Affinity Purification and Mass Spectrometry: An Attractive Choice to Investigate Protein-Protein Interactions in Plant Immunity

Ruiqiang      Chen; Caren      Chang; Mark      L. Tucker; Bret      Cooper

Abstract

The use of affinity purification to isolate protein complexes from biological tissues, followed by mass spectrometry (AP-MS), has ballooned in recent years due to improvements in affinity purification protocols, sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, and technological advances in mass spectrometry. Plant biologists are now exploiting AP-MS to identify plausible protein-protein interactions crucial to plant defense systems. As a result, knowledge of protein interactions in plants has grown. For example, new protein partners have been found to interact with RIN4 and RPS2, two plasma membrane-bound proteins critical for defense responses in Arabidopsis thaliana. Moreover, a nuclear protein complex in A. thaliana that includes the defense signaling protein MOS4 has been affinity purified and many of the identified protein partners found to be conserved with those in a protein complex previously characterized in yeast and humans. In another example, several proteins were identified that interact with a defense signaling GTPase, Rac1, in rice (Oryza sativa). Clearly, AP-MS is an important technique that will continue to provide novel insight into protein-protein interaction networks in plants. Here we review some of these recent discoveries and summarize the different techniques of AP-MS that have been used successfully to identify some of the interacting proteins in the plant defense response to pathogen attack.

Keywords: Plant-pathogen interaction, plant immunity, protein-protein interaction, protein complex, affinity purification, mass spectrometry, 2-D polyacrylamide gel electrophoresis, Multidimensional Protein Identification Technology, Mud-PIT, Arabidopsis thaliana, hemagglutinin, biotin carboxy carrier protein domains, suppressor of npr1-1, constitutive 1, MOS4-Associated Complex, MAC, Nineteen Complex, NTC, Magnaporthe grisea, Calmodulin-binding protein, Glutathione S-transferase, Liquid chromatography-tandem mass spectrometry, Modifier of snc1, Nonrace-specific disease resistance, Pleiotropic regulartor locus 1, Receptor for activated C-kinase 1, RPM1 Interacting Protein 4, Tandem affinity purification