title: Functional analysis of the plant disease resistance gene Pto using DNA shuffling.
abstract: Pto is a serine/threonine kinase that mediates resistance in tomato to strains of Pseudomonas syringae pv. tomato expressing the (a)virulence proteins AvrPto or AvrPtoB. DNA shuffling was used as a combinatorial in vitro genetic approach to dissect the functional regions of Pto. The Pto gene was shuffled with four of its paralogs from a resistant haplotype to create a library of recombinant products that was screened for interaction with AvrPto in yeast. All interacting clones and a representative sample of noninteracting clones were sequenced, and their ability to signal downstream was tested by the elicitation of a hypersensitive response in an AvrPto-dependent or -independent manner in planta. Eight candidate regions important for binding to AvrPto or for downstream signaling were identified by statistical correlations between individual amino acid positions and phenotype. A subset of the regions had previously been identified as important for recognition, confirming the validity of the shuffling approach. Three novel regions important for Pto function were validated by site-directed mutagenesis. Several chimeras and point mutants exhibited a differential interaction with (a)virulence proteins in the AvrPto and VirPphA family, demonstrating distinct binding requirements for different ligands. Additionally, the identification of chimeras that are both constitutively active as well as capable of binding AvrPto indicates that elicitation of downstream signaling does not involve a conformational change that precludes binding of AvrPto, as previously hypothesized. The correlations between phenotypes and variation generated by DNA shuffling paralleled natural variation observed between orthologs of Pto from Lycopersicon spp.
authors.affiliation: Department of Plant Pathology, University of California, Davis, 95616, USA.
authors.forename: Adriana J
authors.initials: AJ
authors.lastname: Bernal
authors.forename: Qilin
authors.initials: Q
authors.lastname: Pan
authors.forename: Jeff
authors.initials: J
authors.lastname: Pollack
authors.forename: Laura
authors.initials: L
authors.lastname: Rose
authors.forename: Alexander
authors.initials: A
authors.lastname: Kozik
authors.forename: Neil
authors.initials: N
authors.lastname: Willits
authors.forename: Yao
authors.initials: Y
authors.lastname: Luo
authors.forename: Muriel
authors.initials: M
authors.lastname: Guittet
authors.forename: Elena
authors.initials: E
authors.lastname: Kochetkova
authors.forename: Richard W
authors.initials: RW
authors.lastname: Michelmore
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D032621:DNA Shuffling; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D005821:Genetic Techniques; D007113:Immunity, Innate / Q000235:genetics*; D018551:Solanum lycopersicum / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D044224:Pseudomonas syringae / Q000378:metabolism; D011995:Recombination, Genetic; D012231:Rhizobium / Q000378:metabolism; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D014774:Virulence
chemical_list: D010940:Plant Proteins; D004247:DNA; C084354:Pto protein, Lycopersicon; D017346:Protein Serine-Threonine Kinases