Purification and characterization of protein complexes containing the Arabidopsis disease resistance protein RPS2

Plants can sense pathogen (or microbe)-associated molecular patterns (PAMPs or MAMPs) to trigger PMAP-triggered immunity (PTI). To counter PTI, gram negative bacterial pathogens deliver effector proteins into plant cells through the type III secretion system (TTSS) to target specific host proteins. Plant disease resistance (R) proteins are able to detect specific effectors either directly or indirectly and trigger a stronger immune response, known as effector-triggered immunity (ETI). Identifying new components of the R protein complexes will help our understanding of early signaling in ETI and possibly also in PTI since both immune responses may share signaling components.;R proteins, such as Arabidopsis RPS2, are generally expressed in low abundance. In addition, RPS2 is localized in the plasma membrane (PM). A method for purification of RPS2 protein complexes was developed, which utilizes a novel affinity purification tag, the HPB tag. The HPB tag contains an in vivo biotinylation site, and the tagged protein can be purified using the strong biotin-streptavidin interaction. Multiple proteins tagged with the HPB tag were functional and efficiently biotinylated in Arabidopsis, suggesting broad applicability of the HPB tag. Using RPS2::HPB transgenic plants, RPS2 protein complexes were purified and the putative complex components were identified using a mass spectrometry-based method.;Two Arabidopsis hypersensitive induced reaction (AtHIR) proteins, AtHIR1 and AtHIR2, were identified as putative RPS2 protein complex components. The ability of AtHIR1 and AtHIR2 to form a protein complex with RPS2 in vivo was confirmed by pulldown from plant protein extracts. The AtHIR1-RPS2 interaction was further confirmed by fluorescence resonance energy transfer (FRET) analysis. All four HIR protein members in Arabidopsis can form homo- and hetero-oligomers in N.benthamiana, suggesting the importance of oligomerization for function and possible functional redundancy. At least some AtHIRs were induced by P. syringae infection, which is consistent with the notion that they play a role in plant immunity and are involved in RPS2-mediated ETI.;The Arabidopsis RNA-binding protein-defense related 1(AtRBP-DR1) was also identified as a putative RPS2 complex component. Atrbp-dr1 mutants were more susceptible to P. syringae and overexpression lines were more resistant to P. syringae. In overexpression lines the salicylic acid (SA) pathway seemed to be constitutively activated. Thus, AtRBP-DR1 is a novel positive regulator of defense against P. syringae, which likely acts on the SA pathway at a post-transcriptional level.;In conclusion, this thesis research developed a new tool for studying R protein complexes as well as other kinds of protein complexes in plants and discovered new players in plant innate immunity through characterization of putative RPS2 protein complex components.