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Combinatorial requirement of W- and WT-boxes in microbe-associated molecular pattern-responsive synthetic promoters

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Abstract

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The WT-box GGACTTTC belongs to a novel class of MAMP-responsive cis-regulatory sequences that are part of combinatorial elements.

Abstract

Microbe-associated molecular pattern (MAMP)-responsive synthetic promoters were generated with two cis-regulatory modules (CRM1 and CRM2) from the Arabidopsis thaliana WRKY30 promoter. Both modules harbour two W-boxes and one WT-box. Mutation analysis of the synthetic promoters and transient gene expression analysis in parsley protoplasts underline the importance of the W- and WT-boxes for MAMP-responsive gene expression and reveal the combinatorial requirement of at least two boxes for full MAMP responsivity. In the context of the native promoter, CRM1 is required for MAMP responsivity, while CRM2 alone is not sufficient. Yeast one-hybrid screenings using CRM1 with a transcription factor (TF) only prey library select only WRKY factors. Selection of WRKY26, 40, 41, and 70 requires the W-boxes. The WT-box is also required for selection of WRKY26 and 41 in yeast. In plant cells, WRKY26, 40, and 41 act as repressors of MAMP-responsive gene expression, whereas WRKY70 is an activator. To investigate whether the WT-box is also required for WRKY26 and 41 mediated gene expression in plant cells, both were converted into transcriptional activators by adding the GAL4 activating domain (AD). In contrast to yeast, transient gene expression in parsley protoplasts shows that only the W-boxes from CRM1 are required for WRKY41AD-activated reporter gene activity but not the WT-box. In addition, WRKY70-activated reporter gene activity in parsley cells does not require the WT-box of CRM1. The results demonstrate the importance of the WT-box as a new cis-regulatory sequence for MAMP-responsive gene expression. Based on these and earlier results, two types of WT-boxes are proposed.

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Acknowledgements

We would like to thank Elke Faurie for excellent technical assistance. The TF-only yeast one-hybrid library of A. thaliana was obtained from the National Institute of Advanced Industrial Science and Technology (AIST), Japan. This work was supported by the German Ministry of Education and Research (BMBF) through Hochschulpakt 2020 (KK) and by the Vietnamese Ministry of Agriculture and Rural Development (DXD).

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Authors and Affiliations

  1. Institut für Genetik, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany

    Konstantin Kanofsky, Ann-Kathrin Bahlmann, Reinhard Hehl & Do Xuan Dong

  2. Laboratory of Plant Cell Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam

    Do Xuan Dong

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  1. Konstantin Kanofsky
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  2. Ann-Kathrin Bahlmann
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  3. Reinhard Hehl
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  4. Do Xuan Dong
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Correspondence to Reinhard Hehl.

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Communicated by Zhanyuan Zhang.

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Kanofsky, K., Bahlmann, AK., Hehl, R. et al. Combinatorial requirement of W- and WT-boxes in microbe-associated molecular pattern-responsive synthetic promoters. Plant Cell Rep 36, 971–986 (2017). https://doi.org/10.1007/s00299-017-2130-3

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