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

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — APIP5 binds to OsLSD1 and OsRac1 mRNAs and regulates their turnover. (A) Binding activity of MBP-APIP5 with OsLSD1-3′UTR in a REMSA. The experiment was repeated three times (biological replicates) with similar results, and the representative data from one replicate are shown. (B) Binding activity of MBP-APIP5 with OsRac1-3′UTR in a REMSA. The experiment was repeated three times (biological replicates) with similar results, and the representative data from one replicate are shown. (C) qRT-PCR analysis of OsLSD1 and OsRac1 mRNA enrichment in in vitro RIP experiments. Relative enrichment was calculated as the fold change of RNA abundance following pull-down by MBP-APIP5 vs. MBP. The experiment was repeated twice (biological replicates) with similar results, and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). (D) In vivo RIP assays of GFP-APIP5 binding to OsLSD1 and OsRac1 mRNAs in GFP-APIP5 transgenic plants. The experiment was repeated twice (biological replicates) with similar results, and the representative data from one replicate are shown. Values are means ± SE (n = 3, technical repeats). In the diagram of OsLSD1 and OsRac1 mRNA structures, the line represents the untranslated region (UTR), and the black box represents the coding sequence (CDS) region. A, B, C and D are the sequence regions used for the RIP assays. (E) OsLSD1 mRNA turnover rates in Cas9-apip5, GFP-APIP5 and NPB plants. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from three biological samples (one leaf disc each). (F) OsRac1 mRNA turnover rates in Cas9-apip5, GFP-APIP5 and NPB plants. Values are means ± SE (n = 3, technical repeats). The mean and SE values were obtained from three biological samples (one leaf disc each). (G) A working model illustrating the molecular mechanism of APIP5-mediated cell death and immunity to M. oryzae in rice. See details in the Discussion section. Asterisks represent significance levels determined by Student’s t-test (**P < 0.01) compared to the negative controls or NPB.