title: Dynamic hydrolase activities precede hypersensitive tissue collapse in tomato seedlings.
abstract: Hydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins. We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity. ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs. In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse.
authors.affiliation: Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors.forename: Daniela
authors.initials: D
authors.lastname: Sueldo
authors.forename: Ali
authors.initials: A
authors.lastname: Ahmed
authors.forename: Johana
authors.initials: J
authors.lastname: Misas-Villamil
authors.forename: Tom
authors.initials: T
authors.lastname: Colby
authors.forename: Wladimir
authors.initials: W
authors.lastname: Tameling
authors.forename: Matthieu H A J
authors.initials: MH
authors.lastname: Joosten
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
mesh_terms: D002963:Cladosporium / Q000502:physiology; D005110:Extracellular Space / Q000378:metabolism; D006813:Humidity; D042541:Intracellular Space / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000276:immunology* / Q000382:microbiology; D036226:Seedlings / Q000201:enzymology* / Q000276:immunology* / Q000382:microbiology; D057057:Serine Proteases / Q000378:metabolism*; D013696:Temperature
chemical_list: D057057:Serine Proteases
keywords: Hsr203; P69; activity-based protein profiling (ABPP); papain-like cysteine protease; programmed cell death (PCD); tomato (Solanum lycopersicum); vacuolar processing enzyme (VPE)