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MOESM4 of Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis

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posted on 30.11.2019, 04:55 by Neha Kachewar, Vishal Gupta, Ashish Ranjan, Hitendra Patel, Ramesh Sonti
Additional file 4: Fig. S2. Transient overexpression of OsPUB41 and its mutant forms (OsPUB41C40A and OsPUB41V51R) in rice leaves (Confirmation by qPCR and Western blotting). Leaves (n = 20) of 10–15 days old TN-1 rice plants were infiltrated with suspension of Agrobacterium strain (100 μl per leaf), containing either pMDC7-OsPUB41, pMDC7-OsPUB41C40A or pMDC7-OsPUB41V51R, with the inducer (40 μM 17-β-estradiol dissolved in 0.1% DMSO) or without the inducer (0.1% DMSO) using needleless 1 ml syringes. After 12 h, leaves were collected, crushed and processed for either western blotting or qPCR. OsActin was used as internal control for qPCR. The graph represents relative fold change (2-∆∆Ct) using expression values of induced over uninduced samples (A). Student’s two-tailed t-test for independent means was performed on delta Ct values to test for significance (p < 0.05). Each sample was split into two: first half was loaded in Gel 1 and remaining half of the same sample was loaded in Gel2. The OsPUB41 protein and its mutant forms were detected (Gel1) using anti-OsPUB41Pep3 antibody (approximate size is 47 kDa). Rabbit polyclonal Histone (H3, Abcam) antibody (1: 50,000) was used to detect Histone (Gel2: loading control, approximate size: 17 kDa; represented by lower panel of Fig. S2B) in these samples. Leaf sample of transgenic Arabidopsis expressing OsPUB41 was used as a positive control (B).


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