de Vries Jeroen S, Andriotis Vasilios M E, Wu Ai-Jiuan, Rathjen John P
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK.
Plant J. 2006 Jan;45(1):31-45. doi: 10.1111/j.1365-313X.2005.02590.x.
Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.
番茄（Lycopersicon esculentum）的Pto激酶赋予植株对由表达avrPto或avrPtoB的丁香假单胞菌番茄致病变种引起的细菌性斑点病的抗性。Pto直接与这些III型分泌效应蛋白相互作用，从而引发包括过敏反应（HR）在内的防御反应。Pto的信号传导需要核苷酸结合位点富含亮氨酸重复序列（NBS-LRR）蛋白Prf。尽管Avr依赖性激活需要激酶活性，但对于Pto在刺激之前或期间是如何被调控的，人们了解甚少。在这里，我们证明了N端在Pto信号传导中的作用。激酶结构域之外的N端残基是在本氏烟草中诱导HR所必需的。N端也有助于AvrPto结合和磷酸化能力。Pto的第1至10位残基构成了一个共有基序，用于将肉豆蔻酸（一种疏水的14碳饱和脂肪酸）共价连接到Gly-2残基上。几条证据表明这个基序对Pto功能很重要。一个异源的N-肉豆蔻酰化基序补充了Pto的N端缺失突变体的Prf依赖性信号传导。野生型和突变型Pto的信号传导严格依赖于Gly-2残基。Pto的N-肉豆蔻酰化基序补充了AvrPto的同源基序的无毒功能和膜结合。此外，Pto在体内的肉豆蔻酰化依赖于Gly-2的存在。Pto的亚细胞定位独立于N-肉豆蔻酰化，表明N-肉豆蔻酰化是除膜亲和力之外的某些功能所必需的。与此观点一致，还发现AvrPtoB是一种可溶性蛋白。这些数据表明肉豆蔻酰化的N端在Pto信号传导中起重要作用。
