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

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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 ~15 kD. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis, which also revealed a partial glutamine cyclization at the N-terminus of each protein. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by western 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.
Shunwen Lu , Justin D. Faris , Robert Sherwood , Michael C. Edwards
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
Journal of plant physiology 2013 v.170
Journal Articles, USDA Authors, Peer-Reviewed
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