SUBCELLULAR LOCALISATION OF THE Cf-9 RESISTANCE PROTEIN

M BENGHEZAL and DA JONES

Plant Cell Biology, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra ACT 2601, Australia

Background and objectives
In gene-for-gene interactions the activation of the plant's defences is initiated by the recognition of specific molecules (elicitors) encoded by pathogen avirulence genes. The tomato Cf-9 resistance gene encodes a membrane-anchored, extracytoplasmic protein composed largely of leucine-rich repeats [1]. The interaction between Cf-9 and the Cladosporium fulvum Avr9 gene is not yet fully understood, nor are the mechanisms by which this recognition process activates plant defence.

Our objective is to investigate the cellular localization of Cf-9. Assuming that Cf-9 is the receptor for the Avr9 avirulence peptide, which is secreted into the apoplasm of infected tomato leaves, it is reasonable to infer a plasma membrane localization of Cf-9. Surprisingly, the C-terminus of Cf-9 has the mammalian and yeast KKXX motif for the retrieval of membrane-bound proteins from the Golgi apparatus to the endoplasmic reticulum [2]. To test whether Cf-9 is retrieved to the ER and whether the KKXX motif is functional in plants, the green fluorescent protein of jellyfish (GFP) has been fused to Cf-9 and six different constructs, one of which contains a KSXX motif, have been introduced into Arabidopsis and tomato.The subcellular localization of these different fusion proteins will be tested in the transgenic plants by confocal light microscopy. These fusion proteins have also been expressed transiently in tobacco protoplasts and inducibly in yeast.

Results and conclusions
In yeast cells and tobacco protoplasts, we observe an ER pattern for the GFP-tagged Cf-9 protein. In contrast, the KSXX mutation directs the fusion protein to the plasma membrane. The functionality of the fusion proteins for three of our constructs has already been tested in tomato, by injecting the Avr9 peptide into the apoplastic space of transgenic plants and looking at the resistance response. Both N-terminal and C-terminal GFP fusions to Cf-9 remain biologically active, but the fusion containing GFP close to the transmembrane domain on the cytosolic side is inactive. An analysis of Cf-9 localization in transgenic tomato and Arabidopsis by confocal microscopy will be reported.

The KKXX motif of Cf-9 is functional in yeast and tobacco cells, which would argue firstly, that Cf-9 behaves as a retrievable protein, and secondly, that plants, like mammals and yeast, use this retrieval motif. Hence, if Cf-9 is also localized in the ER of tomato, it would render the hypothesis of direct interaction of Avr9 peptide with Cf-9 protein on the cell surface more unlikely, and we may have to postulate an interaction elsewhere or even an indirect interaction.

References
1. Jones DA, Thomas CM, Hammond-Kosack KE, Balint-Kurti PJ, Jones JDG, 1994. Science 266:789-793.
2. Jackson et al., 1993. Journal of Cell Biology 121: 317-33.