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. 2022 May 7;50(9):5064–5079. doi: 10.1093/nar/gkac316

Figure 3.

Figure 3.

Targeting and suppression of OsWAK5 by APIP5 and the disease phenotypes of OsWAK5 overexpression and knock out plants. (A) Binding of MBP-APIP5 to the P1 and P2 probes in the OsWAK5 promoter in an EMSA. MBP alone served as a control. The experiment was repeated three times (biological replicates) with similar results, and the representative data from one replicate are shown. (B) ChIP-qPCR of the binding of GFP-APIP5 and GFP-APIP5nes to the OsWAK5 promoter in transgenic plants at the tillering stage. Similar results were obtained from three independent experiments (biological replicates), and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). (C) Luciferase reporter assays of APIP5nes-induced suppression of OsWAK5Pro expression in N. benthamiana. LUC activity was measured by normalizing to REN signal. Similar results were obtained from three independent experiments (biological replicates) with similar results, and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). (D) qRT-PCR analysis of OsWAK5 expression in GFP-APIP5nls and GFP-APIP5nesand plants. UBIQUITIN (UBI) was used as an internal control. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from three biological samples (one leaf disc each). (E and F) Disease symptoms and relative fungal biomass of representative leaves of OsWAK5-overexpression and NPB plants after punch inoculation with M. oryzae isolate RO1-1. The images were taken at 14 d after inoculation. Similar results were obtained from three independent experiments (biological replicates), and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from five samples. OsWAK5-OE plants represent OsWAK5-overexpression plants. (G and H) Disease symptoms and relative fungal biomass of representative leaves of Cas9-oswak5and NPB plants after punch inoculation with M. oryzae isolate RO1-1. The images were taken at 14 d after inoculation. Similar results were obtained from three independent experiments (biological replicates), and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from five samples. (I) Lignin contents of OsWAK5-overexpression and NPB plants. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from more than five biological samples. (J and K) Disease symptoms and relative fungal biomass of representative leaves of GFP-APIP5, OsWAK5-OE, GFP-APIP5/OsWAK5-OE and NPB plants after punch inoculation withM. oryzae isolate RO1-1. Asterisks represent significance levels determined by Student’s t-test (**P < 0.01) compared to the negative controls.