late blight disease. We found that vesicle-associated proteins, transmembrane proteins and RxLR effectors, which are delivered into host cells to suppress immunity, were enriched in the EV proteome. By contrast, the EV-independent secreted proteome was enriched in cell wall modifying enzymes and apoplastic effectors which act outside plant cells. Two proteins, each containing two tetraspanning MARVEL domains, PiMDP1 and PiMDP2, were associated with P. infestans EVs. PiMDP1 and PiMDP2 were co-buoyant with RxLR effectors in sucrose density fractions containing EVs and co-localised frequently with each other and with RxLRs at vesicles within pathogen hyphae grown in vitro and during infection. Interestingly, PiMDP2, which is up-regulated during the early biotrophic phase of infection, accumulates at the haustorial interface, a major site of effector secretion during infection. We argue that PiMDP1 and PiMDP2 are molecular markers that will facilitate studies of the biogenesis and secretion of infection-associated P. infestans EVs.

Keywords: MARVEL domain; Phytophthora infestans; RxLR; extracellular vesicles; pathogen effector.

FIGURE 1

Phytophthora infestans secretes extracellular vesicle like particles into growth media. (a) Schematic representation of the ultracentrifugation protocol used for nanoparticle isolation to generate the crude pellet from P. infestans inoculated media. Schematic made in BioRender. (b) Nanoparticle tracking (NTA) data showing the size distribution of particles in the crude nanoparticle pellet from P. infestans‐inoculated media and the pellet from uninoculated control media. Results from three independent replicates of each inoculated and uninoculated control are shown in the line graph. (c) Transmission electron microscopy negative stain images showing vesicles present within the nanoparticle crude pellet isolated from P. infestans inoculated media. Top image scale bar represents 0.5 µm. Dashed box represents the location of the higher magnification image below where the scale bar represents 100 nm.

FIGURE 2

Overview of the P. infestans crude vesicle pellet proteome. (a) Venn diagram showing the distribution of proteins between the supernatant and pellet samples after crude extracellular vesicle pellet isolation and LC‐MS/MS analysis from P. infestans‐inoculated media. (b) Volcano plot showing the statistical distribution of proteins within the pellet versus supernatant samples. Pink spots indicate RxLR motif‐containing effector proteins, blue spots represent apoplastic effector proteins. Black arrows highlight RxLR proteins of interest. (c) The same volcano plot with green spots indicating cell wall degrading enzymes (CWDE), orange spots indicating vesicle‐associated proteins. Black arrows highlight CWDE and vesicle‐associated proteins of interest. CWDE were identified using dbCAN3 (https://bcb.unl.edu/dbCAN2/). (d–k) Immunoblots of crude vesicle pellet and supernatant isolation from multiple transgenic P. infestans lines expressing mRFP, mCherry or mCitrine tagged proteins of interest. The membranes were probed with αRFP for mRFP or mCherry fusions, or αGFP for mCitrine fusions. These western blots show mycelia (Myc) and culture filtrate (CF) samples prior to any centrifugation, 100,000 × g pellet (100P) and supernatant samples after 100,000 × g spin (100S). These western blots confirm the distribution of the proteins based on the observations from the volcano plots (b and c). The proteins of interest expressed in the P. infestans isolates are (d) RxLR effector Pi09216‐mCitrine. (e) RxLR effector Pi04314‐mCherry. (f) Apoplastic effector PiEPIC1‐mRFP. (g) CWDE Pectinesterase 1 (PiPE1‐mCitrine) and RxLR effector Pi04314‐mCherry. (h) Vesicle associated protein Guanine nucleotide‐binding protein subunit β‐2‐like (PiGNB2L)‐mCitrine. (i) Vesicle associated protein PiRab7‐mCherry. (j) Vesicle associated protein PiCoronin‐mRFP. (k) The control Golgi marker protein PiManI‐eGFP (Ah‐Fong and Judelson 2011).

FIGURE 3

MARVEL‐domain containing proteins are found exclusively in the crude vesicle pellet sample and are expressed during P. infestans infection. (a) Volcano plot showing the statistical distribution of proteins within the pellet versus supernatant samples. Green spots represent transmembrane (TM) domain containing proteins. Black arrows indicate the location of PiMDP1 and PiMDP2. TM domains were identified using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM). (b) Predicted structures of PiMDP1 and PiMDP2 produced using Alphafold2. Protein structures are colour‐coded to show plDDT confidence levels as shown in the key. (c) Gene expression of PiMDP1, PiMDP2 and the RxLR Pi04314 over a time‐course of P. infestans isolate 3928A infecting Maris Piper potato leaves. Expression was normalised to the geometric mean of 3 housekeeping genes (ACTIN, Caesin kinase, Kelch domain repeat) using the 2D/D Ct method. Error bars show St Error. (d–e) Crude vesicle pellet and supernatant isolation from transgenic P. infestans isolates expressing (d) PiMDP1‐mCitrine tagged protein, (e) PiMDP2‐mCitrine tagged protein. These western blots show mycelia (M) and culture filtrate (CF) samples prior to any centrifugation, 100,000 × g pellet (100P) and supernatant samples after 100,000 × g spin (100S). These western blots confirm the localisation of the proteins based on the observations from the volcano plot in (a). (f) −/+ 1% Triton treatment of culture filtrate from PiMDP1‐mCitrine expressing P. infestans isolate. This western blot shows mycelia (M) and culture filtrate (CF) samples prior to any centrifugation and 100,000 × g pellet (100P) −/+ 1% Triton X‐100. The band intensity graph shows the relative intensity of 100P −/+ 1% Triton bands from six independent replicates. Statistical analysis was done using a Paired t‐test on SigmaPlot giving the two‐tailed p value of 0.004. (g) −/+ 1% Triton treatment of culture filtrate from PiMDP2‐mCitrine expressing P. infestans isolate. The western blot shows mycelia (M) and culture filtrate (CF) samples prior to any centrifugation and 100,000 xg pellet (100P) −/+ 1% Triton. The band intensity graph shows the relative intensity of 100P −/+ 1% Triton bands from five independent replicates. Statistical analysis was done using a Paired t‐test on SigmaPlot giving the two‐tailed p value of 0.002. (h) Transmission electron microscopy negative stain image showing vesicles present within a – Triton sample from a transgenic P. infestans isolate expressing both PiMDP1‐mCitrine and Pi04314‐mCherry. Asterisks indicate vesicles, scale bar represents 500 nm. (i) Transmission electron microscopy negative stain image indicating the loss of vesicles in an equivalent + Triton sample. Scale bar represents 500 nm.

FIGURE 4

MARVEL‐domain and RxLR effector proteins are detected within the same sucrose density fraction. Crude vesicle pellets were top‐loaded onto a discontinuous sucrose gradient consisting of 10%, 20%, 30%, 40%, 50%, 60%, and 70% layers. After centrifugation at 100K × g for 16 h, six fractions of 1.8 mL each were collected and processed with further ultracentrifugation to obtain a pure vesicle pellet. All membranes were dual probed with αGFP and αRFP. The density (g/cm³) of each fraction is detailed under each blot. (a) Six fractions isolated using a top‐loaded crude pellet from a P. infestans isolate expressing both Pi09216‐mCitrine and Pi04314‐mCherry. (b) Graph showing band intensity for Pi09216‐mCitrine (green) and Pi04314‐mCherry (pink) from each fraction shown in (a). (c) Fractions isolated from P. infestans expressing both PiMDP1‐mCitrine and Pi04314‐mCherry. (d) Graph showing band intensity for PiMDP1‐mCitrine (green) and Pi04314‐mCherry (pink) from each fraction shown in (c). (e) Fractions isolated from P. infestans expressing both PiMDP2‐mCitrine and Pi04314‐mCherry. (f) Graph showing band intensity for PiMDP2‐mCitrine (green) and Pi04314‐mCherry (pink) from each fraction shown in (e). (g) Fractions isolated from P. infestans expressing both PiMDP2‐mCitrine and mCherry‐PiMDP1. (h) Graph showing band intensity for PiMDP2‐mCitrine (green) and mCherry‐PiMDP1 (pink) from each fraction shown in (g). (i) Nanoparticle tracking (NTA) data showing the number and size distribution of particles in each sucrose fraction. Results show the average from three independent replicates for each sample. (j) Transmission electron microscopy negative stain image of a sample of fraction 1 isolated from P. infestans isolate expressing both PiMDP1‐mCitrine and Pi04314‐mCherry. (k) Transmission electron microscopy negative stain image showing a range of vesicles in fraction 4 isolated from the same isolate. Asterisks indicate EV's, scale bars in I and j represent 200 nm.

FIGURE 5

PiMDP1/PiMDP2 and RxLR proteins colocalise in vesicle‐like structures in hyphae grown in vitro. Projection images from confocal z series images of hyphae from transformed P. infestans transgenic lines expressing fluorescent protein fusions grown on microscope slides. The mCitrine (green) and mCherry (magenta) channels are shown separately and merged with three numbered arrows indicating transects drawn using single optical sections to produce the three numbered fluorescence intensity plots to the right. The transgenic lines were co‐expressing: (a) apoplastic effector PiPE1‐mCitrine and RxLR Pi04314‐mCherry; (b) RxLRs Pi09216‐mCitrine and Pi04314‐mCherry; (c) PiMDP1‐mCitrine which localises to the PM and also partially co‐localises to vesicles with Pi04314‐mCherry; (d) PiMDP2‐mCitrine which localises to the PM and also partially co‐localises to vesicles with Pi04314‐mCherry; (e) PiMDP2‐mCitrine which partially co‐localises to vesicles with mCherry‐PiMDP1; both are observed at the PM. Scale bars are 10 µm. Fluorescence intensity plots show relative fluorescence on the y axis against distance in µm on the x axis. (f) Stacked bar chart shows quantification of the percentage of vesicles showing mCitrine only, mCherry only or colocalisation of both fluorophores. Results are the average counts from at least 12 images; percentages are shown on the chart; n indicates the total number of vesicles counted for each transformant (PiPE1‐mCitrine & Pi04314‐mCherry n = 612; Pi09216‐mCitrine & Pi04314‐mCherry n = 350; PiMDP1‐mCitrine & Pi04314‐mCherry n = 237; PiMDP2‐mCitrine & Pi04314‐mCherry n = 303; PiMDP2‐mCitrine & mCherry‐PiMDP1 n = 293).

FIGURE 6

PiMDP and RxLR proteins co‐localise in vesicle‐like structures in hyphae during infection. Projection images from confocal z series of hyphae from transformed P. infestans isolates expressing fluorescent protein fusions infecting N. benthamiana leaves. The mCitrine (green) and mCherry (pink) channels are shown separately and merged with three numbered arrows indicating transects drawn using single optical sections to produce the three numbered fluorescence intensity plots below. (a) PiMDP1‐mCitrine localises to the plasma membrane (PM) and partially co‐localises to vesicles with Pi04314‐mCherry. (b) PiMDP2‐mCitrine localises to the PM and partially co‐localises to vesicles with Pi04314‐mCherry. The mCherry intensities were low in this hypha and thus are plotted with the right‐hand vertical axis to make the patterns more obvious. Asterisks indicate haustoria and scale bars are 10 µm. Fluorescence intensity plots show relative fluorescence on the y axis against distance in µm on the x axis.

FIGURE 7

PiMDP2‐mCitrine accumulates in the mature haustorial membrane during infection. Confocal projection images from z series images of P. infestans hyphae and haustoria during plant infection showing (a) PiMDP1‐mCitrine localised to the plasma membrane (PM), haustorial membrane (HM) and vesicle‐like bodies in the hypha, with 04314‐mCherry localised around the haustoria and, (b) PiMDP2‐mCitrine accumulates more strongly in the HM with little PM fluorescence, in addition to vesicle‐like bodies in the hypha, with 04314‐mCherry localised around the haustoria. Asterisks indicate haustoria, arrowheads indicate the PM and the scale bars are 10 µM. (c) Boxplot shows quantification of the ratio of the relative fluorescence intensity in regions of the HM compared to the PM measured from single optical sections. The points indicate the 5th and 95th percentile outliers and the lines show the median values. Statistical analysis was done using a Mann‐Whitney Rank Sum Test on SigmaPlot giving the p value of < 0.001 (n ≥ 19).