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. 2020 Jul 4;18(11):2354–2363. doi: 10.1111/pbi.13400

Figure 5.

Figure 5

APIP4 interacts with Piz‐t, and knocking out APIP4 affects Piz‐t immunity to a virulent strain. (a, b) APIP4 protein levels in NPB and Piz‐t‐HA plants after inoculation with isolate RB22‐AvrPiz‐t. The leaf tissue was harvested at 0, 24, 48, 72, 96 and 120 h after inoculation. APIP4 protein levels were determined by immunoblotting using the anti‐APIP4 antibody. The HSP protein was used as the internal control. (c) Co‐IP assay of Piz‐t and APIP4 in NPB and Piz‐t‐HA transgenic plants. Total protein extracted from the NPB and Piz‐t‐HA plants was immunoprecipitated with the anti‐HA antibody. Immunoblot analysis was performed using the anti‐APIP4 antibody. As a valid negative control, we used inoculated NPB plants and non‐inoculated Piz‐t‐HA plants to ensure the high concentration of APIP4 protein extracted from NPB. (d) Quantification of the interaction between Piz‐t‐NT/NBS/LRR fragments and APIP4 by the LCI assay. N. benthamiana leaves were co‐infiltrated with Agrobacterium strains containing APIP4/AvrPiz‐t‐NLuc and CLuc‐Piz‐t‐NT/NBS/LRR. The combinations of the co‐infiltrations are indicated below the panel. AvrPiz‐t‐NLuc was used as the negative control. Error bars indicate the SEM (**P < 0.01; n = 3). (e) Punch inoculation of apip4/Piz‐t‐HA plants. Leaves of eight‐week‐old rice plants were inoculated with the virulent isolate RB22. The leaves were photographed 14 dpi. (f, g) Relative lesion area (left) and relative fungal biomass (right) were measured 14 dpi. Data shown are means of three replications. Error bars indicate the SEM (**P < 0.01; n = 3).