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. 2017 Jul 18;7:5739. doi: 10.1038/s41598-017-06016-2

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© The Author(s) 2017

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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BIL4 is localized to punctate structures and the vacuolar membrane and interacts with BRI1. (a) BIL4pro::BIL4–GFP partially co-localizes with the vacuolar membrane marker mRFP–VAM3 (unmerged puncta are marked by white arrows). (b) BIL4pro::BIL4–GFP partially colocalizes with FM4-64. Seedlings were treated with FM4-64 for 5 min and then incubated in water for 40 min. (c) Seedlings were pretreated with FM4-64 for 5 min, incubated in water for 20 min and then treated with 50 μM BFA for 20 min. (d–f) BIL4pro::BIL4–GFP partially co-localizes with the TGN/EE marker mRFP–SYP41 (d), the LE/MVB marker mRFP–ARA7 (e) and BRI1pro::BRI1–Venus in the endosome (f). Merged structures in b–f are marked by yellow arrows. Scale bar in (a–f): 5 µm. (g) BiFC assay of the interactions between BIL4 and BRI1 in cultured Arabidopsis cells. Scale bars, 10 μm. The white arrowheads indicate characteristic interactions. (h) Co-immunoprecipitation of BRI1 and BIL4. Wild-type (Col-0) and transgenic plants coexpressing BRI1pro::BRI1–GFP and 35 S::FLAG–BIL4 were grown for 7 days. FLAG–BIL4 was immunoprecipitated by anti-FLAG antibody, and the immunoblots were probed with anti-GFP or anti-FLAG antibody.