Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome

Abstract

Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen's effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets.

Keywords: Fusarium graminearum; Triticum aestivum; effectoromics; in planta; plant–fungus interaction; susceptibility factors; transcriptomics.

Figure 1

In planta expression signature of F. graminearum genes coding for putative secreted proteins. Barplots represent the structure of the gene sets coding for putative secreted proteins expressed in planta for the HostV (A) and the PathoV (B) experiments. HostV barplot displays the number of genes expressed by the strain MDC_Fg1 in all the infected hosts, in some hosts (Accessory) and in only a specific host: ‘Arche’ (ARC) specific, ‘Courtot’ (COU) specific, ‘Chinese Spring’ (CS) specific, ‘Recital’ (REC) specific or ‘Renan’ (REN) specific. PathoV barplot displays the number of genes expressed by the three strains MDC_Fg1, MDC_Fg13 and MDC_FgU1, by two strains only (Accessory) and by only one strain (MDC_Fg1 specific, MDC_Fg13 specific, MDC_FgU1 specific). The Venn diagram (C) represents the intersection of the gene sets expressed in all the hosts (HostV) and expressed in all the strains (PathoV).

Figure 2

Number of secretome genes from the HostV (A) and the PathoV (B) experiments significantly impacted by the different effects tested in the differential expression (DE) analysis. For each factor of the DE analysis, the Venn diagrams indicate the number of genes displaying significant expression variations. Significance threshold: p-value corrected by Benjamini–Hochberg method < 0.001.

Figure 3

Structure of the HostV (A) and PathoV (B) effectome gene sets. These sets gather the genes significantly regulated along infection progress and coding for putative secreted proteins. (A) The flower plot displays the number of genes expressed by the strain MDC_Fg1 in all the infected hosts (center circle), in some hosts (annulus) and in only a specific host (petals). (B) The flower plot displays the number of genes expressed by the three strains MDC_Fg1, MDC_Fg13 and MDC_FgU1 (center circle), by two strains only (annulus) and by only one strain (petals).

Figure 4

Discrimination of HostV (A) and PathoV (B) effectome gene sets expressed in all the hosts or by all the strains according to the experimental conditions. PLS-DA method was applied on the 433 genes expressed in all the hosts for HostV (A) to predict the host–infection progress combinations and on the 682 genes expressed by all the strains for PathoV (B) to predict the strain–infection progress combinations. The plots of the individuals extracted from the PLS-DA are represented on the two first components. For each condition, confidence ellipses are plotted to highlight discrimination strength (level set to 95%).

Figure 5

Expression regulation patterns of the core effectome genes along with the infection progress in HostV (A) and PathoV (B) data sets. The structure of gene and sample data sets were determined by HAC based on Ward’s minimum variance method using the z-score transformed gene expression values. Heatmap color scales represent the z-score transformed expression values of the genes from the core effectome gene set for each sample. The clustering on top of the heatmap represents the experimental conditions which are labeled according to the factors Infection Progress and Host for the HostV experiment, and Infection Progress and Strain for the PathoV experiment. The clustering on the right side of the heatmap represents the genes, which are colored according to their cluster membership.

Figure 6

Localization within the host of the fungal proteins encoded by the core effectome gene set according to the expression timing during the infection progress. Barplots represent the frequencies of the predicted localizations within the host of the proteins coded by F. graminearum genes expressed at the early stages of the infection (Early Expression), at intermediate stages of infection (Intermediate Expression), at latter stages of infection (Late Expression) in both HostV and PathoV experiments or expressed with different dynamics between the HostV and PathoV experiments.

Figure 7

Model summarizing the conserved and complex F. graminearum infection strategy on wheat spikes. As a whole, 357 effector genes were identified as the key drivers of FHB infection expressed by all the strains and in all the infected hosts; they represent the F. graminearum core effectome. These genes were expressed at very specific infection stages in a per-wave manner, including genes highly expressed at the very beginning of the interaction with the wheat tissues and others highly expressed in the later stages of the infection. The timing of gene expression was mostly conserved independently of the strain or the host. Targeted processes within the host are highly diverse with a wide array of targeted compartments and predicted functions.