Functional analysis of the plant disease resistance gene Pto using DNA shuffling

Adriana J. Bernal, Qilin Pan, Jeff Pollack, Laura Rose, Alexander Kozik, Neil Willits, Yao Luo, Muriel Guittet, Elena Kochetkova, Richard W Michelmore

Research output: Contribution to journalArticle

20 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)23073-23083
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number24
DOIs
StatePublished - Jun 17 2005

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DNA Shuffling
Plant Diseases
Functional analysis
Disease Resistance
Lycopersicon esculentum
Virulence
Clone Cells
Genes
Pseudomonas syringae
Phenotype
Mutagenesis
Protein-Serine-Threonine Kinases
DNA
Site-Directed Mutagenesis
Yeast
Haplotypes
Libraries
Proteins
Yeasts
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bernal, A. J., Pan, Q., Pollack, J., Rose, L., Kozik, A., Willits, N., ... Michelmore, R. W. (2005). Functional analysis of the plant disease resistance gene Pto using DNA shuffling. Journal of Biological Chemistry, 280(24), 23073-23083. https://doi.org/10.1074/jbc.M500992200

Functional analysis of the plant disease resistance gene Pto using DNA shuffling. / Bernal, Adriana J.; Pan, Qilin; Pollack, Jeff; Rose, Laura; Kozik, Alexander; Willits, Neil; Luo, Yao; Guittet, Muriel; Kochetkova, Elena; Michelmore, Richard W.

In: Journal of Biological Chemistry, Vol. 280, No. 24, 17.06.2005, p. 23073-23083.

Research output: Contribution to journalArticle

Bernal, AJ, Pan, Q, Pollack, J, Rose, L, Kozik, A, Willits, N, Luo, Y, Guittet, M, Kochetkova, E & Michelmore, RW 2005, 'Functional analysis of the plant disease resistance gene Pto using DNA shuffling', Journal of Biological Chemistry, vol. 280, no. 24, pp. 23073-23083. https://doi.org/10.1074/jbc.M500992200
Bernal AJ, Pan Q, Pollack J, Rose L, Kozik A, Willits N et al. Functional analysis of the plant disease resistance gene Pto using DNA shuffling. Journal of Biological Chemistry. 2005 Jun 17;280(24):23073-23083. https://doi.org/10.1074/jbc.M500992200
Bernal, Adriana J. ; Pan, Qilin ; Pollack, Jeff ; Rose, Laura ; Kozik, Alexander ; Willits, Neil ; Luo, Yao ; Guittet, Muriel ; Kochetkova, Elena ; Michelmore, Richard W. / Functional analysis of the plant disease resistance gene Pto using DNA shuffling. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 24. pp. 23073-23083.
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