title: An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP.
abstract: Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.
authors.affiliation: The School of Biological Sciences, Flinders University, Adelaide, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Williams
authors.forename: Pradeep
authors.initials: P
authors.lastname: Sornaraj
authors.forename: Emma
authors.initials: E
authors.lastname: deCourcy-Ireland
authors.forename: R Ian
authors.initials: RI
authors.lastname: Menz
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Ellis
authors.forename: Peter N
authors.initials: PN
authors.lastname: Dodds
authors.forename: Peter A
authors.initials: PA
authors.lastname: Anderson
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019597:Flax / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D012231:Rhizobium
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate