Transcriptomics and experimental proof that compound appressoria are arsenals of Fusarium graminearum

Fusarium graminearum is one of the most destructive plant pathogens. Its infection of cereals causes significant losses due to yield reduction and contamination with mycotoxins, e.g. deoxynivalenol (DON). F. graminearum infects the floral leaf by defined structures: runner hyphae are formed for colonization of the floral surface and compound appressoria, e.g. infection cushions, are developed to facilitate multiple penetrations of the plant cell wall. In this study, we created und compared RNAseq data sets of three different fungal cell types, i.e. infection cushions and runner hyphae (isolated via laser capture microdissection from the surface of paleae), and in culture grown mycelium. Validation of the data by qRT-PCR showed similar gene regulation patterns. The analysis showed that 3916 genes (about one-third of all genes) are specifically regulated during the infection of the plant compared to mycelium. In total 653 genes are exclusively transcribed: 235 in mycelium, 77 in runner hyphae and 341 in infection cushions. In particular, we show that 1. in runner hyphae, the highly expressed fungal pigment aurofusarin acts as an antibiotic agent against bacteria and fungi, 2. infection cushions are arsenals of virulence factors, harboring plant cell wall degrading enzymes, specific metabolites like DON, and putative effector proteins, 3. the putative effector protein FgEF1 is localized in the plant cell after colonization by F. graminearum

The bZIP Transcription Factor Fgap1 Mediates Oxidative Stress Response and Trichothecene Biosynthesis But Not Virulence in <i>Fusarium graminearum</i>

<div><p>Redox sensing is of primary importance for fungi to cope with oxidant compounds found in their environment. Plant pathogens are particularly subject to the oxidative burst during the primary steps of infection. In the budding yeast <i>Saccharomyces cerevisiae</i>, it is the transcription factor Yap1 that mediates the response to oxidative stress via activation of genes coding for detoxification enzymes. In the cereal pathogen <i>Fusarium graminearum</i>, Fgap1 a homologue of Yap1 was identified and its role was investigated. During infection, this pathogen produces mycotoxins belonging to the trichothecenes family that accumulate in the grains. The global regulation of toxin biosynthesis is not completely understood. However, it is now clearly established that an oxidative stress activates the production of toxins by <i>F. graminearum</i>. The involvement of <i>Fgap1</i> in this activation was investigated. A deleted mutant and a strain expressing a truncated constitutive form of <i>Fgap1</i> were constructed. None of the mutants was affected in pathogenicity. The deleted mutant showed higher level of trichothecenes production associated with overexpression of <i>Tri</i> genes. Moreover activation of toxin accumulation in response to oxidative stress was no longer observed. Regarding the mutant with the truncated constitutive form of <i>Fgap1</i>, toxin production was strongly reduced. Expression of oxidative stress response genes was not activated in the deleted mutant and expression of the gene encoding the mitochondrial superoxide dismutase MnSOD1 was up-regulated in the mutant with the truncated constitutive form of <i>Fgap1</i>. Our results demonstrate that <i>Fgap1</i> plays a key role in the link between oxidative stress response and <i>F. graminearum</i> secondary metabolism.</p> </div

Get ready for infection: Transcriptional profiling reveals virulence-specific traits inside of infection cushions of Fusarium graminearum

International audienceThe fungal pathogen Fusarium graminearum forms specialized infection cushions (ICs) essential for penetration of wheat floral-leaf cells. To understand the molecular basis of IC development, ICs and non-invasive runner hyphae (RH) were isolated by laser capture microdissection and subjected to RNAseq. Quantitative expression analysis show marked differences in gene expression patterns between RH and ICs:1. The majority of known and putative secondary metabolite gene clusters, including those responsible for trichothecene and butenolide production, are significantly up-regulated in ICs, 2. Carbohydrate-modifying enzymes (CAZymes) with proven capacities for cell-wall degradation are exclusively present in ICs. In total, 174 genes encoding for CAZymes are differentially expressed (42 in RH, 132 in ICs), 3. Genes encoding for enzymes involved in reactive-oxygen species metabolism reside in the upper ranks of differentially expressed genes (DEGs). Secreted ROS-related enzymes (SREs), presumably involved in plant-defense response, are relatively enriched in ICs, 4. We identified a large subset of transcripts encoding for putative effector proteins. By use of this novel transcriptional profiling of runner hyphae and infection cushions from a fungal plant pathogen obtained under in planta conditions, we gain new insights in the initial infection process of F. graminearum on wheat. Complementary to this approach, functional characterization of genes and histological analyses are ongoing. First results will be presented. We conclude that infection cushions serve as an armory of virulence factors

Get ready for infection: Transcriptional profiling reveals virulence-specific traits inside of infection cushions of Fusarium graminearum

International audienceThe fungal pathogen Fusarium graminearum forms specialized infection cushions (ICs) essential for penetration of wheat floral-leaf cells. To understand the molecular basis of IC development, ICs and non-invasive runner hyphae (RH) were isolated by laser capture microdissection and subjected to RNAseq. Quantitative expression analysis show marked differences in gene expression patterns between RH and ICs:1. The majority of known and putative secondary metabolite gene clusters, including those responsible for trichothecene and butenolide production, are significantly up-regulated in ICs, 2. Carbohydrate-modifying enzymes (CAZymes) with proven capacities for cell-wall degradation are exclusively present in ICs. In total, 174 genes encoding for CAZymes are differentially expressed (42 in RH, 132 in ICs), 3. Genes encoding for enzymes involved in reactive-oxygen species metabolism reside in the upper ranks of differentially expressed genes (DEGs). Secreted ROS-related enzymes (SREs), presumably involved in plant-defense response, are relatively enriched in ICs, 4. We identified a large subset of transcripts encoding for putative effector proteins. By use of this novel transcriptional profiling of runner hyphae and infection cushions from a fungal plant pathogen obtained under in planta conditions, we gain new insights in the initial infection process of F. graminearum on wheat. Complementary to this approach, functional characterization of genes and histological analyses are ongoing. First results will be presented. We conclude that infection cushions serve as an armory of virulence factors

Expression of genes encoding antioxidant activities in the wild-type strain and in the <i>ΔFgap1</i> and <i>Fgap1</i>^c_trunc mutants.

<p>(A) Antioxidant genes expression (expression ratio treated/not treated in log2 scale) in the wild-type strain in 5-day old cultures. The star indicates a significant difference between treated <i>vs</i>. not treated (p<0.05). ND stands for not detected. (B) Antioxidant genes expression in the mutants strain compared to wild-type strain (expression ratios mutant/wild-type strain in log2 scale) in 5-day old cultures. The star indicates a significant difference for mutant compared to wild-type (p<0.05).</p

<i>Fgap1</i> plays a role in toxin accumulation and <i>Tri</i> genes expression.

<p>(A) DON + 15ADON yields (in ng of toxins produced per mg of dry fungal biomass) in the wild-type and the mutant strains after 3, 5 and 14 days of growth, ND stands for not detected. Error bars represent the standard deviation of three biological replicates. (B) Expression ratio mutant/wild-type (in log2 ratio scale) for <i>Tri</i> genes after 5 days of culture. The star indicates a significant difference compared to the wild-type (p<0.05).</p

Toxin production and <i>Tri</i> genes expression in the wild-type strain and in the <i>ΔFgap1</i> and <i>Fgap1</i>^c_trunc mutants after exposure to oxidative stress by H₂O₂.

<p><i>F. graminearum</i> wild-type or mutants were cultured up to 14 days in liquid medium supplemented or not supplemented with 0.5 mM H₂O₂. (A) DON + 15ADON yield (ng of toxins per mg of dry fungal biomass) after 3, 5 or 14 days of growth. Error bars represent the standard deviation of three biological replicates. The star indicates a significant difference for the treated condition compared to the control condition (p<0.05). (B) <i>Tri</i> genes expression (expression ratios treated/not treated in log2 scale) in 5 day-old cultures. <i>Tri4, Tri5, Tri6, Tri10, Tri12</i>, and <i>Tri101</i> were considered. The star indicates a significant difference of expression between treated <i>vs</i>. not treated (p<0.05).</p

Wheat ears inoculated with <i>F. graminearum</i> wild-type strain, <i>ΔFgap1, ΔFgap1:ap1</i>, and <i>Fgap1</i>^c_trunc.

<p>Wheat ears 21 days after point inoculation of two central spikelets. Bleaching indicates successful infection. Infection assays were performed with 10 replicates for each strain. For each strain, calculated disease index (% of infected spikelets) was: WT = 90.2 ± 25.6, <i>ΔFgap1</i> = 94.2 ± 11.1, <i>ΔFgap1:ap1</i> = 90.0 ± 22.4, <i>Fgap1</i>^<i>c</i>_trunc = 77.9 ± 25.2. </p

Radial growth assay to evaluate stress tolerance in <i>F. graminearum</i> wild-type strain and mutants.

<p><i>F. graminearum</i> wild-type strain, <i>ΔFgap1</i>, <i>ΔFgap1:ap1</i>, and <i>Fgap1</i>^<i>c</i>_<i>trunc</i> were grown 7 days under oxidative stress with cadmium chloride 150 µM (C_dCl₂) or H₂O₂ (15 mM), osmotic stress with sorbitol (1 M) or NaCl (1 M). </p