Positive selection of the Hrp pilin HrpE of the plant pathogen Xanthomonas

Abstract

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. The TTS system is necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. At the cell surface, the TTS system is associated with an extracellular filamentous structure, the Hrp pilus, which serves as a conduit for the transfer of bacterial proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads. Previous work showed that HrpE contains two regions: a hypervariable surface-exposed domain, including the N-terminal secretion signal, and a C-terminal polymerization domain. In this study, the evolutionary rate of the hrpE gene was analyzed. Twenty-one alleles were cloned, sequenced, and compared with five known hrpE alleles. The ratio of synonymous (K(s)) and nonsynonymous (K(a)) substitution rates shows that parts of the HrpE N terminus are subjected to positive selection and the C terminus is subjected to purifying selection. The trade-off between positive and purifying selection at the very-N terminus allowed us to ascertain the amphipathic alpha-helical nature of the TTS signal. This is the first report of a surface structure from a plant-pathogenic bacterium that evolved under the constraint of positive selection and hints to the evolutionary adaptation of this extracellular appendage to avoid recognition by the plant defense surveillance system.

FIG. 1.

UPGMA tree of hrpE sequences from 26 Xanthomonas strains. Numbers on branches indicate the number of times (percent) that the node was supported by 1,000 replicates of the bootstrap analysis (only values greater than 80% are shown).

FIG. 2.

Nonredundant amino acid sequence alignment of HrpE. HrpE sequences of group I and II xanthomonads were aligned using CLUSTAL X (26). Residues which are identical in at least 90% of the sequences are shown in white on a black background. Proposed functional domains are indicated on top of the alignment (29).

FIG. 3.

Type III secretion signal of the X. campestris pv. vesicatoria 85-10 HrpE in a helical wheel representation. Conserved amino acids and hydrophobic residues are shaded in gray and black, respectively. The N-terminal proline residue is oriented to the bottom.

FIG. 4.

Mean pairwise diversity plots of K_s and K_a values. The plots were constructed for windows of 15 nucleotides sliding along the hrpE sequence alignment. The first codon of group I and the first four codons of group II sequences are not included. (A) Comparison of all hrpE sequences. (B) Group I sequence comparison. (C) Group II sequence comparison. Filled boxes indicate the windows (step size of 9 nucleotides) in which nonsynonymous differences significantly exceed synonymous differences (one-tailed Z test, P < 0.05). White boxes indicate positions in the sequence where the reverse condition applies (K_s > K_a; P < 0.05) or sequences which are identical. Solid line, mean K_a; dashed line, mean K_s.

FIG. 5.

Positively selected parts of HrpE are surface exposed. Mean hydrophobicity plots of group I (dashed line) and group II (solid line) HrpE proteins were calculated over a sliding window of five amino acid residues, using the Kyte and Doolittle hydrophobicity scale (16). Values below 0.4 are considered hydrophilic. Positively selected parts in groups I and II are indicated by open and filled boxes, respectively.