Identification of innate immunity elicitors using molecular signatures of natural selection

Abstract

The innate immune system is an ancient and broad-spectrum defense system found in all eukaryotes. The detection of microbial elicitors results in the up-regulation of defense-related genes and the elicitation of inflammatory and apoptotic responses. These innate immune responses are the front-line barrier against disease because they collectively suppress the growth of the vast majority of invading microbes. Despite their critical role, we know remarkably little about the diversity of immune elicitors. To address this paucity, we reasoned that hosts are more likely to evolve recognition to "core" pathogen proteins under strong negative selection for the maintenance of essential cellular functions, whereas repeated exposure to host-defense responses will impose strong positive selective pressure for elicitor diversification to avoid host recognition. Therefore, we hypothesized that novel bacterial elicitors can be identified through these opposing forces of natural selection. We tested this hypothesis by examining the genomes of six bacterial phytopathogens and identifying 56 candidate elicitors that have an excess of positively selected residues in a background of strong negative selection. We show that these positively selected residues are atypically clustered, similar to patterns seen in the few well-characterized elicitors. We then validated selected candidate elicitors by showing that they induce Arabidopsis thaliana innate immunity in functional (virulence suppression) and cellular (callose deposition) assays. These finding provide targets for the study of host-pathogen interactions and applied research into alternative antimicrobial treatments.

Fig. 1.

Estimation of the nonsynonymous (K_a) to synonymous substitution (K_S) ratio (ω) at each amino acid position using the Bayes Empirical Bayes (BEB) estimates from PAML/codeml model M8. Crosses above the sequence denote sites with moderate to high probability of positive selection (P > 0.50). Solid gray shading shows regions for which candidate peptides were synthesized for functional analysis, whereas the dark gray shading in PSPTO5086 indicates the region used for synthesizing the negative candidate peptide. Hatched shading indicates the region in EF-Tu (elf18) and flagellin (Flg22) previously identified as an elicitor of innate immunity in A. thaliana.

Fig. 2.

Functional validation of candidate elicitors. (A) Virulence suppression activity of putative elicitor peptides in A. thaliana. A. thaliana Col-0 leaves were infiltrated with treatment peptides, followed by inoculation with the strong pathogen P. syringae pv. tomato DC3000 (PtoDC3000) 24 h later. Peptides that induce innate immunity should suppress the growth of PtoDC3000. Average bacterial growth (±SE) was assayed immediately after inoculation (white bars) and again 1 d later (gray bars). Asterisks indicate that the day 1 growth is significantly different (P < 0.05) than the water control by an unpaired, homoscedastic t test. (B) Representative images analyzed for callose deposition in A. thaliana Col-0 leaves. Callose deposits are indicated by dark spots. Images have been converted to grayscale and inverted. (Scale bar: 200 μm.) (C) Average proportion of the field of view (2.9 mm²) occupied by callose deposits for each treatment peptide (±SE). Significant comparisons with the water control are designated (*P < 0.05 and **P < 0.005).