Fusarium graminearum SnodProt proteins protect fungal cell wall polysaccharides from enzymatic degradation

The genome of Fusarium graminearum, a necrotrophic fungal pathogen causing Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, contains five genes putatively encoding for proteins similar to cerato platanins, which contribute to the virulence of the fungal pathogens Botrytis cinerea and Magnaporthe grisea. Two of them (FGSG_10212 and FGSG_11205) belong to the class of SnodProt proteins, with reported phytotoxic activity. To verify the contribution of the two F. graminearum SnodProt proteins to the infection process, single and double gene knock-out mutants were produced but no reduction in symptoms severity was observed compared to the wild type strain on both soybean and wheat spikes. Histological analysis performed by fluorescence microscopy on wheat spikelets infected with mutants constitutively expressing the dsRed confirmed that the F. graminearum SnodProt proteins do not contribute to fungal virulence. In particular, the formation of compound appressoria on wheat glumes was unchanged. Looking for other functions of these proteins, the double mutant was characterized by in vitro experiments. Wild type and mutants were similarly inhibited by salt and H2O2 stress. Surprisingly, the mutant grew better than wild type on carboxymethyl cellulose, while no difference was observed on glucose. Furthermore, conidia and mycelium of the mutant were more affected by treatments with chitinase and \u3b2-1,3-glucanase, thus indicating that the F. graminearum SnodProt proteins could protect fungal cell wall polysaccharides from enzymatic degradation

Involvement of the Fusarium graminearum cerato-platanin proteins in fungal growth and plant infection

The genome of Fusarium graminearum, a necrotrophic fungal pathogen causing Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, contains five genes putatively encoding for proteins with a cerato-platanin domain. Cerato-platanins are small secreted cysteine-rich proteins possibly localized in the fungal cell walls and also contributing to the virulence. Two of these F. graminearum proteins (FgCPP1 and FgCPP2) belong to the class of SnodProt proteins which exhibit phytotoxic activity in the fungal pathogens Botrytis cinerea and Magnaporthe grisea. In order to verify their contribution during plant infection and fungal growth, single and double gene knock-out mutants were produced and no reduction in symptoms severity was observed compared to the wild type strain on both soybean and wheat spikes. Histological analysis performed by fluorescence microscopy onwheat spikelets infected with mutants constitutively expressing the dsRed confirmed that FgCPPs do not contribute to fungal virulence. In particular, the formation of compound appressoria on wheat paleas was unchanged. Looking for other functions of these proteins, the double mutant was characterized by in vitro experiments. The mutant was inhibited by salt and H2O2 stress similarly to wild type. Though no growth difference was observed on glucose, the mutant grew better than wild type on carboxymethyl cellulose. Additionally, the mutant's mycelium was more affected by treatments with chitinase and b-1,3- glucanase, thus indicating that FgCPPs could protect fungal cell wall polysaccharides from enzymatic degradation

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

Infection cushions of Fusarium graminearum are fungal arsenals for wheat infection

International audienceFusarium graminearum is one of the most destructive plant pathogens worldwide, causing fusarium head blight (FHB) on cereals. F. graminearum colonizes wheat plant surfaces with specialized unbranched hyphae called runner hyphae (RH), which develop multicelled complex appressoria called infection cushions (IC). IC generate multiple penetration sites, allowing the fungus to enter the plant cuticle. Complex infection structures are typical for several economically important plant pathogens, yet with unknown molecular basis. In this study, RH and IC formed on the surface of wheat paleae were isolated by laser capture microdissection. RNA-Seq-based transcriptomic analyses were performed on RH and IC and compared to mycelium grown in complete medium (MY). Both RH and IC displayed a high number of infection up-regulated genes (982), encoding, among others, carbohydrate-active enzymes (CAZymes: 140), putative effectors (PE: 88), or secondary metabolism gene clusters (SMC: 12 of 67 clusters). RH specifically up-regulated one SMC corresponding to aurofusarin biosynthesis, a broad activity antibiotic. IC specifically up-regulated 248 genes encoding mostly putative virulence factors such as 7 SMC, including the mycotoxin deoxynivalenol and the newly identified fusaoctaxin A, 33 PE, and 42 CAZymes. Furthermore, we studied selected candidate virulence factors using cellular biology and reverse genetics. Hence, our results demonstrate that IC accumulate an arsenal of proven and putative virulence factors to facilitate the invasion of epidermal cells. K E Y W O R D S effector proteins, Fusarium graminearum, infection cushion, runner hyphae, secondary metabolites, transcriptome, wheat infectio