Zeiss7 duo nlo lsm880 confocal laser microscope

Cy3-labeled oligonucleotide probes specifically targeting MISSEN were designed and synthesized by RiboBio corporation (China). Root hair cells of WT and MISSEN-OX were collected fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.5% Triton X-100 for 30 min. Cells were incubated with Cy3-labeled FISH probes dissolved by 50% formamide in 2×SCC at 37 °C overnight. After hybridization, the nuclei were counterstained with DAPI. Cells were observed on a Zeiss7 DUO NLO LSM880 confocal laser microscope (Carl Zeiss, Germany).

For immunolocalization of tubulin in endosperm cells during mitosis, caryopsis of WT, MISSEN-OX, and hefp were used at 3 DAP. Caryopsis were fixed in 4% (w/v) paraformaldehyde in microtubule-stabilizing buffer/DMSO (MTSB; 50 mM PIPES, 5 mM EGTA, 5 mM MgCl₂, and 5% DMSO, pH 6.7–7.0) at room temperature under vacuum for 1 h and then overnight at 4 °C. Fixed caryopsis were dehydrated in an ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) and then washed twice in absolute ethanol for 30 min. Embedded in melted Steedman’s wax^{58 (link)}, and sectioned to a thickness of 8 μm with a microtome (Leica Microsystems RM2145). After dewaxing with ethanol, hydrated the sample with MTSB. The plasma membrane was permeabilized with 1% (v/v) Triton X-100 in 10% (v/v) DMSO-MTSB for 30 min at room temperature. Blocking with 1% (w/v) BSA in MTSB for 1 h at room temperature, anti-alpha tubulin DM1A (Abcam, USA) was diluted 1:50 in 3% (w/v) BSA in MTSB and incubated overnight at 4 °C. After washing in MTSB for three times, anti-mouse Fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Invitrogen, CA, USA) was used at 1:300 dilution in 3% (w/v) BSA in MTSB. DNA was stained with 1 mg/mL DAPI (Sigma, USA), endosperm cells were observed on a Zeiss7 DUO NLO LSM880 confocal laser microscope (Carl Zeiss, Germany).

We modified BiFC vectors pUC19-YN and pUC19-YC to generate TriFC assays vectors with a MS2 system^{32 (link),56 (link)}. Briefly, MSCP was fused to pUC19-YN, HeFP was fused to pUC19-YC, 12×MS2-MISSEN-S or 12×MS2-MISSEN-AS was fused to pUC19-YN without the sequence of nYFP. PEG-mediated transfections were performed as previously described^{57 (link)}. Briefly, Aliquots of protoplasts (200 μL) were transferred into a 2-mL round-bottom microcentrifuge tube and mixed gently with 20 μg plasmid DNA. Transfected protoplasts were collected by centrifugation for 5 min at 100×g, resuspended and then incubated at 28 °C in the dark for 20 h. Protoplasts were observed using a Zeiss7 DUO NLO LSM880 confocal laser microscope (Carl Zeiss, Germany) with excitation/emission wavelengths of 514/519–563 nm.