Comparative Genomics of Eight Fusarium graminearum Strains with Contrasting Aggressiveness Reveals an Expanded Open Pangenome and Extended Effector Content Signatures

Abstract

Fusarium graminearum, the primary cause of Fusarium head blight (FHB) in small-grain cereals, demonstrates remarkably variable levels of aggressiveness in its host, producing different infection dynamics and contrasted symptom severity. While the secreted proteins, including effectors, are thought to be one of the essential components of aggressiveness, our knowledge of the intra-species genomic diversity of F. graminearum is still limited. In this work, we sequenced eight European F. graminearum strains of contrasting aggressiveness to characterize their respective genome structure, their gene content and to delineate their specificities. By combining the available sequences of 12 other F. graminearum strains, we outlined a reference pangenome that expands the repertoire of the known genes in the reference PH-1 genome by 32%, including nearly 21,000 non-redundant sequences and gathering a common base of 9250 conserved core-genes. More than 1000 genes with high non-synonymous mutation rates may be under diverse selection, especially regarding the trichothecene biosynthesis gene cluster. About 900 secreted protein clusters (SPCs) have been described. Mostly localized in the fast sub-genome of F. graminearum supposed to evolve rapidly to promote adaptation and rapid responses to the host's infection, these SPCs gather a range of putative proteinaceous effectors systematically found in the core secretome, with the chloroplast and the plant nucleus as the main predicted targets in the host cell. This work describes new knowledge on the intra-species diversity in F. graminearum and emphasizes putative determinants of aggressiveness, providing a wealth of new candidate genes potentially involved in the Fusarium head blight disease.

Keywords: Fusarium graminearum; Fusarium head blight; aggressiveness; intra-species genomic diversity; pangenome; proteinaceous effectors; secreted protein clusters.

Figure 1

Structure and function of the F. graminearum pangenome. (A) The flower plot displays the number of core proteins (in the center) and accessory proteins (in the annulus), including the singletons (in the petals), encoded by the genome of 20 F. graminearum strains. (B) The F. graminearum pangenome accumulation curve is depicted with turquoise boxplots, indicating the number of accessory proteins discovered with the sequential addition of new genomes. The purple boxplots illustrate the number of core proteins discovered with the sequential addition of new genomes. (C) The density plot depicts the functional enrichment of the core and accessory F. graminearum genomes. Enriched GO terms belonging to biological processes, molecular functions and cellular components are presented. The color of the circle represents the statistical significance of the enriched GO terms. The size of the circles represents the number of occurrences of the corresponding GO term.

Figure 2

Phylogenetic relationship of 20 F. graminearum strains from different geographic origins. A Maximum likelihood bootstrapped cladogram was generated from 7640 single-copy protein sequences found in the 20 F. graminearum strains and from 3 other species of the Fusarium genus used as an outgroup (F. pseudograminearum, F. culmorum and F. Fujikuroi). Numbers at nodes indicate a bootstrap value greater than 70%, performed with 1000 replications. The country of origin is shown on the right. Names of the 8 strains characterized in this study are shown in red.

Figure 3

Genome-wide genetic variant distributions. (A) Pie charts represent the percentage distribution of the effects of mutations in different genomic regions (inter- and intra-genic), including different types of identified mutations in coding sequences. All the effects expected from the mutations identified in the eight F. graminearum genomes, relative to the PH-1 reference sequence, are combined. (B) Count distribution of synonymous and non-synonymous mutations per gene is depicted for the eight F. graminearum strains. The colored dots correspond to each of the eight genomes. (C) Density and localization of genes with a high rate of non-synonymous mutations are depicted along the four F. graminearum chromosomes. Only genes with at least six non-synonymous mutations and exhibiting a non-synonymous/(synonymous + non-synonymous) ratio greater than 0.55 were included. The size of the chromosomes is in Mb, in 2 Mb increments.

Figure 4

Abundances of selected metabolites in the eight F. graminearum strains. Ion abundances were derived from extracted ion chromatography (XIC) profiles obtained from mass spectrometry analysis (LC/MS) of the eight strains grown on PDA medium. Different detected ions corresponding to trichothecene biosynthesis (see Supplementary Figure S2 for details, mass-to-charge ratio (m/z): 163.30, 203.30, 203.42, 203.18 235.13, 235.17, 221.30, 221.41, 237.30), fusarin isoforms (retention time 19.8 min and m/z values of 396.31 for M-2xH2O+H, 414.24 for M-H2O+H, 432.27 for M + H), zearalenone and its intermediates (retention times 21.1, 23.5 and 34.3 min with m/z values of 319.20, 337.10, 367.34, respectively), Butenolide (retention time 2.9 min with m/z value of 164.17) and Fusaristatin A (retention time 27.9 min and m/z value of 659.47) are reported.

Figure 5

Symptom severity produced by the eight F. graminearum strains in bread wheat spikes during the FHB disease progress. The eight strains of F. graminearum were inoculated on the Recital wheat cultivar and symptoms were monitored at 72 hpi according the scoring described by Fabre et al. [56]. The Y-axis represents the symptom severity scale ranging from 0 to a maximum of 4. The error bars are the means ± SE of five biological replicates, and each replicate was characterized by the average value (represented as black dots) computed from six spikelets of three inoculated spikes. The different letters indicate significant differences at p ≤ 0.05 (Duncan’s multiple range test for post-hoc ANOVA mean separation).

Figure 6

Characterization of the F. graminearum pan-secretome. (A) Categorization of putative secreted protein clusters (SPCs) of the eight F. graminearum strains into core, accessory and singleton secreted proteins. In each of the three categories, the percentage of SPCs assigned to the fast and slow sub-genome is reported. (B) The bar graph depicts the number of SPCs encoding CAZymes, proteases and putative proteinaceous effectors (according to EffectorP v2.0) in each of the three pan-secretome categories. (C) The Venn diagram depicts the number of SPCs displaying one or more labels (among CAZyme, protease or effector), as well as those with no predicted functions. (D) Density and localization of the genes encoding secreted CAZymes, secreted proteases, secreted effectors and genes potentially involved in the host–pathogen interaction (according to PHI-base) along the four F. graminearum chromosomes. The size of the chromosomes is in Mb, in 2 Mb increments.

Figure 7

Expected subcellular targeting of SPCs in the plant cell. Bar graphs represent the predicted subcellular localization of putative secreted proteins (light gray), including the secreted putative effectors (dark gray). The pipeline used for the prediction of the secretion characteristics is described in Supplementary Figure S3.