Japan Society for the Promotion of Science, Tokyo, Japan.
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, 305-8566, Japan.
Plant Mol Biol. 2022 Aug;109(6):799-821. doi: 10.1007/s11103-022-01277-6. Epub 2022 May 11.
Comprehensive yeast and protoplast two-hybrid analyses illustrated the protein-protein interaction network of the TALE homeodomain protein family, KNOX and BLH proteins, in tomato leaf and fruit development. KNOTTED-like (KNOX, KN) proteins and BELL1-like (BLH) proteins, which belong to the same TALE homeodomain family, act together by forming KNOX-BLH heterodimer modules. These modules play crucial roles in regulating multiple developmental processes in plants, like organ differentiation. However, despite the increasing knowledge about individual KNOX and BLH functions, a comprehensive view of their functional protein-protein interaction (PPI) network remains elusive in most plants, including tomato (Solanum lycopersicum), an important model plant to study fruit and leaf development. Here, we characterized eight tomato KNOX genes (SlKN1 to SlKN8) and fourteen tomato BLH genes (SlBLH1 to SlBLH14) by expression profiling, co-expression analysis, and PPI network analysis using two-hybrid techniques in yeasts (Y2H) and protoplasts (P2H). We identified 75 pairwise KNOX-BLH interactions, including ten novel interactors of SlKN2/TKN2, a primary class I KNOX protein, and nine novel interactors of SlKN5, a primary class II KNOX protein. Based on these data, we classified KNOX-BLH modules into several categories, which made us infer the order and combination of the KNOX-BLH modules involved in differentiation processes in leaf and fruit. Notably, the co-expression and interaction of SlKN5 and fruit preferentially expressing BLH1-clade paralogs (SlBLH5/SlBEL11 and SlBLH7) suggest their important roles in regulating fruit differentiation. Furthermore, in silico modeling of the KNOX-BLH modules, sequence analysis, and P2H assay identified several residues and a linker region potentially influencing the affinity of BLHs to KNOXs within their conserved dimerization domains. Together, these findings provide insights into the regulatory mechanism of KNOX-BLH modules underlying tomato organ differentiation.
综合酵母双杂交和原生质体双杂交分析阐明了番茄叶片和果实发育过程中 TALE 同源域蛋白家族、KNOX 和 BLH 蛋白的蛋白质-蛋白质相互作用网络。属于同一 TALE 同源域家族的 KNOTTED 样（KNOX、KN）蛋白和 BELL1 样（BLH）蛋白通过形成 KNOX-BLH 异源二聚体模块协同作用。这些模块在调节植物的多个发育过程中发挥着关键作用，例如器官分化。然而，尽管人们对单个 KNOX 和 BLH 功能的了解越来越多，但在大多数植物中，包括番茄（Solanum lycopersicum），一种研究果实和叶片发育的重要模式植物，它们的功能蛋白质-蛋白质相互作用（PPI）网络的综合视图仍然难以捉摸。在这里，我们通过表达谱分析、共表达分析以及使用酵母（Y2H）和原生质体（P2H）的双杂交技术对 8 个番茄 KNOX 基因（SlKN1 到 SlKN8）和 14 个番茄 BLH 基因（SlBLH1 到 SlBLH14）进行了表征。我们鉴定了 75 对 KNOX-BLH 相互作用，包括主要 I 类 KNOX 蛋白 SlKN2/TKN2 的 10 个新相互作用因子和主要 II 类 KNOX 蛋白 SlKN5 的 9 个新相互作用因子。基于这些数据，我们将 KNOX-BLH 模块分类为几个类别，这使我们推断出参与叶片和果实分化过程的 KNOX-BLH 模块的顺序和组合。值得注意的是，SlKN5 和果实特异性表达 BLH1 类基因（SlBLH5/SlBEL11 和 SlBLH7）的共表达和相互作用表明它们在调节果实分化中具有重要作用。此外，通过对 KNOX-BLH 模块进行计算机建模、序列分析和 P2H 测定，鉴定出几个残基和一个连接区，这些残基和连接区可能影响其保守二聚化结构域内 BLH 与 KNOX 的亲和力。总之，这些发现为番茄器官分化中 KNOX-BLH 模块的调控机制提供了新的见解。
