Fusarium graminearum forms mycotoxin producing infection structures on wheat

<p>Abstract</p> <p>Background</p> <p>The mycotoxin producing fungal pathogen <it>Fusarium graminearum </it>is the causal agent of Fusarium head blight (FHB) of small grain cereals in fields worldwide. Although <it>F. graminearum </it>is highly investigated by means of molecular genetics, detailed studies about hyphal development during initial infection stages are rare. In addition, the role of mycotoxins during initial infection stages of FHB is still unknown. Therefore, we investigated the infection strategy of the fungus on different floral organs of wheat (<it>Triticum aestivum </it>L.) under real time conditions by constitutive expression of the <it>dsRed </it>reporter gene in a <it>TRI5prom</it>::<it>GFP </it>mutant. Additionally, trichothecene induction during infection was visualised with a green fluorescent protein (GFP) coupled <it>TRI5 </it>promoter. A tissue specific infection pattern and <it>TRI5 </it>induction were tested by using different floral organs of wheat. Through combination of bioimaging and electron microscopy infection structures were identified and characterised. In addition, the role of trichothecene production for initial infection was elucidated by a Δ<it>TRI5</it>-<it>GFP </it>reporter strain.</p> <p>Results</p> <p>The present investigation demonstrates the formation of foot structures and compound appressoria by <it>F. graminearum</it>. All infection structures developed from epiphytic runner hyphae. Compound appressoria including lobate appressoria and infection cushions were observed on inoculated caryopses, paleas, lemmas, and glumes of susceptible and resistant wheat cultivars. A specific trichothecene induction in infection structures was demonstrated by different imaging techniques. Interestingly, a Δ<it>TRI5</it>-<it>GFP </it>mutant formed the same infection structures and exhibited a similar symptom development compared to the wild type and the <it>TRI5prom</it>::<it>GFP </it>mutant.</p> <p>Conclusions</p> <p>The different specialised infection structures of <it>F. graminearum </it>on wheat florets, as described in this study, indicate that the penetration strategy of this fungus is far more complex than postulated to date. We show that trichothecene biosynthesis is specifically induced in infection structures, but is neither necessary for their development nor for formation of primary symptoms on wheat.</p

Nanoscale enrichment of the cytosolic enzyme trichodiene synthase near reorganized endoplasmic reticulum in Fusarium graminearum

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms

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

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