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. 2022 Oct 4;13:5856. doi: 10.1038/s41467-022-33516-1

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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. 2 — a Plant phenotypes of transplastomic lines expressing fAMP combinations of 3, 6 or 9 different AMPs connected by 5 or 15 amino acid linkers (length of linker is indicated on the left of the pictures; cf. Supplementary Table 1, Supplementary Data 1 and 2). Plants incapable of photoautotrophic growth on soil are shown in tissue culture boxes. Photos were taken three months, or 4.5 months (6-MW-15) or six months (3-W-5) after transfer of (apical) shoot cuttings to fresh medium. 3‐N‐5 was the only fAMP line capable of growing on soil. The photo was taken five months after transfer from tissue culture. b Phenotypes of transplastomic plants expressing single AMPs. Shown are (from left to right) a CXCL9 plant three months after transfer of a shoot into a new box with synthetic medium, HA‐WAM1, HA‐Novispirin and a wild‐type plant one month after sowing on soil, and a wild‐type plant after one month of growth on synthetic medium. Scale bars: 2.5 cm. For images of HA-Novispirin, HA-WAM1 and wild-type plants two months after sowing, see Supplementary Fig. 4. c Northern blot analysis to compare mRNA accumulation for AMP and fAMP transgenes in transplastomic tobacco lines (see Methods section). MB: methylene blue staining as loading control. The blot was performed twice with similar results. d Western blot analysis of AMP accumulation (see Methods section). Samples of total protein extracts (20 µg) were loaded, and fAMPs were immunodetected with an anti-HA antibody. Note that CXCL9 is not tagged. The Coomassie-stained membrane is shown below the immunoblot as a loading control. Three independent blots were performed with similar results. Plant material for northern and western blots was produced in sterile culture to enable side-by-side comparison to lines that do not survive on soil. C: CXCL9, N: HA-Novispirin, W: HA-WAM1, 3M: 3‐Molluxubin, 3W: 3‐Wamdeposin, 3N: 3‐Novescandin, 6MW: 3M + 3W, 9MNW: 3M + 3N + 3W. Source data are provided as a Source Data file.