López-Gómez Pablo, De La Mora-Franco Daniela, Herrera-Ubaldo Humberto, Díaz-Quezada Corina, Brieba Luis G, de Folter Stefan
Advanced Genomics Unit (UGA-Langebio), Center for Research and Advanced Studies (Cinvestav), Irapuato 36824, Mexico.
Rosario Izapa Experimental Field of National Institute of Forestry, Agriculture and Livestock Research (INIFAP), Chiapas 30870, Mexico.
Plants (Basel). 2025 Jun 8;14(12):1756. doi: 10.3390/plants14121756.
The aim of this study was to investigate the biological relevance of predicted sites involved in protein-protein interaction formation by bHLH transcription factors associated with gynoecium development in Arabidopsis (). We used AlphaFold2 to generate three-dimensional protein structures of the bHLH proteins SPATULA (SPT), HECATE1 (HEC1), and INDEHISCENT (IND). These structures were subjected to molecular docking using the HawkDock server, enabling the identification of potential interaction sites. PCR-based site-directed mutagenesis was used to modify the predicted interaction sites, followed by testing for protein-protein interaction formation using Bimolecular Fluorescence Complementation (BiFC) assays. Furthermore, these modified versions were overexpressed in Arabidopsis to observe whether gynoecium and fruit development would be affected. BiFC assays with the modified versions revealed a complete loss of the SPT-HEC1 interaction and a strong reduction in the SPT-IND interaction. The overexpression experiments in Arabidopsis showed that the line exhibited strong phenotypes in the development of the medial tissues of the gynoecium, resulting in reduced seed number and shorter fruits. In the line, a reduced seed number and shorter fruits were also observed, but no other obvious defects were observed. Finally, the line was less affected than the line. In the latter, medial tissue development was strongly affected, while in the line, it was only slightly affected; however, a reduced seed number and shorter fruits were observed. In summary, the predicted interaction sites are relevant and, when modified, affect gynoecium development in Arabidopsis. The findings demonstrate that predictive computational tools represent a viable strategy for a deeper understanding of protein-protein interactions.
本研究的目的是调查拟南芥中与雌蕊发育相关的bHLH转录因子在蛋白质-蛋白质相互作用形成中涉及的预测位点的生物学相关性。我们使用AlphaFold2生成了bHLH蛋白SPATULA（SPT）、HECATE1（HEC1）和INDEHISCENT（IND）的三维蛋白质结构。这些结构使用HawkDock服务器进行分子对接，从而能够识别潜在的相互作用位点。基于PCR的定点诱变用于修饰预测的相互作用位点，随后使用双分子荧光互补（BiFC）分析测试蛋白质-蛋白质相互作用的形成。此外，这些修饰版本在拟南芥中过表达，以观察雌蕊和果实发育是否会受到影响。对修饰版本进行的BiFC分析显示，SPT-HEC1相互作用完全丧失，SPT-IND相互作用大幅减少。拟南芥中的过表达实验表明， 系在雌蕊内侧组织发育中表现出强烈的表型，导致种子数量减少和果实变短。在 系中，也观察到种子数量减少和果实变短，但未观察到其他明显缺陷。最后， 系受到的影响小于 系。在后者中，内侧组织发育受到强烈影响，而在 系中，仅受到轻微影响；然而，观察到种子数量减少和果实变短。总之，预测的相互作用位点是相关的，经修饰后会影响拟南芥的雌蕊发育。这些发现表明，预测性计算工具是深入理解蛋白质-蛋白质相互作用的可行策略。
