Location: Cereal Crops Research
Title: Dimerization and protease resistance: new insight into the function of PR-1 Authors
Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 4, 2012
Publication Date: January 1, 2013
Citation: Lu, S., Faris, J.D., Sherwood, R., Edwards, M.C. 2013. Dimerization and protease resistance: new insight into the function of PR-1. Journal of Plant Physiology. 170:105-110. Interpretive Summary: Plants produce a group of proteins known as type -1 pathogenesis-related (PR-1) proteins in response to attacks by microbial pathogens or insects. These proteins are believed to have anti-microbial activities or play roles in plant cell suicide as defense mechanisms against invading pathogens. The first PR-1 protein was isolated from tobacco as early as in 1970, followed by dozens from other plant species, but how PR-1 proteins work in defense is still unknown because of the lack of knowledge about their biochemical functions. The objective of this study was to produce two wheat PR-1 proteins (called PR-1-1 and PR-1-5) in a yeast-based expression system and characterize the recombinant proteins in order to gain insights into the biochemical functions. We obtained a high yield and good quality for both yeast-expressed PR-1 proteins and confirmed their identities by immunological tests and mass spectrometry. We found that PR-1-1 exists primarily as a monomer (a single polypeptide) whereas PR-1-5 undergoes dimerization to form homodimers (consisting of two identical polypeptides of the same protein). We also discovered that both PR-1 proteins are resistant to digestion by certain proteases that degrade a broad spectrum of protein substrates. Finally, we determined, by site-specific mutagenesis, that several putative active sites (conferring catalytic activities in enzyme proteins) in PR-1 proteins are required for dimerization and protease resistance, and some of these active sites are reminiscent of those of caspases (substrate-specific proteases) known to be involved in cell suicide processes in human/animal systems. We hypothesize that PR-1 proteins may have substrate-specific protease-like activities that contribute to cell suicide pathways for mediating disease resistance in plants.
Technical 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 ~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.