K+ channels of Cf‐9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor‐dependent signal transduction

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Abstract
Summary: 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 μM staurosporine and 100 μM 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.