The population genetic test Tajima's D identifies genes encoding pathogen-associated molecular patterns and other virulence-related genes in Ralstonia solanacearum

Abstract

The detection of pathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor-Tu (EF-Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum-plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non-neutrally evolving genes, including not only EF-Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence-related genes in plant pathogen genomes.

Keywords: PAMPs; Ralstonia solanacearum; evolution; virulence.

Figure 1

Sequence alignment of the csp22 epitope. The csp22 epitope of the consensus sequence according to Felix and Boller (2003) and in Ralstonia solanacearum strains clustered into five groups based on amino acid polymorphism. Allele 1 includes 10 strains (FQY4, FJAT‐1458, FJAT‐91, Rs‐09–161, SD54, YC45, GMI1000, Rs‐10–244, Y45 and CMR15). Allele 2 includes only PSI07. Allele 3 includes four strains (CFBP2957, Greada9‐1, CFBP3858 and 58_RSOL). Allele 4 includes 14 strains (K60, UW163, IBSBF1900, UW179, 23‐10BR, Po82, CFBP7014, CIP417, B50, MolK2, P673, CFBP6783, IBSBF1503 and CFBP1416). Allele 5 includes eight strains (RS2, CPPB909, UW551, UY031, IPO1609, CFIA906, CPPB282 and POPS2).

Figure 2

Peptides corresponding to both the csp22 consensus and csp22 of Ralstonia solanacearum allele 5 elicit the production of reactive oxygen species (ROS) in leaves of 4‐week‐old tomato (A) and 6‐week‐old Nicotiana benthamiana (B), whereas Xup25_Rs is not recognized, using a luminol‐based ROS detection assay. All peptides were tested at a concentration of 1 μm. n = 8 for all plant–peptide combinations in all experiments. Error bars represent standard errors. Similar results were obtained in three independent experiments.