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. 2022 Jul 29;13:4392. doi: 10.1038/s41467-022-32026-4

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic diagrams of constructs for AvrRppK gene screening in transient protoplast expression assays. EV is the empty vector; Rp1-D21 was used as the positive control; PPG genes from P. polysora were cloned by using mRNA isolated from P. polysora-infected leaves; ΔSP indicates deletion of the signal peptide region; and the LUC construct was used to evaluate cell viability in the transient protoplast expression assay. b PPG1259ΔSP expression in the protoplasts of RppK transgenic plants induced an HR but its expression in the protoplasts of nontransgenic plants did not. PPG1259ΔSP was coexpressed with the 35s:LUC construct in the protoplasts of RppK transgenic plants or nontransgenic plants; the Rp1-D21 gene or empty vector (EV) was coexpressed with the 35s:LUC construct in this assay as a positive or negative control, respectively. Relative LUC levels were measured to indicate the viability of protoplasts. Values are means ± SDs (for expression of PPG1259ΔSP, n = 8; for the others, n = 6; P = 0.4466, 3.4E-10, 1). **P < 0.01; ns, not significant (two-tailed Student’s t-test). c The coinfiltration of the construct carrying the RppK genomic DNA sequence with the 35S:PPG1259ΔSP construct induced an HR in N. benthamiana. The coinfiltration of the construct carrying the RppK genomic DNA construct or the 35S:PPG1259ΔSP construct with the empty vector were performed as negative controls and the infiltration of Rp1-D21-3×HA was taken as the positive control. The infiltration sites were labeled with “*”. d Infiltration of the PPG1259ΔSP protein into RppK transgenic plants and nontransgenic plants. GST-PPG1259ΔSP and GST-PPG348ΔSP proteins were expressed in E. coli and purified. After digestion with the 3C PPase enzyme to cleave the GST tag, PPG1259ΔSP and PPG348ΔSP were infiltrated into the leaves of 3-leaf stage RppK transgenic plants and nontransgenic plants, respectively. In this assay, PPG348ΔSP, which did not induce a cell death phenotype in protoplasts of RppK transgenic plants or nontransgenic plants, was used as the negative control. Source data are provided as a Source Data file.