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. 2017 Jul 7;7:4879. doi: 10.1038/s41598-017-04853-9

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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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HpaB_Xcv can functionally control RipP1 secretion/translocation in R. solanacearum and in X. campestris pv. vesicatoria and is able to bind RipP1. (a) Lack of secretion of RipP1 and RipP2 in R. solanacearum hpaB mutant is partially restored when complementing with the Xcv _85-10 hpaB gene. R. solanacearum secretion assays were performed in type III inducing conditions, for the GMI1000 wild-type strain, the hrcV mutant, the hpaB mutant and the hpaB mutant complemented with the hpaB gene from R. solanacearum GMI1000 or the hpaB gene from Xcv _85-10. Total proteins from bacterial pellets and supernatants were analyzed by Western-blot using specific antibodies or total proteins were stained by silver nitrate. (b) HpaB_Xcv binds R. solanacearum RipP1 T3E. Glutathione S-transferase (GST) and GST-RipP1 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing 6His-HpaB_Xcv. Total extract (TE) and eluted proteins were analyzed using antibodies directed against the 6His epitope and GST respectively. Bands corresponding to GST and GST fusion protein are marked by asterisks. (c) RipP1 R. solanacearum T3E is heterogously translocated by Xcv, in an HpaB dependent manner. For this experiment, the wild-type strain Xcv _85-10 (85*) and the hpaB _Xcv mutant (85*ΔhpaB) both carry hrpG*, a mutated version of the key regulatory gene hrpG in the bacterial chromosome (Büttner et al., 2004). Xcv 85* and 85*ΔhpaB strains, and derivatives containing RipP1-AvrBs3Δ2 construct, were infiltrated into leaves of ECW-10R (carrying Bs1 resistance gene) and ECW-30R (carrying Bs3 resistance gene) pepper plants. Bacteria were infiltrated at OD₆₀₀ of 0.2 (corresponds to 2 × 10⁸ cfu/ml). Leaves were bleached 2 dpi for better visualization of the HR. Dashed lines indicate the infiltrated areas. For protein analysis, bacteria were grown ON in NYG medium. Equal amounts of cell extracts (adjusted according to OD) were analyzed by SDS-PAGE and immunoblotting, using an AvrBs3-specific antibody. For (a), (b) and (c), two biological replicates were done with similar results.