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.

Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

“…The reason for this may be that these mutations have a deleterious effect on folding of Pto kinase. Notably, substitutions at other buried residues (Leu-252, Ala-256, and Val-257) around L1 loop resulted in no expression of Pto in plant cells (Wu et al, 2004), whereas mutations of certain solvent-exposed residues in this region (Glu-248 and Glu-254) had no effect on interaction with either AvrPtoB or AvrPto (Bernal et al, 2005). Our current data support that the buried residues from this region are important for proper folding of Pto as suggested before (Bernal et al, 2005).…”

“…Our data indicate that AvrPtoB likely uses a similar mechanism for activation of plant immunity, although these two effector proteins exhibit strikingly different tertiary structures. Residues surrounding the Pto P+1 loop are important for negative regulation of immune signaling by Pto, as many substitutions at this region induced the hypersensitive response (HR) independent of AvrPtoB or AvrPto (Rathjen et al, 1999;Wu et al, 2004;Bernal et al, 2005;Xing et al, 2007). The simultaneous substitutions Leu-205/Phe-213 in Pto around the unique AvrPtoB-interacting interface 1 specifically disrupted interaction with AvrPtoB ( Figure 5B) and led to a CGF phenotype ( Figure 6A), indicating that the region flanking these two residues also negatively regulates Prf signaling.…”

Natural Variation in the Pto Disease Resistance Gene Within Species of Wild Tomato (Lycopersicon). II. Population Genetics of Pto

“…Of the $300 residues, 12 are essentially invariant among kinases, and these 12 sites are also invariant in the collection of 48 Pto alleles described here. Furthermore, functional studies of the Pto protein using site-directed and random mutagenesis have specifically characterized the functional consequences of variation at 66 positions (20%) of the protein (Salmeron et al 1994;Scofield et al 1996;Frederick et al 1998;Rathjen et al 1999;Sessa et al 2000;Xiao et al 2003;Wu et al 2004;Bernal et al 2005;de Vries et al 2006). The vast majority (.86%) of the positions that knock out Avr recognition and/or signaling are invariant among the alleles collected from these natural populations.…”

“…This dual specificity of Pto may give us some insight into the maintenance of amino acid differentiation among Pto alleles. For the most part, these two Avr proteins appear to bind and activate Pto in a similar way (Kim et al 2002;Bernal et al 2005). However, we recently used a DNA-shuffling approach to generate chimeric Pto proteins and tested these chimeric molecules for AvrPto and AvrPtoB recognition ability .…”

The evolution of resistance genes in multi-protein plant resistance systems

“…Using this technique, Bernal et al [59] fragmented four paralogs of the tomato Pto gene and reannealed the fragments to generate a library of Pto homolog chimeras, similar to chimeras produced by natural sequence exchanges, and retrieved 56 nonredundant combinatorial clones that interacted with AvrPto in Y2H. The study focused on dissecting Pto functional domains, rather than generating novel specificities; however, shuffling has previously been used to enhance protein performance [60], and has the advantage of recombining natural sequence polymorphisms which may constitute functional domains.…”