Evidence for a Common Toolbox Based on Necrotrophy in a Fungal Lineage Spanning Necrotrophs, Biotrophs, Endophytes, Host Generalists and Specialists

The Sclerotiniaceae (Ascomycotina, Leotiomycetes) is a relatively recently evolved lineage of necrotrophic host generalists, and necrotrophic or biotrophic host specialists, some latent or symptomless. We hypothesized that they inherited a basic toolbox of genes for plant symbiosis from their common ancestor. Maintenance and evolutionary diversification of symbiosis could require selection on toolbox genes or on timing and magnitude of gene expression. The genes studied were chosen because their products have been previously investigated as pathogenicity factors in the Sclerotiniaceae. They encode proteins associated with cell wall degradation: acid protease 1 (acp1), aspartyl protease (asps), and polygalacturonases (pg1, pg3, pg5, pg6), and the oxalic acid (OA) pathway: a zinc finger transcription factor (pac1), and oxaloacetate acetylhydrolase (oah), catalyst in OA production, essential for full symptom production in Sclerotinia sclerotiorum. Site-specific likelihood analyses provided evidence for purifying selection in all 8 pathogenicity-related genes. Consistent with an evolutionary arms race model, positive selection was detected in 5 of 8 genes. Only generalists produced large, proliferating disease lesions on excised Arabidopsis thaliana leaves and oxalic acid by 72 hours in vitro. In planta expression of oah was 10–300 times greater among the necrotrophic host generalists than necrotrophic and biotrophic host specialists; pac1 was not differentially expressed. Ability to amplify 6/8 pathogenicity related genes and produce oxalic acid in all genera are consistent with the common toolbox hypothesis for this gene sample. That our data did not distinguish biotrophs from necrotrophs is consistent with 1) a common toolbox based on necrotrophy and 2) the most conservative interpretation of the 3-locus housekeeping gene phylogeny – a baseline of necrotrophy from which forms of biotrophy emerged at least twice. Early oah overexpression likely expands the host range of necrotrophic generalists in the Sclerotiniaceae, while specialists and biotrophs deploy oah, or other as-yet-unknown toolbox genes, differently

<i>In planta</i> normalized <i>oah</i> transcript copy number at sampling times 1, 2, 4, 8, 12, 24, 48 and 72 hours post-inoculation.

<p>All <i>oah</i> gene expression values are represented normalized to the housekeeping gene, <i>actin</i>. Error bars on both graphs represent the standard error among 3 biological replicates. Asterisks represent values that are significantly different using a split plot factorial repeated measures analysis. <b>A.</b> The first trial included the necrotrophic generalists, <i>Botrytis cinerea</i> and <i>Sclerotinia sclerotiorum</i>, a host specialist of unknown trophic type, <i>S. glacialis</i>, and the biotrophic specialists, <i>Myriosclerotinia scirpicola</i> and <i>Myriosclerotinia duriaeana</i>. <b>B.</b> The second trial included the necrotrophic generalists, <i>Botrytis cinerea</i> and <i>Sclerotinia sclerotiorum</i>, the necrotrophic specialists, <i>B. tulipae</i>, <i>Monilinia fructicola</i>, and <i>Monilinia aucupariae</i>, and the biotrophic specialist, <i>Myriosclerotinia curreyana</i>.</p

Site-specific likelihood analyses for eight pathogenicity-related genes and two housekeeping genes.

<p>A likelihood ratio test (LRT) was performed to compare the likelihood scores (lnL) of the two nested models: a model that accounts for sites with ω>1 (M8) and a null model that does not (M7). <i>P</i>-values that are statistically significant are indicated with an asterisk.</p

Branch-specific likelihood analyses for lineage comparisons.

<p>The null model fixes the <i>d</i>N/<i>d</i>S ratio across all lineages in the phylogeny, while the alternative model allows for a different ω value for the foreground branch(es). <i>P</i>-values that are statistically significant are indicated with an asterisk.</p>A<p>Comparisons of the biotrophic lineage(s) to all other necrotrophic lineages.</p>B<p>Comparisons of all host-specialist lineages to host-generalist lineages in the Sclerotiniaceae.</p

Phylogeny of the Sclerotiniaceae inferred from 3 housekeeping loci showing incongruencies with pathogenicity-related genes trees, and results of acid production on indicator plates.

<p><b>A.</b> Housekeeping gene phylogeny of the Sclerotiniaceae from the concatenated sequence of <i>hsp60</i>, <i>g3pdh</i> and <i>cal</i>. The putative obligate biotroph, <i>Ciborinia whetzelii</i>, was added based on the placement of Holst-Jensen <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029943#pone.0029943-HolstJensen2" target="_blank">[52]</a>. Thick branches represent well-supported nodes with >90% support from 1000 maximum likelihood (ML) bootstrapped pseudoreplicates and >0.95 posterior probabilities. An asterisk represents the node with >75% ML bootstrap support and >0.90 posterior probabilities. Trophic type is designated by text color: necrotrophs in red, biotrophs in green, and taxa with unknown life histories in black. <b>B.</b> Incongruence between the housekeeping gene phylogeny in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029943#pone-0029943-g001" target="_blank">Figure 1A</a> and the phylogeny of each pathogenicity-related gene (<i>acp1</i>, <i>asps</i>, <i>pg1</i>, <i>pg3</i>, <i>pg5</i>, <i>pg6</i>, <i>pac1</i>, <i>oah</i>). Horizontally aligned with the housekeeping phylogeny, red fill designates a species incongruent with respect to the gene indicated at the top of the column and the housekeeping phylogeny; grey fill indicates congruence; white fill represents a gene that failed to amplify with the primers employed. <b>C.</b> Results from bromophenol blue plates indicating acid production as a proxy for oxalic acid production at two time points: 72 hours post-plating, and a final time point if acid production was delayed. Purple fill represents no color change indicating no acid production or production below the limit for detection, bright yellow fill represents a strong color change qualitatively indicating higher production, and a light yellow fill represents a less intense color change. Isolates not tested are n/a.</p

Relative virulence of fungal strains on detached <i>Arabidopsis thaliana</i> leaves.

<p>Disease lesions were measured as a percentage of total leaf area at 16, 24, 48, 72 and 96 hours post-inoculation using ImageJ. Error bars on both graphs represent the standard deviation among 3 biological replicates. Necrotrophic host generalists are shown in warm colors (reds and oranges), while necrotrophic and biotrophic host specialists are shown in cool colors (blues and greens, respectively). Strains that did not produce disease lesions on <i>A. thaliana</i> (<i>Monilinia aucupariae</i>, <i>Monilinia fructicola</i>, <i>S. glacialis</i>, and <i>Sclerotium cepivorum</i>) are not shown.</p