A condensates-to-VAPVs conversion pathway regulates autophagy degradation in plants

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Pharmacological assays Stock solutions of various chemicals were prepared as follows: 50 mM menadione (Coolaber, Cat#CV11881-5g) , 2 mM AZD8055 (APExBIO, Cat#A8214) , 100 mM oryzalin (Sigma-Aldrich; Cat # 36182), 0.5 mM Latrunculin B (LatB) (Abcam; Cat #ab144291), 1mM LysoTrackerTM Red (Invitrogen, Cat#L-7528), 50 mM 2,4-Dinitrophenol (DNP) (Sigma-Aldrich, Cat#137731-5g), 5 µg/ml tunicamycin (TM) (APExBIO, Cat#B7417), 10 mM methyl viologen dichloride hydrate (MV) (Sigma-Aldrich, Cat#856177), and 2 mM bafilomycin A1 (MedChemExpress, Cat#HY-100558). These chemicals were dissolved in dimethyl sulfoxide (DMSO). All treatments were conducted in liquid half strength MS media with 1:1000 dilution of the stock solutions. DMSO was used as the solvent control. For autophagy induction, 6-day-old seedlings were treated with menadione, AZD8055, 300 mM mannitol, MV, DNP and TM for 1 h, 1 h, 1 h, 24 h, 1 h and 6 h, respectively. LatB was cotreated for 1 h together with the autophagy inducers except for MV and TM. For MV and TM, the co-treatment was carried out in the last 1.5 h. For the analysis of autophagic bodies within the vacuole lumen, seedlings were pretreated with 2 µM Bafilomycin A1 for 6h before cotreatment with AZD80555 or menadione for 1 h, respectively. For visualizing autophagosomes under normal growth condition by MDC staining, seedlings were stained by immersion in 0.05 mM monodansylcadaverine (MDC) (Cayman, Cat#15571) in PBS for 10 min, followed by two washes using PBS before observation according to previous publication40. Live-cell imaging Live-cell images were collected by Leica TCS SP8 confocal laser-scanning microscope via consistent laser format of 1.0 pinhole and 100 gain value. The lasers were set up to 10%–20% of the maximum intensity. The GFP signals were excited at 488 nm and the emission wave length was collected at 505–545 nm. YFP signals were excited at 514 nm and the emission wave length was collected at 520–545 nm. The RFP, mCherry and Lysotracker Red signals were excited at 552 nm and the emission wave length was collected at 570–620 nm. For MDC detection, the MDC signals were excited at 405 nm and the emission was collected at 480–530 nm. Electron microscopy (EM) analysis The standard procedures for high-pressure freezing (HPF), freeze substitution, ultra-thin sectioning, immuno-gold labeling, and imaging have been described previously21,65. Briefly, root tips of 5- or 6-day-old seedlings were dissected and rapidly frozen in a high-pressure freezing apparatus (Leica EM ICE High Pressure Freezer, Leica), followed by freeze substitution in dry acetone containing 0.1% uranyl acetate at -85°C. Infiltration with HM20, embedding, and UV polymerization were performed stepwise at -45°C. For immuno-gold labeling, the GFP antibody (Takara, JL-8) was used at 1:10 dilution and ATG8 antibody (Agrisea, AS142769) at 1:15. Sections were examined using a Hitachi HT7700 transmission electron microscope (Hitachi High-Technologies).

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