Purification and Characterization of Serine Proteases That Exhibit Caspase-Like Activity and Are Associated with Programmed Cell Death in <em>Avena sativa</em>
Abstract
Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.
Rice subtilisin, putative subtilisin-like protease (accession number {"type":"entrez-protein","attrs":{"text":"BAB89803","term_id":"20160864","term_text":"BAB89803"}}BAB89803). P69A, P69B, and P69C, subtilisin-like proteases from L. esculentum (accession numbers {"type":"entrez-protein","attrs":{"text":"CAA76724","term_id":"4200334","term_text":"CAA76724"}}CAA76724, {"type":"entrez-protein","attrs":{"text":"CAA76725","term_id":"4200336","term_text":"CAA76725"}}CAA76725, and {"type":"entrez-nucleotide","attrs":{"text":"T06577","term_id":"317726","term_text":"T06577"}}T06577, respectively). ARA12, subtilisin-like protease from Arabidopsis (accession number {"type":"entrez-protein","attrs":{"text":"NP_569048","term_id":"18425181","term_text":"NP_569048"}}NP_569048). Cucumisin, subtilisin-like protease form Cucumis melo (accession number {"type":"entrez-protein","attrs":{"text":"A55800","term_id":"7428210","term_text":"pir||A55800"}}A55800). Identical amino acids are in bold.
Partially purified enzymes assayed 1 h in indicated salt with the substrate Z-VAD-AFC. Activity is expressed as relative fluorescence units.
Z-VAD-AFC was used as the 100% control.
Z, benzyloxycarbonyl; Ac, acetyl; AFC, 7-amido-4-(trifluoromethyl)coumarin.
Z-VAD-AFC was used as the 100% control.
Z, benzyloxycarbonyl; Boc, tert-butyloxycarbonyl; Suc, N-succinyl; AFC, 7-amido-4-(trifluoromethyl)coumarin; AMC, 7-amido-4-methylcoumarin.
Enzymes were preincubated with 200 μM inhibitor for 2 h and then assayed for Z-VAD-AFC hydrolytic activity for 1 h.
Z, benzyloxycarbonyl; Ac, acetyl; B, biotin.
Purified SAS-1 and SAS-2 were preincubated with 200 μM inhibitor for 2 h, except PMSF (1 mM) and aprotinin (1 μg/mL), and then assayed for Z-VAD-AFC hydrolytic activity for 1 h.
Acknowledgments
We thank Lilo Barofsky for her help with the mass spectrometry analysis and Donald Buhler for the use of the fluorometer. We are also grateful to Jennifer Lorang, Teresa Sweat, and Marc Curtis for their scientific discussion and technical assistance. This work was supported in part by grants from the USDA National Research Initiative Competitive Grants Program (1999-02404 and 2001-35319-10896) and the National Science Foundation (IBN-9631442).
Notes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Thomas J. Wolpert (ude.etatsnogero.ecneics@ttreplow).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017947.