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

doi:10.1074/jbc.m500992200

Adriana J Bernal, Qilin Pan + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m500992200

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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.

Highlights

The outcome of challenges by potential pathogens on a plant is determined by multiple molecular interactions between host and pathogen
Variation occurs in nucleotide binding site-leucine-rich repeat encoding genes, the most common class of resistance genes
This is the case of resistance in tomato to Pseudomonas syringae pv. tomato, where the observed intraspecific variation in specificity is determined by differences in the Pto gene that encodes a kinase, rather than in Prf that encodes a nucleotide binding site-leucine-rich repeat protein [6, 7]

Summary

Introduction

The outcome of challenges by potential pathogens on a plant is determined by multiple molecular interactions between host and pathogen. The most abundant class of resistance genes encodes for proteins with nucleotide binding site and leucine-rich repeat domains [3]. Variation occurs in nucleotide binding site-leucine-rich repeat encoding genes, the most common class of resistance genes. Tomato, where the observed intraspecific variation in specificity is determined by differences in the Pto gene that encodes a kinase, rather than in Prf that encodes a nucleotide binding site-leucine-rich repeat protein [6, 7]. Resistance to Pst strains expressing the avirulence proteins AvrPto or AvrPtoB is conferred by the Pto gene that encodes a functional serine/threonine protein kinase of 321 amino acids [9]. A nucleotide binding site-leucine-rich repeat-encoding gene located within the Pto locus, is required for this resistance [7].

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