A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

Summary The tomato C f‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus C ladosporium fulvum in response to the avirulence protein A vr9. Although the N ‐terminal half of the extracellular leucine‐rich repeat (e LRR ) domain of the C f‐9 protein determines its specificity for A vr9, the C ‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of C f‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in A vr9‐dependent necrosis triggered by C f‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of C f‐9 plays an important role in C f‐9‐mediated necrosis, and two amino acids within this region, a threonine ( T 835) and a proline ( P 838), are particularly important for C f‐9 function. An alanine mutation of T835 had no effect on C f‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced C f‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether C f‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of C f‐9 interacts with the cytosolic domain of tomato VAP 27. This interaction could be disrupted by an alanine mutation of P 838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of C f‐9 may be important for interaction with VAP 27, which may, in turn, be important for C f‐9 function.