The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.尽管植物细胞具有高度的可塑性，但植物仅在极为特定的非生物与生物胁迫下才会引发细胞增生，例如当暴露于甜菜曲顶病毒（Beet curly top virus, BCTV）这类病原体时。甜菜曲顶病毒的C4蛋白足以诱导细胞增生，并改变拟南芥（Arabidopsis）的生长发育。既往研究显示，C4蛋白可与两种拟南芥类Shaggy蛋白激酶AtSK21和AtSK23相互作用，二者为油菜素内酯（brassinosteroid, BR）信号通路的负调控因子。本研究发现，C4蛋白还可与另外5种AtSK家族成员发生相互作用。毕吉宁（Bikinin）——一种可与结合C4的7种AtSK家族成员竞争性结合的抑制剂——可诱导出与C4蛋白相似的细胞增生表型。研究证实，C4蛋白的丝氨酸49（Ser49）残基对其功能至关重要，具体依据如下：1）将Ser49诱变为丙氨酸（Ala）可消除C4蛋白诱导的表型，阻断C4与AtSK的相互作用，并使突变蛋白无法引发油菜素内酯信号通路的变化；2）Ser49残基在植物体内发生磷酸化；3）植物编码的AtSK激酶必须具备催化活性，才能与C4蛋白结合。此外，一种无法定位于质膜且不能诱导表型的C4蛋白N-肉豆蔻酰化位点突变体，仍保留了结合AtSK的能力。综合上述结果，本研究表明定位于质膜的C4蛋白可通过与多种AtSK激酶相互作用并利用它们，促进自身的磷酸化与激活，进而干扰细胞周期调控。