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. 2023 Aug 12;14:4877. doi: 10.1038/s41467-023-40623-0

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© The Author(s) 2023

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. 2 — a Representative leaves showing cell death induced by expression of PsTET3, XEG1 or NPP1 in NbSERK3a/b-silenced (TRV: NbSERK3a/b) or control (TRV: GFP) N. benthamiana leaves. Leaves (n = 9) were photographed three days after agro-infiltration. b RT-qPCR quantification of transcript levels of NbSERK3a/b in VIGS-silenced N. benthamiana leaves, normalized to those in TRV: GFP lines, using the NbEF1α gene as an internal reference. Data are presented as mean values (±SD). Statistical analyses were performed using Two-tailed Student’s t test. c Immunoblot analysis of PsTET1 and PsTET3 fused with an eGFP tag transiently expressed in TRV: GFP or TRV: NbSERK3a/b N. benthamiana leaves for 2 days after agro-infiltration. Ponceau S-stained Rubisco protein is shown as a total protein loading control. d Confocal microscopy images of eGFP-PsTET1/eGFP-PsTET3 and plant extracellular vesicles marker protein RFP-AtTET8 and multivesicular bodies (MVB) marker protein RFP-AtARA6 and RFP as control in N. benthamiana leaves. eGFP-PsTET1 and eGFP-PsTET3 were colocalized with AtTET8 and were partially colocalized with AtARA6. Scale bars, 20 μm. e PsTET1 and PsTET3 are localized to EVs when expressed in N. benthamiana. EVs of N. benthamiana are isolated from apoplastic fluids of leaves, before the development of cell death. AtTET8 is a positive control and AtARA6 is a negative control. Ponceau S-stained Rubisco protein is shown as a total protein loading control. All experiments were repeated three times with similar results. Source data are provided as a Source Data file.