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

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein t hat is membrane anchored near its C-terminus. The protein confers resistanc e in tomato to races of the fungus Cladosporium fulvum expressing the corre sponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene conf ers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the to mato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mec hanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using gu ard cells as a model. Much detail of guard cell ion channels and their regu lation is already known. Measurements were carried out on intact guard cell s in epidermal peels, and the currents carried by inward- (I-K,I-in) and ou tward-rectifying (I-K,I-out) K+ channels were characterized under voltage c lamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold st imulation of I-K,I-out and almost complete suppression of I-K,I-in within 3 -5min. The K+ channel responses were irreversible. They were specific for t he 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 funct ions of Avr9 concentration and were completely blocked in the presence of 0 .5 mu M staurosporine and 100 mu M H7, both broad-range protein kinase anta gonists. These results demonstrate the ability of the Cf-9 transgene to cou ple Avr9 elicitation specifically to a concerted action on two discrete Kchannels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.