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cmi12254-sup-0001-ts1.xlsx52K

Table S1. Inventory of P. infestans genes encoding proteins homologous to the yeast proteins that bind with actin or are known to be associated with actin patches.

cmi12254-sup-0002-fs1-7.pdf745K

Fig. S1. Expression of Lifeact–eGFP in P. infestans does not hamper virulence. Virulence assays on (A) Nicotiana benthamiana and (B) Solanum tuberosum (potato cultivar Impala). (A) Detached leaves inoculated with zoospores of three Lifeact–eGFP transformants (LA-eGFP1–3), the wild-type recipient strain 88069 (WT) and two control transformants (RFP and GFP) at 4 dpi. (B) Detached leaves inoculated with zoospores of the control mRFP expressing transformant and the Lifeact–eGFP expressing transformant LA-eGFP1 at 7 dpi.

Fig. S2. Lifeact–eGFP is present in the Lifeact–eGFP strain. The presence of eGFP-tagged proteins was determined by Western blot analysis using GFP-TRAP_A precipitated protein isolated from a GFP strain, the non-transgenic 88069 strain, and the Lifeact–eGFP strain LA-eGFP1. Proteins were separated on a 12% SDS-PAGE gel and the blot was probed with a GFP antibody. The smaller band (eGFP) has an apparent mass of 27 kDa which is in agreement with the calculated mass of 27.306 kDa. The larger protein (Lifeact–eGFP) has an apparent molecular mass of 29.5 kDa for Lifeact–eGFP which correlates with the expected 29.483 kDa.

Fig. S3. Actin cable organization in a P. infestans hypha visualized in a strain expressing Lifeact–eGFP. A representative image showing the actin bundles in a region more distant from the tip (top panel) and the hyphal tip (bottom panel).

Fig. S4. Apical actin polymerization waves in P. infestans occur at regular intervals. Representative kymograph constructed from time-lapse movies of a growing hyphal tip showing actin polymerization waves. In this time-space-plot actin polymerization waves are detectable as bright spots.

Fig. S5. Disassembling plaques in P. infestans are not internalized. Three representative plaques (a–c) were monitored in a midplane confocal section during 12 s starting with the initial stage of disassembly at T = 0, to full disassembly at T = 12 and with 2 s intervals, the shortest interval for proper monitoring of Lifeact–eGFP fluorescence.

Fig. S6. FM4-64-labelled membranes do not accumulate at the location of actin plaques. Detailed study showing separate channels for Lifeact–eGFP (top; wavelength 488 nm), FM4-64 (middle; wavelength 561 nm) and merge (bottom) derived from Video S10. Microscope slides were prepared in which hyphae were embedded in medium containing FM4-64 (10 μg ml−1). After slide preparation time series were collected, starting approximately 5 min after the initial contact with FM4-64. Bar = 5 μm.

Fig. S7. Actin cytoskeleton organization in a sporangium of a P. infestans transformant expressing Lifeact–eGFP.

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Video S1. Confocal fluorescence time-lapse imaging of actin filament polymerization waves in a hyphal tip of a P. infestans strain expressing Lifeact–eGFP. Avi movie frame rate: five frames per second (1 s movie time is 40 s in real time); bar = 5 μm.

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Video S2. Confocal fluorescence time-lapse imaging of the dynamics of the actin cable network in P. infestans in the apical zone of a hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: 30 frames per second (1 s movie time is 30 s real time); bar = 5 μm.

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Video S3. Confocal fluorescence time-lapse imaging of actin cable network dynamics in P. infestans in the distal part of a hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: 30 frames per second (1 s movie time is 30 s real time); bar = 5 μm.

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Video S4. Confocal fluorescence time-lapse imaging of the actin cytoskeleton in a non-growing P. infestans hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: five frames per second (1 s movie time is 40 s in real time); bar = 5 μm.

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Video S5. Confocal fluorescence time-lapse imaging of the actin cytoskeleton in a slow growing P. infestans hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: seven frames per second (1 s movie time is 42 s in real time); bar = 5 μm.

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Video S6. Confocal fluorescence time-lapse imaging of the actin cytoskeleton in a fast-growing P. infestans hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: five frames per second (1 s movie time is 40 s in real time); bar = 5 μm.

cmi12254-sup-0009-v7.avi20K

Video S7. Confocal fluorescence time-lapse imaging of an appearing plaque in a P. infestans hypha, visualized in a strain expressing Lifeact–eGFP. Avi movie frame rate: five frames per second (1 s movie time is 20 s in real time); movie frame is 2 μm wide.

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Video S8. Confocal fluorescence time-lapse imaging of plaque in the steady-state stage in a P. infestans hypha, visualized in a Lifeact–eGFP expressing strain. Avi movie frame rate: five frames per second (1 s movie time is 20 s in real time); movie frame is 2 μm wide.

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Video S9. Confocal fluorescence time-lapse imaging of a plaque during its disassembly in a P. infestans hypha, visualized in a Lifeact–eGFP expressing strain. Avi movie frame rate: five frames per second (1 s movie time is 20 s in real time); movie frame is 2 μm wide.

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Video S10. FM4-64-labelled membranes do not accumulate at the location of actin plaques. Microscope slides were prepared in which hyphae were embedded in medium containing FM4-64 (10 μg ml−1). After slide production time series were collected, starting approximately 5 min after the initial contact with FM4-64. The movie is 7.5× real time. Bar = 5 μm.

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Video S11. Confocal fluorescence time-lapse imaging of a P. infestans hypha, expressing Lifeact–eGFP, treated with 750 nM latB. Avi movie frame rate: 10 frames per second (1 s movie time is 5 min in real time); bar = 5 μm.

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Video S12. Confocal fluorescence time-lapse imaging of a P. infestans hypha, expressing Lifeact–eGFP, treated with 10 μM latB. Avi movie frame rate: 10 frames per second (1 s movie time is 5 min in real time); bar = 5 μm.

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