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. 2018 Jun 29;8:9842. doi: 10.1038/s41598-018-27869-1

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© The Author(s) 2018

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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Yeast Two Hybrid, Far Western Blot and BiFC assays of the interaction between HrpE and CsGRP. (a) Representative photograph of YTH assay showing that HrpE interacts with CsGRP. Yeast cells were co-transformed with: HrpE-BD/CsGRP-AD, HrpE-BD/empty-AD and empty-BD/CsGRP-AD. Yeast growth (serial 1:10 dilutions) is shown in −WL plates and in −WLHA plates containing 35 mM 3AT. (b) Purification of the Hrp-pilus from XcchrpG⁺ (lane 1) and XcchrpG⁻ (lane 2) grown in XVM2 medium and from XcchrpG⁺ (lane 3) grown in SB. Proteins obtained from pilus preparations were analyzed by Tricine-SDS-PAGE and Western blot revealed with anti-HrpE polyclonal antibody. (c) Far Western blots showing interactions between HrpE present in the Hrp-pilus preparations and CsGRP-GST. Nitrocellulose membranes similar to that showed in (b) were incubated with 50 μg CsGRP-GST or GST as a control and, after washing, probed with anti-GST polyclonal antibody. (d) Confocal laser-scanning micrographs of the abaxial surface of N. benthamiana leaves. BiFC constructs of HrpE-nVenus/CsGRP-cCFP (HrpE+/GRP+), HrpE-nVenus/empty-cCFP (HrpE+/GRP−), empty-nVenus/GRP-cCFP (HrpE−/GRP+), and empty-nVenus/empty-cCFP (HrpE−/GRP−) were co-expressed in N. benthamiana using agroinfiltration. Scale bars represent 25 μm. DIC: Differential Interference Contrast.