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. 2022 Aug 16;34(11):4554–4568. doi: 10.1093/plcell/koac252

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© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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GhTCEE1 enhances the activation activity of GhTCE1 by forming heterodimers. A, Sequences encoding the GhTCE1 C-terminus from aa residues 160–331 without transactivation activity were fused to sequences encoding a GAL4 BD domain and designated GhTCE1-BD. The prey AD was linked to callus cDNAs. Negative represents no growth, positive represents growth. SD-TL/TLHA represents synthetically defined medium dropouts Trp, Leu or Trp, Leu, His, and Ade, respectively. B, BiFC assay between GhTCE1–nYFP and GhTCEE–cYFP in tobacco epidermal cells. Bars = 30 μm. C, LUC assays in tobacco leaves and cotton callus protoplasts. Promoters of GhLTP2 and GhLTP3 were, respectively, fused upstream of LUC. 35S_pro:REN was used as an internal control. GhTCE1 and GhTCEE1 were fused downstream of 35S_pro in pGreenII 62-SK. The pGreenII 62-SK empty vector was used as control named Null. For each tobacco leaf, four constructs were expressed: (i) pGreenII 0800-LUC:GhLTP2_pro+35S_pro:REN+pGreenII 62-SK empty (Null/LTP2_pro): (ii) pGreenII 0800-LUC:GhLTP2_pro+35S_pro:REN+pGEreenII 62-SK:GhTCE1 (GhTCE1/LTP2_pro); (iii) pGreenII 0800-LUC:GhLTP2_pro+35S_pro:REN+pGreenII 62-SK:GhTCEE1 (GhTCEE1/LTP2_pro); and (iv) pGreenII 0800-LUC:GhLTP2_pro+ 35S_pro:REN+pGreenII 62-SK:GhTCE1+GhTCEE1 (GhTCE1+GhTCEE1/LTP2_pro) following Agrobacterium-mediated transfection. D, Activation of LUC expression by GhTCE1/GhTCEE1 in cotton callus protoplasts. Relative activation level represents the ratio of fluorescence intensity of LUC/REN with GhTCE1 to LUC/REN without GhTCE1. Error bars represent ± standard errors of three biological replicates as described in Figure 3E. Statistical significance is indicated by *P < 0.05, **P < 0.01, Supplemental Data Set S2.