Uorescent microscope

According to the manufacturer instructions, 2,000 cells/150 µL of medium were seeded into the 96-well plate per well and cultured for 4 hours. The medium was replaced by 10 µM EdU DNA Cell Proliferation kit ((RiboBio, Guangzhou, China) and cells were cultured for an additional 12 hours. Thereafter, the cells were xed with 4% formaldehyde for 30 min. Next, the cells were exposed to 100 µL per well of Apollo staining for 30 min and incubated with 5 µg/mL of DAPI to cell nuclear staining for 15 min. Finally, the cells were counted using a uorescent microscope (Olympus, Tokyo, Japan).

The durably resistant rice Ziyu44 and the susceptible rice JNXN were grown in a growth chamber at 28°C and 75% humidity in a 12 h light/12 h dark photoperiod. For microscope monitoring of fungal development, M. oryzae strains Zhong-10-8-14 and LP33 were grown on Potato sucrose medium for 2 weeks (at 26°C and natural light), then conidial spores were collected via ooding oat-tomato agar medium with sterile water. 5×10 5 conidia/mL spore suspension were used to inoculate the detached rice sheaths from 4-week-old Ziyu44 and JNXN as described previously [27, (link)28] . The images of conidial germination, appressorium development, and invasive hyphae growth were recorded using an Olympus uorescent microscope. The microscopic evaluation of the infected sheath was performed in three independent experiments, more than 30 cells were counted in each replicate.

Cells were seeded in triplicate wells of 6-well plates at 1 × 10 5 cells per well and treated with cinobufagin.
Mitochondrial membrane potential (MMP) was measured using a commercial Mitochondrial Membrane Potential Assay kit with JC-1 (Beyotime) following manufacturer's instructions. Images were taken using an Olympus uorescent microscope.

Bioinformatics software http://lncatlas.crg.eu/ was applied to predict the subcellular localization of ADAMTS9-AS2.
A slide (10 mm × 10 mm) was placed in a 24-well culture plate and seeded with cells at 6 × 10 3 cells/well. At 24 h post culture, cells were xed with 4°C paraformaldehyde, permeabilized with 0.1% Triton X-100 (Guangzhou RiboBio) and pre-hybridized with the pre-hybridization solution. Then, cells were hybridized with 18S, U6, ADAMTS9-AS2 anti-sense and ADAMTS9-AS2 sense probes (Guangzhou RiboBio) for 16-20 h and rinsed with 2 × sodium citrate buffer. Finally, cells were stained with 4',6-diamidino-2-phenylindole uorescent staining solution in the hybridized area and observed under a uorescent microscope (Olympus, Japan, Tokyo).

The cells were pre-seeded on glass slides. After culture for certain times, the cells were xed with 4% paraformaldehyde for 15 min, permeated with 0.1% TritonX-100 (Beyotime) for 30 min, and blocked with goat serum for 10 min at room temperature. Subsequently, the cells were incubated with antibody against FBP1 (1:100; cat. no. 12842-1-AP, Proteintech) at 4 ℃ overnight, incubated with secondary antibody labeled with Cy3 (1:200; cat. no. A0516, Beyotime) at room temperature in the dark for 60 min, and counterstained with DAPI (Aladdin). Finally, the glass slide were mounted with anti-fading reagent (Solarbio, Beijing, China), and the cells were observed under a uorescent microscope (Olympus, Tokyo, Japan) at 400× magni cation.

Apoptotic cells of the retina were detected using in situ cell death detection kit (DeadEnd™ Fluorometric TUNEL System kit, Promega, Madison, WI) and PI counterstain (Sigma-Aldrich, USA) according to the manufacturer's protocol. All slides were visualized under a uorescent microscope (Olympus, Center Valley, PA, USA) equipped with an Olympus DP70 camera.

The apoptotic neurons in the brain sections were detected using the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. After depara nization and rehydration, the brain sections were permeabilized with proteinase K solution, then exposed to the mixture of biotinylated nucleotide dUTP and recombinant terminal deoxynucleotidyl transferase (TdT) following the instruction manual of TUNEL Apoptosis Assay Kit (Servicebio, Wuhan, China). Staining with 4,6-diamino-2-phenyl indole (DAPI) (Sigma, St. Louis, USA) was performed to visualize nuclei. Images were obtained under a uorescent microscope (Olympus, Center Valley, USA).