Widespread signatures of selection for secreted peptidases in a fungal plant pathogen

Abstract

Background: Fungal plant pathogens secrete a large arsenal of hydrolytic enzymes during the course of infection, including peptidases. Secreted peptidases have been extensively studied for their role as effectors. In this study, we combined transcriptomics, comparative genomics and evolutionary analyses to investigate all 39 secreted peptidases in the fungal wheat pathogen Zymoseptoria tritici and its close relatives Z. pseudotritici and Z. ardabiliae.

Results: RNA-seq data revealed that a majority of the secreted peptidases displayed differential transcription during the course of Z. tritici infection, indicative of specialization for different stages in the life cycle. Evolutionary analyses detected widespread evidence of adaptive evolution acting on at least 28 of the peptidases. A few peptidases displayed lineage-specific rates of molecular evolution, suggesting altered selection pressure in Z. tritici following host specialization on domesticated wheat. The peptidases belonging to MEROPS families A1 and G1 emerged as a particularly interesting group that may play key roles in host-pathogen co-evolution, host adaptation and pathogenicity. Sister genes in the A1 and G1 families showed accelerated substitution rates after gene duplications.

Conclusions: These results suggest widespread evolution of secreted peptidases leading to novel gene functions, consistent with predicted models of "escape from adaptive conflict" and "neo-functionalization". Our analyses identified candidate genes worthy of functional analyses that may encode effector functions, for example by suppressing plant defenses during the biotrophic phase of infection.

Keywords: Adaptive evolution; Escape from adaptive conflict (EAC); Neo-functionalization (NEO-F); Peptidases; Transcriptomics.

Fig. 1

Expression patterns of secreted peptidases in gene families A1 (panel a) and G1 (panel b) in Zymoseptoria tritici. The x-axis shows TMM (trimmed mean of M values) normalized RPKM values (reads per kilo base per million mapped reads) from RNA-seq experiments in Z. tritici. The gene expression bars represent the average from three biological replicates and the error bars are the standard error of the mean. Prot. ID is the protein identification number from the JGI database for the reference strain IPO323. Biotroph, necrotroph and saprotroph correspond to the three life cycle stages of Z. tritici measured at 7 days post inoculation (dpi), 14 dpi and 28 dpi, respectively. All the genes except for the one encoding Prot. ID-91855 were ≥5-fold upregulated during the biotrophic phase, with a FDR-corrected p value ≤0.05

Fig. 2

Differential expression of peptidase families during different life cycle stages. Means of Z-score transformed TMM Normalized log ₂ CPM values for each gene family. Biotroph, necrotroph and saprotroph correspond to the three investigated life cycle stages of Zymoseptoria tritici measured at 7 days post inoculation (dpi), 14 dpi and 28 dpi, respectively

Fig. 3

Scatter plot indicating the 2*Δlnl values resulting from the PAML site model analyses for all 39 secreted peptidases. The black dashed line (--) indicates the threshold value for a significant likelihood ratio test (p < 0.01). Values above this line are indicative of adaptive (accelerated) evolution. The y-axis is represented using a logarithmic scale. The colors represent the major families of secreted peptidases

Fig. 4

Scatter plot indicating the dN/dS ratios (ω) estimated with the PAML branch model analyses for all 39 secreted peptidases in Zymoseptoria species. ω values of the background branch (Z. pseudotritici and Z. ardabiliae) are represented on the x-axis and the ω values of the foreground branch (Z. tritici) are represented on the y-axis. The triangle symbol (Δ) indicates genes under significantly accelerated evolution (Prot. IDs-92645, 10047, 105030 and 90471). The colors correspond to the major classes of secreted peptidases

Fig. 5

Phylogenetic reconstruction used in the PAML analyses to infer branch-specific signatures of selection after gene duplications (indicated by black dots) in the peptidase families G1 and A1. A. maximum likelihood tree depicting the evolutionary relationship of all secreted peptidases in Zymoseptoria tritici, Z. pseudotritici and Z. ardabiliae. B. Enlarged view of peptidase family G1. C. Enlarged view of peptidase family A1. Analyzed branches are indicated by numbers that correspond to Additional file 1: Table A7. Red branches denote significant accelerated evolution. Numbers on the right are protein identification numbers according to Additional file 1: Table S1. The trees are drawn to scale, with branch lengths in units of nucleotide substitutions per site