Evolution of RXLR-Class Effectors in the Oomycete Plant Pathogen <i>Phytophthora ramorum</i>

<div><p><i>Phytophthora</i> plant pathogens contain many hundreds of effectors potentially involved in infection of host plants. Comparative genomic analyses have shown that these effectors evolve rapidly and have been subject to recent expansions. We examined the recent sequence evolution of RXLR-class effector gene families in the sudden oak death pathogen, <i>P. ramorum</i>. We found that <i>P. ramorum</i> RXLR effectors have taken multiple evolutionary paths, including loss or gain of repeated domains, recombination or gene conversion among paralogs, and selection on point mutations. Sequencing of homologs from two subfamilies in <i>P. ramorum</i>’s closest known relatives revealed repeated gene duplication and divergence since speciation with <i>P. lateralis</i>. One family showed strong signatures of recombination while the other family has evolved primarily by point mutation. Comparison of a small number of the hundreds of RXLR-class effectors across three clonal lineages of <i>P. ramorum</i> shows striking divergence in alleles among lineages, suggesting the potential for functional differences between lineages. Our results suggest future avenues for examination of rapidly evolving effectors in <i>P. ramorum</i>, including investigation of the functional and coevolutionary significance of the patterns of sequence evolution that we observed.</p></div

Expression of four RXLR effector genes in Rhododendron leaves infected with <i>P. ramorum</i>.

<p>In lineage NA1 isolate Pr-102, the genes <i>PrAvh60</i>, <i>PrAvh68</i>, <i>PrAvh120</i>, and <i>PrAvh205</i> exhibited induction of expression, on average, at 2, 3, and 5 days after inoculation relative to day 1. Expression of these genes was 20 to 60 times higher on day 2 relative to day 1. <i>PrAvh36</i> and <i>PrAvh121</i> were slightly induced on day 3. <i>PrAvh108</i> was not detected at any time point. The plant internal control (cox) was not induced relative to transcript levels on day 1.</p

<i>Metacoder</i> can be used with any type of data that can be organized hierarchically.

<p>This plot shows the results of the 2016 Democratic primary election organized by region, division, state, and county. The regions and divisions are those defined by the United States census bureau. Color corresponds to the difference in the percentage of votes for candidates Hillary Clinton (green) and Bernie Sanders (brown). Size corresponds to the total number of votes cast. Data was downloaded from <a href="https://www.kaggle.com/benhamner/2016-us-election/" target="_blank">https://www.kaggle.com/benhamner/2016-us-election/</a>.</p

Another alternate use example: Visualizing gene expression data in a GO hierarchy.

<p>The gene ontology for all differentially expressed genes in a study on the effect of a glucocorticoid on airway smooth muscle tissue [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005404#pcbi.1005404.ref019" target="_blank">19</a>]. Color indicates the sign and intensity of averaged changes in gene expression and the size indicates the number of genes classified by a given gene ontology term.</p

<i>Metacoder</i> has an intuitive and easy to use syntax.

<p>The code in this example analysis parses the taxonomic data associated with sequences from the Ribosomal Database Project [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005404#pcbi.1005404.ref009" target="_blank">9</a>] 16S training set, filters and subsamples the data by sequence and taxon characteristics, conducts digital PCR, and displays the results as a heat tree. All functions in bold are from the <i>metacoder</i> package. Note how columns and functions in the taxmap object (green box) can be referenced within functions as if they were independent variables.</p

Relationships among orthologs and paralogs in an effector gene family with recombination.

<p>A family in <i>P. ramorum</i> that is orthologous to the PexRD2 family in <i>P. infestans</i> shows expansion and recombination in <i>P. ramorum</i>. A. Minimum evolution tree inferred using amino acid sequences for Haas et al.’s (2009) Fam6. This family includes the PexRD2 genes in <i>P. infestans</i>. Proteins from <i>P. infestans</i> have a PITG prefix and proteins from <i>P. sojae</i> a PsG prefix. Branch support is shown as a percentage of 500 bootstrap samples. The branch lengths are drawn to scale and measured in the number of substitutions per site. B. Maximum likelihood tree for nucleotide sequences representing the C-terminal region of Haas Fam6 effector genes from <i>P. ramorum</i>, <i>P. lateralis</i>, <i>P. hibernalis</i>, and <i>P. foliorum</i> indicating putative orthologous and paralogous relationships for this region. Branches with high bootstrap support, based on 500 bootstrap samples, are indicated. The branch lengths are drawn to scale and measured in the number of substitutions per site.</p

<i>P. ramorum</i> RXLR effector gene families exhibiting strong evidence of recombination.

<p>Changing evolutionary relationships in different regions of genes illustrate recombination among paralogs. Maximum likelihood trees were constructed for gene fragments on either side of estimated recombination breakpoints inferred using RDP. Bootstrap support for branches is out of 100 samples. The branch lengths are drawn to scale and measured in the number of substitutions per site. A. The phylogenetic relationships among genes in this gene family change between the N- and C-terminal coding regions of the gene. The recombination breakpoint is in the DEER motif. B. Recombination in the C-terminal functional region. <i>PrAvh379</i> has a frameshift mutation leading to a 507 bp truncated gene and thus is not included in the second tree.</p

<i>P. ramorum</i> RXLR effector family with length polymorphism.

<p>Similar domain structures are found in different subclades and stop codons have shortened coding regions. A. Maximum likelihood tree of the targeting region (signal peptide through RXLR-DEER) with indels removed. Branch support is shown as a percentage of 500 bootstrap samples. The branch lengths are drawn to scale and measured in the number of substitutions per site. B. Schematic showing the domain structure of the aligned genes (not to scale). Colors are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079347#pone-0079347-g001" target="_blank">Figure 1</a>. C. Sliding window analysis of nucleotide divergence between selected genes that share similar domain structures showing that the middle sections of the genes are relatively conserved. Sliding window analysis of amino acid replacements per site (Ka) showed a very similar pattern. Total length of alignment was 1825 bp, 1614 bp after indels were removed.</p

Positive selection on codons among <i>PrAvh205</i> orthologs and paralogs, for the nonrecombinant region from site 130 to 330.

<p>Positive selection on codons among <i>PrAvh205</i> orthologs and paralogs, for the nonrecombinant region from site 130 to 330.</p

<i>P. ramorum</i> RXLR effector family with insertion/deletion of domains.

<p>Evolution in the domain structure of effectors is evident by comparing the phylogeny of the genes to their composition. A. The ancestral relationships among the genes inferred from a maximum likelihood tree of the targeting region (signal peptide through RXLR-DEER) with indels removed. Branch support is shown as a percentage of 500 bootstrap samples. The branch lengths are drawn to scale and measured in the number of substitutions per site. B. Schematic showing the aligned domain structure of the genes (not to scale). Gaps in the alignment are indicated using forward slashes. Colors of domains are: black – signal peptide, red – RXLR-DEER, and blue –WY-domain. WY-domains in lighter shade indicate low scoring matches to the HMM. *<i>PrAvh198</i> was at the end of a scaffold and is missing the 3’ end of the gene. </p