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The host response and Pst growth in TaARPC3 know-down plants were observed by light microscopy. Stained leaf segments were fixed and cleared in ethanol/acetic acid (1:1 v/v). Auto-fluorescence of infected mesophyll cells was observed as a necrotic death area with an Olympus BX-51 microscope (Olympus Corp., Tokyo) (excitation filter, 485 nm; dichroic mirror, 510 nm; and barrier filter, 520 nm). Infection sites and lengths of infection hyphae were measured under the blue light excitation. H₂O₂ accumulation around the infection site was detected by using 3,3-diaminobenzidine (DAB; Amresco, Solon, OH, United States) staining, observed by the differential interference contrast optics. Fifty infection sites were randomly selected for each leaf segment, per treatment. Methods were taken as described previously (Wang et al., 2007 (link)), including identification of successful infection sites, hyphae staining and measurement of fungal structures.

Wheat leaves infected with BSMV were sampled at 24, 48, and 120 hpi with Pst and stained as described (Wang et al., 2007 (link)). The fourth leaves pre-infected with BSMV were sampled at 24, 48, and 120 hpi with Pst race CYR23. The areas of necrosis in infected leaves were estimated by auto-fluorescence of mesophyll cells. Hyphal length and hyphal branches were examined under blue light excitation by epi-fluorescence microscopy (excitation filter, 485 nm; dichromic mirror, 510 nm; and barrier filter, 520 nm). H₂O₂ accumulation was detected by staining with 3,3′-diaminobenzidine (DAB; Amresco, Solon, OH, USA) as previously described (Wang et al., 2007 (link)), then viewed by differential interference contrast optics. Only a site where an appressorium had formed over a stoma was considered to be successfully penetrated. At least 50 infection sites were examined on each of five randomly selected leaf segments for each treatment. Necrotic areas, areas of H₂O₂ accumulation, and hyphal length were observed with an Olympus BX-53 microscope (Olympus, Corp., Tokyo) and estimated with DP2-TWAIN/DP2-BSW software. Statistical analysis was performed by Tukey’s HSD test (P < 0.05) with the use of SPSS software (SPSS, Inc., Chicago, IL, USA).

At least six inoculated rice leaves were harvested at 10 days post inoculation (dpi) for histopathological analysis. Rice leaf segments of 4 cm were cut from the center of inoculated leaves. Leaf sections were fixed and decolorized in ethanol/trichloromethane (4:1, v/v) containing 0.15% (w/v) trichloroacetic acid for 2 days, and the fixation solution was replaced with fresh solution twice every other day. The specimens were cleared in saturated chloral hydrate until leaf tissues were translucent. For Calcofluor White (Sigma–Aldrich) staining, the method described by Zhang et al. (2011) (link) was followed. For further visualization of internal infection structures, the wheat germ agglutinin (WGA) conjugated to the Fluorophore Alexa 488 (Invitrogen) staining was used (Ayliffe et al., 2011b (link)). H₂O₂ was detected using the 3, 3-diaminobenzidine (DAB, Amresco, Solon, OH, USA) staining method (Thordal-Christensen et al., 1997 (link)) and observed under differential interference contrast (DIC) optics.

For the SDS Page assay, a transfer sandwich was prepared in a Novex^® Semi-Dry Blotter (Invitrogen, Waltham, MA, USA) and the normalized denatured intracellular and extracellular proteins were run at 65.0 mA for 75 min. After that, the PVDF membrane containing the desired proteins was subjected to the Western blotting assay. The membrane was blocked with 2% (w/v) Bovine Serum Albumin (BSA) dissolved in 1X PBS and 0.1% Tween-20 (PBST) solution for 1 h at RT with agitation and washed. Detection of target protein on the membrane was then performed by incubation with primary antibody (1:1000 mouse anti-His·Tag^® Monoclonal Antibody, Novagen, Madison, WI, USA) at 4 °C overnight without agitation and washed. The membrane was incubated with a secondary antibody (1:1000 horseradish peroxidase (HRP) conjugated goat anti-mouse IgG, Amresco, Cleveland, OH, USA) for another 1 h at RT with agitation and washed. The brown-coloured chromogenic protein bands were finally developed by incubation briefly with a substrate mixture containing a tablet of 3,3’-Diaminobenzidine (DAB) (Amresco, USA) dissolved in 10 mL of PBST solution and a drop of hydrogen peroxide. The membrane was washed and an image was captured.