Refining the Model of Plant Disease Resistance Protein Activation

Author: Emma de Courcy-Ireland

de Courcy-Ireland, Emma, 2015 Refining the Model of Plant Disease Resistance Protein Activation, Flinders University, School of Biological Sciences

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Plant disease resistance (R) proteins act as specific pathogenic sensors within each and every plant cell. Upon recognition of their cognate pathogenic protein (effector or avirulence protein), R proteins initiate the pathogen-specific branch of plant immunity called effector triggered immunity, or ETI. Activation of ETI most often culminates in a form of cell death known as the hypersensitive response. Sacrifice of infected plant cells can save a plant from pathogenic colonisation.

R proteins have a tripartite architecture that consists of an N-terminal effector domain, a C-terminal sensor domain and a central activation domain and can be separated into two main classes on the basis of the form of their effector domain. These domains are either coiled-coil (CC) structures, or domains that share considerable homology with Drosophila Toll and mammalian interleukin-1 receptors and are thus called TIR domains. The central activation domain is a nucleotide binding site (NBS) domain that has been proven, in some R proteins, to bind adenosine nucleotides and catalyse an ATP hydrolysis reaction.

The molecular mechanisms that drive R protein activation remain poorly understood. The classical model of activation for these proteins describes a molecular switch that is turned on through exchange of an ADP molecule for ATP and switched off by hydrolysis of that ATP molecule. More recent evidence from the flax M TIR disease resistance protein suggests that, at least for this protein, activation of the HR maybe dependent on the hydrolysis of ATP. The aim of this study was to refine, redefine and/or expand the current model of R protein activation, with a focus on the M protein. Expression of R proteins other than M was also trailled so that their activation mechanisms could be studied too.

Expression of the Arabidopsis thaliana R proteins RPS2 and RPP1B was trialled with the Pichia pastoris-based system developed for the M protein. RPS2 expression was also tested in a baculovirus/insect cell system. Despite testing a number of truncated expression constructs, no RPS2 or RPP1B expression was detected in P. pastoris; however, insoluble full-length RPS2 protein was detected in the baculovirus/insect cell system. Additional work is warranted and may result in the production of soluble RPS2 protein.

In order to further investigate the mechanism of M protein activation, mutations were made within the highly conserved acidic residues of the Walker B motif within the NBS domain. These acidic residues are believed to play roles in coordinating and catalysing the ATP hydrolysis reaction. The functional consequence of each mutation was established with an in planta Agrobacterium tumefaciens-mediated transient expression assay. Mutants that had a function dissimilar to that of the M protein were subsequently recombinantly expressed in P. pastoris and purified using affinity chromatography. The identity and quantity of any nucleotide bound within these mutant proteins was determined with a bioluminescent ATP quantification assay. ATP hydrolysis assays were also conducted with a subset of mutants. Results from this study corroborate the proposition that ATP hydrolysis is required for M to activate the HR.

Is a simple ATP hydrolysis reaction performed by the M protein, or does it have other biochemical functions? To investigate this possibility the M protein was tested for its potential to act as a nucleotide phosphatase and for its ability to perform protein phosphorylation. A functionally inactive mutant was also subjected to the same assays. A potential phosphorylation signal that was both time and M protein dependent was observed in SDS-PAGE after reaction of the M protein with [γ-32P]ATP. The intensity of this signal was reduced in reactions containing the inactive mutant that has an impaired nucleotide binding capacity. A similar signal was not observed in reactions of M proteins with [α-32P]ATP, suggesting that M is not a nucleotide phosphatase. Further study is required for the exact identity of this signal to be determined, but its presence does hint that the mechanism of M protein activation is more complex that first hypothesised and may involve protein phosphorylation.

It is evident from the results presented in this study that the mechanisms underlying R protein activation are likely to be more complex than first proposed. It is possible that some R proteins can perform biochemical reactions other than simple β-γ ATP hydrolysis, and that such activity may be necessary for the initiation of an effective immune response.

Keywords: plant disease resistance, R protein, flax, flax rust, R protein activation, biochemical characterisation

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Biological Sciences
Supervisor: Dr Peter Anderson