Author: Simon John Williams
Williams, Simon John, 2010 Molecular Insight into the Activation of a Plant Disease Resistance Protein, Flinders University, School of Biological Sciences
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A plant’s ability to detect an invading pathogen and circumvent a subsequent disease state is essential for its survival. Disease resistance, and the mechanisms behind it, are thus of critical importance. The pioneering work of Harold Flor, using the interaction between flax and the flax rust fungus, Melampsora lini, demonstrated that this ability to detect and resist the infection of a specific pathogen rests with two critical genes; a resistance (R) gene in the plant and a corresponding avirulence (Avr) gene in the pathogen. This, so called ‘gene-for-gene’ model, has subsequently been shown to apply in many other plant-pathogen interactions and has spawned considerable research efforts directed towards understanding the molecular basis of host-pathogen interactions and the consequential disease resistance response. Using the flax-flax rust pathosystem, and utilising a biochemical approach, this research has endeavoured to further the current understanding of the molecular basis of the interaction between plants and pathogens, with a particular focus on R protein function. Chapter 3 describes the production of soluble, recombinant flax R proteins, M and L6, using the Pichia pastoris expression system. These flax R proteins can be purified from total cell lysates utilising a number of chromatography techniques. Following nickel affinity chromatography, concentration of protein in the presence of imidazole, leads to aggregation. This, however, can be alleviated by lowering the imidazole concentration prior to the protein concentration step. This fine tuning of the purification protocol enabled the expression and enrichment of near full-length and truncated versions of M and L6, and rational point mutations of M. Utilising this expression and purification system, Chapter 4 presents a detailed functional study of the flax M protein, with particular focus on mutations that cause autoactivity and inactivity. These mutations were generated in the NB-ARC region of M with predicted loss- or gain-of-function consequences, as determined from the results of the in planta phenotypes of analogous mutations in other R proteins, in particular the flax L6 protein. Nucleotide quantification of purified wild type M and L6 demonstrated that these proteins are associated with ADP. Analysis of proteins with mutations within the NB-ARC domain demonstrated that this ADP binding is dependent on a functional P-loop in the NB subdomain. Mutations within the MHD motif and motif VIII that are predicted to result in an autoactive in planta phenotype, have more ATP associated with purified protein preparations in comparison to wild type. Taken together, these results further support the model that R proteins act as a molecular switch, whereby the inactive form of the protein is ADP bound, while the active conformation of the protein is ATP bound. Prior to this study yeast two hybrid analysis had demonstrated that a direct interaction between M and AvrM occurs. To investigate the interaction, and the consequence of interaction, between M and AvrM proteins in vitro, an expression and purification protocol was generated for AvrM (and variants) in Chapter 5. Here, a direct protein-protein interaction was supported by co-immunoprecipitation of purified M and AvrM proteins. The interaction that M has with AvrM is dependent on a functional P-loop and therefore presumably requires the presence of a bound nucleotide. The preferred model of R protein activation suggests that interaction with an effector causes the R protein to exchange its bound nucleotide from ADP to ATP. To determine if AvrM could induce nucleotide exchange, ADP/ATP exchange assays were performed, however, the results of this study were inconclusive. It is possible that nucleotide exchange is not the mechanism of activation of the flax M protein; although, it is equally likely that the conditions in the in vitro assay were not conducive for exchange to occur or that other proteins are needed to facilitate the exchange event. Whilst this study adds further proof to the theory of a direct interaction between flax rust effectors and their corresponding R proteins, the molecular effect that this event has on the R protein is yet to be understood. In summary, only a small number of biochemical investigations of R proteins have been published, nevertheless, they have provided highly revealing information regarding R protein function. Utilising an in vitro approach, the results from this thesis provide further insight into the function and interaction between flax R proteins, and their effectors. It is hoped that the techniques developed and presented in this thesis will assist, and inspire, future in vitro investigations of flax R protein molecular function, and thus contribute to a wider understanding of plant disease resistance.
Keywords: Resistance proteins,plant disease,NB-ARC-LRR
Subject: Biological sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Peter Anderson