Plant hormone cytokinin signals through histidine-aspartic acid (H-D) phosphorelay to regulate plant growth and development. While it is well known that the phosphorelay involves histidine kinases, histidine phosphotransfer proteins (HPs), and response regulators (RRs), how this process is regulated by external components remains unknown. Here we demonstrate that protein phosphatase with kelch-like domains (PPKL1), known as a signaling component of steroid hormone brassinosteroid, is actually a cryptic inhibitor of cytokinin phosphorelay in rice (Oryza sativa). Mutation at a specific amino acid D364 of PPKL1 activates cytokinin response and thus enlarges grain size in a semi-dominant mutant named s48. Overexpression of PPKL1 containing D364, either with the deletion of the phosphatase domain or not, rescues the s48 mutant phenotype. PPKL1 interacts with OsAHP2, one of authentic HPs, and D364 resides in a region resembling the receiver domain of RRs. Accordingly, PPKL1 can utilize D364 to suppress OsAHP2-to-RR phosphorelay, whereas mutation of D364 abolishes the effect. This function of PPKL1 is independent of the phosphatase domain that is required for brassinosteroid signaling. Importantly, editing of the D364-residential region produces a diversity of semi-dominant mutations associated with variously increased grain sizes. Further screening of the edited plants enables the identification of two genotypes that confer significantly improved grain yield. Collectively, our study uncovers a noncanonical cytokinin signaling suppressor and provides a robust tool for seed rational design.

植物激素细胞分裂素通过组氨酸-天冬氨酸 (HD) 磷酸层传递信号来调节植物的生长和发育。众所周知，磷酸化过程涉及组氨酸激酶、组氨酸磷酸转移蛋白 (HP) 和反应调节剂 (RR)，但外部成分如何调节这一过程仍然未知。在这里，我们证明了具有 kelch 样结构域的蛋白磷酸酶 (PPKL1)，被称为类固醇激素油菜素内酯的信号成分，实际上是水稻 ( Oryza sativa ) 中细胞分裂素磷酸层的隐蔽抑制剂。PPKL1 的特定氨基酸 D364 的突变激活细胞分裂素反应，从而在名为s48的半显性突变体中扩大晶粒尺寸. 含有 D364 的 PPKL1 的过表达，无论是否缺失磷酸酶结构域，都拯救了s48突变表型。PPKL1 与OsAHP2（一种真正的HP）相互作用，而D364 位于类似于RR 的接收域的区域中。因此，PPKL1 可以利用 D364 抑制 OsAHP2 到 RR 的磷酸化，而 D364 的突变消除了这种作用。PPKL1 的这一功能独立于油菜素类固醇信号转导所需的磷酸酶结构域。重要的是，对 D364 居住区的编辑产生了与各种增加的晶粒尺寸相关的多种半显性突变。进一步筛选经过编辑的植物能够鉴定出两种基因型，这些基因型可显着提高谷物产量。总的来说，我们的研究揭示了一种非经典的细胞分裂素信号抑制因子，并为种子合理设计提供了一个强大的工具。