PubAg

Main content area

Dimerization and protease resistance: New insight into the function of PR-1

Author:
Lu, Shunwen, Faris, Justin D., Sherwood, Robert, Edwards, Michael C.
Source:
Journal of plant physiology 2013 v.170 no.1 pp. 105
ISSN:
0176-1617
Subject:
Pichia pastoris, Triticum aestivum, Western blotting, active sites, apoptosis, bovine serum albumin, cysteine proteinases, dimerization, fungal diseases of plants, host-pathogen relationships, hypersensitive response, mutation, pathogenesis-related proteins, polyacrylamide gel electrophoresis, protein synthesis, proteolysis, sequence analysis, subtilisin, wheat, yeasts
Abstract:
The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.
Agid:
58109
Handle:
10113/58109