Natural variation in the Pto pathogen resistance gene within species of wild tomato (Lycopersicon). I. Functional analysis of Pto alleles

Abstract

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the cultivated tomato, Lycopersicon esculentum, and the closely related L. pimpinellifolium is triggered by the physical interaction between plant disease resistance protein, Pto, and the pathogen avirulence protein, AvrPto. To investigate the extent to which variation in the Pto gene is responsible for naturally occurring variation in resistance to Pst, we determined the resistance phenotype of 51 accessions from seven species of Lycopersicon to isogenic strains of Pst differing in the presence of avrPto. One-third of the plants displayed resistance specifically when the pathogen expressed AvrPto, consistent with a gene-for-gene interaction. To test whether this resistance in these species was conferred specifically by the Pto gene, alleles of Pto were amplified and sequenced from 49 individuals and a subset (16) of these alleles was tested in planta using Agrobacterium-mediated transient assays. Eleven alleles conferred a hypersensitive resistance response (HR) in the presence of AvrPto, while 5 did not. Ten amino acid substitutions associated with the absence of AvrPto recognition and HR were identified, none of which had been identified in previous structure-function studies. Additionally, 3 alleles encoding putative pseudogenes of Pto were isolated from two species of Lycopersicon. Therefore, a large proportion, but not all, of the natural variation in the reaction to strains of Pst expressing AvrPto can be attributed to sequence variation in the Pto gene.

Figure 1.

Map of western coast of South America indicating locations of populations used in this study. The populations are labeled by an abbreviated form of the species name and an accession number corresponding to the accession numbers in Table 1.

Figure 2.

Variable amino acids in the alleles transiently expressed in the susceptible N. benthamiana line containing avrPto. The numbers at the top indicate the corresponding codon position in the predicted amino acid sequence of the Pto allele (LA400) from L. pimpinellifolium. Positions invariant among these 16 alleles are removed. A dot indicates an amino acid residue matching the LA400 sequence, while a dash indicates a gap. (A) The first 11 alleles conferred an avrPto-dependent HR (hypersensitive response). These alleles were designated A+ alleles. (B) The last five alleles did not confer an HR. These alleles were designated A− alleles. Amino acid substitutions limited to the alleles that did not confer an HR are underlined. The reaction of the genotypes from which each allele was derived when inoculated with Pst expressing avrPto is indicated to the right of the allele sequence (Res, resistant; Sus, susceptible; and n.t., not tested).

Figure 3.

Summary of results from plants tested for resistance to Pst expressing AvrPto and from which the alleles tested in the transient assay were derived. The top number is the number of observations per category and the bottom number is the percentage of observations per category. Open blocks show a 1:1 correspondence between the resistance assay and transformation assay. Shaded blocks indicate those observations that suggest that additional loci are involved in disease resistance.

Figure 4.

One of 100 equally most parsimonious trees of the nucleotide sequences of alleles of Pto. The tree was rooted using the Pth2, Pth4, and Fen genes. Alleles are named by a short abbreviation of the species name followed by an arbitrary number. The source of the allele according to population is indicated further to the right by the LA and PI accession numbers. The responses of alleles tested in the transient assay are indicated by HR (hypersensitive response) or no HR. The resistance responses to Pst expressing AvrPto of the plants from which each allele was derived are indicated (R, resistant; S, susceptible).

Figure 5.

Southern blot hybridization of 26 accessions of Lycopersicon and Solanum species using the entire open reading frame of Pto as a probe. See materials and methods for details. Above each lane is the abbreviated name of the species and the population or individual number of each plant sample. Pim (76R) is the L. esculentum cultivar containing the Pto region introgressed from L. pimpinellifolium. F₁ (LA400 × LA114) is an F₁ individual of a cross between the LA400 and LA114 individuals.