Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: (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 vs 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 x g pellet (100P) and supernatant samples after 100,000 x g spin (100S). These western blots confirm the distribution of the proteins based on the observations from the volcano plots (b-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 .

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transgenic Assay, Expressing, Centrifugation, Binding Assay, Control, Marker

Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: (a) Volcano plot showing the statistical distribution of proteins within the pellet vs 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 x g pellet (100P) and supernatant samples after 100,000 x 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 x g pellet (100P) -/+ 1% Triton X-100. The band intensity graph shows the relative intensity of 100P -/+ 1% Triton bands from 6 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 x g pellet (100P) -/+ 1% Triton. The band intensity graph shows the relative intensity of 100P -/+ 1% Triton bands from 5 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.

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Produced, Gene Expression, Expressing, Isolation, Transgenic Assay, Western Blot, Centrifugation, Two Tailed Test, Transmission Assay, Electron Microscopy, Staining

Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: 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 x g for 16 h, six fractions of 1.9 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/cm 3 ) 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.

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Centrifugation, Isolation, Expressing, Transmission Assay, Electron Microscopy, Staining

Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: 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).

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Transformation Assay, Transgenic Assay, Expressing, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: 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.

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Transformation Assay, Expressing, Fluorescence, Membrane

Journal: bioRxiv

Article Title: Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

doi: 10.1101/2025.04.11.648357

Figure Lengend Snippet: 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 5 th and 95 th 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).

Article Snippet: Primary antibodies for RFP/ mCherry (Chromotek, 58F) and GFP/ mCitrine (Roche, 11814460001) were incubated overnight at 4 °C in blocking buffer at a dilution of 1:4000 or 1:2000 respectively.

Techniques: Infection, Membrane, Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Pupylation-based proximity labeling reveals regulatory factors in cellulose biosynthesis in Arabidopsis

doi: 10.1038/s41467-025-56192-3

Figure Lengend Snippet: a , b Volcano plots show differently enriched proteins in the PafA-CC1 and PafA-LTI6B groups from experiments in N. benthamiana ( a ) and Arabidopsis seedlings ( b ), based on a two-sided t-test with permutation-based FDR (FDR = 0.05, S0 = 1). Orange dots represent proteins unique to the PafA-CC1 group, maroon dots represent high-abundance proteins, gray dots represent proteins with no significant differences, and blue dots represent proteins more abundant in the control. CSC and BEN1-related proteins are marked with larger dots. c Co-localization of BEN1-mCherry and EGFP-CC1 in stable transgenic Arabidopsis. Scale bar = 10 µm. d Fluorescence intensity along the transect in ( c ). Images were taken from cells in the root tip area of 5-day-old seedlings. e BEN1-mCherry was labeled with FLAG when co-expressed with FLAG-Pup(E) and PafA-CC1 in N. benthamiana . RFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-FLAG antibodies for western blot. p19-only samples served as control. f BEN1-mCherry was co-immunoprecipitated with EGFP-CC1 in N. benthamiana . GFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-GFP antibodies for western blot. EGFP samples served as the negative control, and p19-only samples as the empty control. Arrowheads point to proteins of interest: BEN1-mCherry (white), EGFP (yellow) and EGFP-CC1 (red). g CC1 is a transmembrane protein with its N terminal facing the cytosol and C terminal in the apoplast, C terminal truncation (∆C), N terminal truncation (∆N). h Membrane split-ubiquitin Y2H was used to detect interactions between BEN1 and CC1, with CC1 constructs designed as shown in ( g ). Colony growth percentages on selection media from three replicates. Values are mean + SD. Significance was determined by one-way ANOVA followed by a Tukey’s test ( p < 0.05). i BiFC assay assessed the interaction between BEN1 and CC1, with CC1 constructs designed as shown in ( g ). Scale bar = 50 µm. j Relative YFP to RFP ratio was measured along the cell outlines in BiFC images. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test ( p < 0.05, n = 15).

Article Snippet: For RFP (mCherry) detection, membranes were blocked for 2 h at room temperature, incubated with primary antibody α-RFP (mCherry) (Chromotek, 6G6, 1:2000) overnight at 4 °C, and secondary antibody α-mouse-HRP (Agilent, P0260, 1:5000) for 1 h at room temperature on a shaking device.

Techniques: Control, Transgenic Assay, Fluorescence, Labeling, Immunoprecipitation, Western Blot, Negative Control, Membrane, Construct, Selection, Bimolecular Fluorescence Complementation Assay