title: 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.
abstract: The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-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 Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function.
authors: Division of Plant Sciences, Research School of Biology, The Australian National University, 134 Linnaeus Way, Canberra, ACT, 2601, Australia.
authors: Apratim
authors: A
authors: Chakrabarti
authors: Division of Plant Sciences, Research School of Biology, The Australian National University, 134 Linnaeus Way, Canberra, ACT, 2601, Australia.
authors: Thilaga
authors: T
authors: Velusamy
authors: Division of Plant Sciences, Research School of Biology, The Australian National University, 134 Linnaeus Way, Canberra, ACT, 2601, Australia.
authors: Choon Yang
authors: CY
authors: Tee
authors: Division of Plant Sciences, Research School of Biology, The Australian National University, 134 Linnaeus Way, Canberra, ACT, 2601, Australia.
authors: David A
authors: DA
authors: Jones
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry*; D002963:Cladosporium / Q000502:physiology; D003600:Cytosol / Q000737:chemistry; D004252:DNA Mutational Analysis; D060467:Disease Resistance; D005656:Fungal Proteins / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000378:metabolism* / Q000382:microbiology; D009154:Mutation / Q000235:genetics; D009336:Necrosis; D010935:Plant Diseases / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D000072417:Protein Domains; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D010940:Plant Proteins; C073605:AVR9 protein, Cladosporium fulvum
keywords: Cladosporium fulvum; Solanum lycopersicum; endocytosis motif; leucine-rich repeat receptor-like protein; protein phosphorylation; protein trafficking