Anti rabbit hrp conjugated secondary antibody

The vascular sections were incubated with anti-TGF-β1 antibody (1:200; Bioss, Beijing, China; cat. no. bs-0086R) for 1 h at 37°C, following which anti-rabbit rhodamine-conjugated secondary antibody (1:500; CWbiotech, Beijing, China; cat. no. CW2035M) was added for 1 h at room temperature. Images were captured under a fluorescent microscope (magnification, ×200). The red fluorescence indicated TGF-β1 expression. Immunohistochemical staining was also performed to assess the expression of Smad3. The vascular sections were incubated with rabbit anti-Smad3 antibody (1:100; Abcam, Cambridge, UK; cat. no. ab40854) for 1 h at 37°C. Anti-rabbit HRP-conjugated secondary antibody (CWbiotech; cat. no. CW0159S) was then added for 30 min at 37°C. The expression of Smad3 was evaluated according to the size of the dark brown-stained region in the entire section. Images were captured (magnification, ×200) with a microscope linked to a computerized imaging system (cellSens version 1.7, Olympus Corporation). The mean optical density values of immunofluorescence and immunohistochemistry were calculated using Image-Pro Plus 6.0 software.

Seven‐day‐old rice seedlings were pretreated with 10 μM DEX and mock solution overnight, and were then treated with 1 µM flg22, 10 µg/mL chitin or sterile H₂O plus 0.01% Silwet L‐77 (GE Healthcare, Amersham, UK). The seedlings were collected at 6 h after treatment for detection of defence‐marker gene expression and at 15 min for MAPK activation assay. Total RNAs were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the leaves of treated seedlings following the manufacturer’s instructions. The synthesis of cDNA was performed with the Reverse Transcription System (Takara, Dalian, China) using oligo‐d(T)₁₈ as primers. The transcript levels of defence‐marker genes were analysed using the SYBR PrimeScript RT‐PCR kit (Takara) on an ABI PRISM 7500 system and normalized to the reference gene OsActin. The proteins were isolated from rice seedlings and MAPK activation was detected as described (Wang et al., 2015). Phospho‐serine/‐threonine was detected using anti‐Phospho‐p44/42 MAPK antibody (1:2000 dilution) (Cell Signaling Technology, Danvers, MA, USA) in 4 °C overnight, followed by anti‐rabbit‐HRP conjugated secondary antibody (CWBio) at 1:5000 dilution.