Mesophyll Protoplast Immunostaining and Imaging

The preparation of mesophyll protoplast and the transient gene expression were carried out following the published protocol^{46 (link)}. We performed the immunostaining and imaging of YFP-XIP-M and γ-H2A.X in protoplasts^{25 (link)}. Briefly, protoplasts were cultured at room temperature for 20 hours for protein expression, and at the last 3 hours, BLM was added to a final concentration of 2.5 μM to induce γ-H2A.X. After that, protoplasts were collected and fixed for at least 3 hours using 4% PFA/PBS. After extensive washes by PBS at room temperature, protoplasts were mounted on slides covered with poly-lysine. Anti-γ-H2A.X (rabbit)^{25 (link)} and anti-GFP (mouse) (Abmart, China; Code No, 20004 L) were added at 1:200 dilution ratio. Fluorescent secondary antibodies (Invitrogen, Code No, A11001, A21428) was added at 1:1000 dilution ratio. Confocal images were acquired by using a LSM880 microscope (Zeiss).
Differential interference contrast (DIC) images were taken with an Imager A2 microscope (Zeiss)^{47 (link)}. For PI staining, all the root tips were transferred to liquid medium with or without 2.5 μM BLM for 6 hours. Confocal images were acquired by using a LSM880 microscope (Zeiss). At least 15 root tips per sample were used in these observations.