Imager a2

Growth of the A. fumigatus strains was carried out onto solid CM or MM at 37 °C. For morphological observation, 2 × 10⁸ spores were inoculated into 200 ml liquid CM and incubated at 37 °C at 200 rpm, then stained with 10 μg/ml Calcofluor white (Sigma) and 1 mg/ml 4′-6-diamidino-2-phenylindole (DAPI; Sigma) as previously described^{36 (link)}, prior to fluorescence microscopy using a Zeiss Imager A2 (Zeiss, Japan). For propidium iodide (PI) staining, 2 × 10⁸ conidia were inoculated in 200 ml of CM and incubated at 37 °C with 200 rpm. The mycelia were collected, stained with PI, and then examined under the fluorescence microscope.
For scanning electron microscopy (SEM) or transmission electron microscopy (TEM), the mycelia cultivated in liquid CM at 37 °C were collected and fixed as previously described^{11 (link),36 (link)} prior to examining the sections with a Tecnai Spirit (120 kV) transmission electron microscope (FEI, USA). For chemical analysis of the cell wall, cell wall components were isolated and determined as previously described^{11 (link)}.

To observe the H₂O₂ accumulation in rice plants, three-leaf-stage seedlings were inoculated with M. oryzae strain Guy11 at the concentration of 5 × 10⁵ conidia mL⁻¹. At 40 hpi, leaves were collected and incubated in 1 mg/mL DAB (Sigma, Merck Life Science Co., Ltd. Shanghai, China) at 22 °C for 8 h at illumination. The DAB-stained leaves were cleaned in 95% ethanol and then observed under a microscope (Zeiss imager A2, Carl Zeiss (Chengdu) Co. Ltd, China).

The Agrobacterium strain GV3101 carrying the individual construct in the binary vector pCAMBIA1300 was incubated in LB media containing specific antibiotics (rifampin, 50 μg/mL; kanamycin, 50 μg/mL) at 28°C incubation. The bacteria were collected at 5,000 rpm for 5 min and resuspended in MMA buffer (10 mM MES, 10 mM MgCl₂, and 100 μM AS). The Agrobacteria containing the expression constructs were infiltrated into leaves of N. benthamiana for transient expression assay. Leaves were examined for image acquisition by using the Zeiss fluorescence microscope (Zeiss imager A2) between 36 and 72 hpi. Western blot analyses were performed following a previous protocol (Li et al., 2017 (link)). Total proteins were extracted from equal amounts of leaves with the protein extraction buffer with 1 × loading buffer (0.05 mg/mL Bromophneil blue; 0.065 M Tris-HCl, pH 6.8; 0.02 g/mL SDS; 0.05 mL/mL 2-mercaptoethanol; 0.1 mL/mL glycerol; 1 × protease inhibitor cocktail EDTA-free, Bimake, b14002). Total proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore) by using Trans-Blot Turbo (BIO-RAD). Protein blot was hybridized with the rabbit anti-GFP to determine YFP accumulation, and membranes were stained with Ponceau S as the loading control.

Formaldehyde-fixed tissue was embedded in paraffin wax for further immunohistochemical examination. Sections were deparaffinized in xylene and passed through descending alcohol series. The antigen retrieval process was performed in citrate buffer solution (pH 6.0) for 15 min in a microwave oven at 700 W. Sections were allowed to cool at room temperature for 30 min and washed twice in phosphate buffered saline (PBS) for 5 min. Endogenous peroxidase blockage was performed in a 3% hydrogen peroxide solution for 7 min. The washed samples were incubated in Ultra V block for 8 min. Blocking solution was removed from the sections and allowed to incubate overnight at +4 °C with primary antibodies epidermal growth factor, (catalog no: ab9695, Abcam, Cambridge, UK) and fibroblast growth factor (catalog no: ab92337, Abcam, Cambridge, UK). After washing the sections in PBS, secondary antibody was applied for 20 min. The sections were washed in PBS for 2×5 min and then exposed to streptavidin-peroxidase for 20 min. Sections washed with PBS were allowed to react with 3,3′-diaminobenzidine chromogen. Counterstaining with hematoxylin was applied and after washing, the preparations were mounted. Sections were examined under a light microscope (Zeiss Imager A2, Jena, Germany)18
^-20 .

The renal cross sections were subjected to haematoxylin and eosin (H&E) and periodic acid‐Schiff (PAS) staining. The tissue damage was examined and scored based on the percentage of damaged tubules as follows: 0, no damage; 1, <25%; 2, 25%‐50%; 3, 50%‐75%; 4, >75%. The samples were blinded to the assessor. The mean tubular injury scores for each mouse represented the average score of the 20 fields examined.28, 29, 30 The expression levels of CD3, Ki67 and CDK (Santa Cruz Biotechnology) in the kidney tissues were evaluated by immunohistochemical (IHC) staining. The digital images of stained sections were captured under a light microscope (Zeiss Imager A2).