Blatt M R, Grabov A, Brearley J, Hammond-Kosack K, Jones J D
Laboratory of Plant Physiology and Biophysics, Wye College, University of London, Wye, Kent, UK.
Plant J. 1999 Aug;19(4):453-62. doi: 10.1046/j.1365-313x.1999.00534.x.
The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (IK,in) and outward-rectifying (IK,out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of IK,out and almost complete suppression of IK,in within 3-5 min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 microM staurosporine and 100 microM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.
Cf-9基因编码一种胞外富含亮氨酸重复序列（LRR）的蛋白质，该蛋白质在其C末端附近锚定在膜上。该蛋白质赋予番茄对表达相应无毒基因Avr9的番茄叶霉病菌株的抗性。在烟草中，Cf-9转基因赋予对Avr9激发子的敏感性，并在激发后引发一系列防御反应，这些反应在性质上与番茄宿主中通常观察到的反应相似。在天然宿主和转基因宿主中，最早的反应之一是受感染组织中的钾盐流失。然而，这种溶质通量及其控制的潜在机制尚不清楚。我们以保卫细胞为模型，研究了Avr9对Cf-9转基因烟草的作用。保卫细胞离子通道及其调节的许多细节已经为人所知。在表皮条带中的完整保卫细胞上进行测量，并在电压钳制下对内向（IK,in）和外向整流（IK,out）钾通道携带的电流进行表征。暴露于含Avr9的提取物会在3-5分钟内导致IK,out的2.5至3倍刺激，并几乎完全抑制IK,in。钾通道反应是不可逆的。它们对Avr9激发子具有特异性，在缺乏Cf-9转基因的烟草保卫细胞中未观察到，并且从动力学分析来看，可归因于通道门控的变化。发现两种钾通道反应都是Avr9浓度的饱和函数，并且在0.5 microM星形孢菌素和100 microM H7（两种广谱蛋白激酶拮抗剂）存在的情况下完全被阻断。这些结果证明了Cf-9转基因能够将Avr9激发特异性地与对两个离散钾通道的协同作用偶联起来，并且它们表明蛋白质磷酸化在导致运输控制的Avr9/Cf-9信号转导中起作用。
