add autophagic activity assay and protocol

assays/AutophagicActivityAssay/protocols/Autophagic-Activity.md

+## Autophagic activity assay
+
+Growth medium (0.5 × MS (Duchefa, ref. M0222); MES 10 mM (Duchefa, ref. M1503); 1% sucrose; pH 5.8; 0.8% Plant agar (Duchefa, ref. P1001)) was autoclaved for 20 min at 120 °C, cooled to approximatively 60 °C and pipetted under sterile conditions into either 4–wells Ibidi coverslips (cat # 80,421 #1.5 polymer coverslip, hydrophobic, sterilized, Ibidi, Germany) for the experiment presented in Fig. 5 or into RoPod v24 for the experiment presented in Supplementary figure S2. 1 ml of medium was used per well of Ibidi chamber and 4 ml were used for the RoPod v24. 
+
+*Arabidopsis thaliana* seeds were surface sterilized in 70% EtOH, 0.05% TritonX-100 for 20 min, washed three times with 96% EtOH, air dried in a sterile bench and transferred into the chambers using a sterile toothpick. The chambers were then placed under long day growth conditions (16 h, 150 µmol m−2.s−1 light at 22 °C, 8 h dark
+at 20 °C). Typically, for the first four days the chambers were kept horizontally allowing the roots to reach the bottom cover slip. After this, the chambers were placed vertically to guide the root growth along the cover slip.
+
+Chemical compounds were diluted in 0.5 × MS liquid medium (the same composition as the growth medium, but lacking Plant agar) to a final concentration of 1 µM AZD8055 (Sigma, ref. ADV465749178, CAS number 1009298-09-2), 1 µM ConA (Sigma, ref. C9705, CAS number 80890-47-7) or 0.1% DMSO for the vehicle control. The solutions were pipetted into the wells of the chambers mounted on the confocal stage in 1:1 ratio (1 volume of the medium in the well: 1 volume of the drug solution. Drug final concentration: 500 nM) right before the start of imaging.
+
+Imaging was performed using CLSM Leica SP5 or SP8 (Leica Microsystems, Germany), 63× water immersion objective, NA = 1.2, excitation light 488 nm, emission range of 498–541 nm and system-optimized pinhole size or and Zeiss CLSM 800 Objective 40x/1.2W and similar scanning settings. For this study, pHusion tag was used solely as a fluorescent reporter and ot as a pH indicator. Four to six biological replicates were imaged for wildtype and *atg* KO backgrounds in each experiment. The experiment was performed four times in two independent laboratories. Images were acquired at the beginning of the root differentiation zone, collecting approximatively 100 µm deep z-stack starting at the surface of a root (pixel dimensions 0.29 µm.px−1, image size 848 × 848 px, 8-bit). Each replicate was imaged every 10 to 28 min for a period of 12 to 18 h.
+
+Images in Fig. 5 were analyzed with *Fiji* and Ilastik44. The detailed protocol and the macro used for the analysis are presented in the Supplementary File S3 and Supplementary Methods. The number of vesicles was counted for each cell file type using data obtained from two independent experiments. 1 to 5 roots and 2 to 15 cell files per type were analyzed in total. The fluorescence intensity was measured for each vesicle counted, for a total population per cell file of 8 to 2339 vesicles, depending on the genotype, the treatment, the cell file type and the time of the record considered. The mean intensity has been corrected with the background signal and normalized to the first time point of the record. As the image acquisition rate varied between experiments due to different number of biological replicates, each presented time point actually corresponds to an interval of up to 15 min. 
+
+For quantification of autophagic bodies in the complete cell volume, seeds of the transgenic line co-expressing spL-RFP and GFP-ATG8a were sterilized using the above described Chlorin method and plated either on Petri plates or in the RoPod v24. 5 days old seedlings were treated with concanamycin A for 12 h as described above and imaged using Zeiss CLSM 800 Objective 40x/1.2W to obtain tiled, z-stack encompassing complete root hair cells. Puncta quantification was performed using *Fiji* and dedicated macro as described in the Supplementary File S4.
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