Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Abstract

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.

Figure 1.

Features of Validated RXLR Effectors. (A) Position distribution of signal peptide cleavage site (SP), RXLR motif start position (RxLR), and EER start position (EER) among a control set of 43 RXLR effectors. (B) Consensus sequence pattern of the RXLR/RXLR-EER motif was calculated using WebLogo based on an alignment of the 43 control sequences. The bigger the letter, the more conserved the amino acid site. This consensus served as a basis for the HMM described in the text.

Figure 2.

The RXLR Sequence Is Overrepresented in the Secretome of Phytophthora Relative to Other Eukaryotic Proteomes. The RXLR motif distribution in proteomes of eukaryotes is shown. RXLR distribution is shown in nonextracellular proteins (non-PEX proteins) in the top panels and in putative extracellular proteins (PEX proteins) in the bottom panels. RXLR motifs were counted only if they fell within the first 10 to 110 amino acids (left panels) or the first 30 to 60 amino acids (right panels) starting from the N termini of the proteins. The frequency for each eukaryote was calculated as a percentage of either all non-PEX proteins or all PEX proteins. RXLR frequency for each eukaryote was calculated as a percentage of either all non-PEX proteins or all PEX proteins. A full list of species is shown in Supplemental Table 1 online. AN, animals; FG, fungi; AM, amoebozoa; EX, excavata; AV, alveolates; ST, stramenopiles; AP, archaeplastida.

Figure 3.

The RXLR Sequence Is Positionally Constrained in the Secretome of Phytophthora. Positional distribution of the RXLR motif within the first 10 to 110 amino acids (aa) in non-PEX proteins (left panels) and PEX proteins (right panels) of selected eukaryotes.

Figure 4.

An Algorithm for ab Initio Mining of RXLR Effectors from Oomycete Genomes.

Figure 5.

Higher Rates of Closely Related Paralogs among the RXLR Effectors of P. ramorum. (A) Distribution of percentage identity of the closest paralog among the RXLR effectors of P. ramorum (Pr), P. sojae (Ps), and H. parasitica (Hp). (B) Closest paralog identity among the RXLR-EER effectors.

Figure 6.

Elevated d_N:d_S Ratios in the C-Terminal Domain of the RXLR Effectors. (A) Comparison of d_N:d_S (ω) values calculated for the full-length sequences versus the N-terminal region. (B) d_N:d_S plots for the full-length sequences versus the C-terminal region. (C) d_N:d_S plots for the N-terminal region versus the C-terminal region. The lines demark a d_N:d_S value of 1, above which positive selection is inferred.

Figure 7.

The Positively Selected Ps PGG20 Consists of Two Functional Genes, Avr1b-1 and Avh1b, with Distinct Effector Activities. (A) Percentages of wound-inoculation sites showing R3a-dependent hypersensitive cell death at 3 to 7 d after inoculation of N. benthamiana leaves transiently expressing R3a with Agrobacterium strains expressing AVR3a^KI (green circles), Avh1b (gray squares), and Avr1b-1, Avr1b^AAM20939, AVR3a^EM, and Avh1b^T101K mutants (purple asterisks; no response). Percentages are based on the averages of two replicated experiments with 18 and 36 wound-inoculated sites per construct. Error bars indicate se. dpi, days after inoculation. (B) Symptoms of R3a hypersensitive cell death at wound-inoculated sites of N. benthamiana leaves transiently expressing R3a. Photographs were taken at 7 d after inoculation. (C) Symptoms of R3a hypersensitive cell death at wound-inoculated sites of N. benthamiana leaves from a transgenic line expressing R3a.

Figure 8.

RXLR Effector Gene Clusters. Gene clusters in P. ramorum (A), P. sojae (B), and H. parasitica (C). The gene names start with the scaffold/supercontig number. The PGG groups are shown at right. Only clusters corresponding to positively selected genes in regions of <100 kb are shown.

Figure 9.

H. parasitica Proteins with Similarity to CRN Effectors and Overlapping RXLR and LXLFLAK Motifs. (A) Multiple alignment of the H. parasitica CRN-like RXLR proteins and P. infestans CRN1 to CRN16. The signal peptide region is indicated by a gray line. The LXLFLAK typical of CRN proteins is indicated by a black line. Note that the H. parasitica sequences contain the overlapping motif RXLRLFLAK. (B) Consensus sequence pattern illustrating the LXLFLAK motif was calculated using WebLogo based on an alignment of the 105 oomycete CRN-like sequences. The bigger the letter, the more conserved the amino acid site. (C) Neighbor-joining tree illustrating the relationship between the H. parasitica CRN-like proteins and the P. infestans CRNs. Bootstrap values corresponding to the neighbor-joining, ML, and parsimony methods are indicated above the branch separating the two major clusters.