Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material

Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.     

Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory,Variability, and Virulence

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers.     

Targeting the pattern-triggered immunity pathway to enhance resistance to Fusarium graminearum

Fusarium head blight (FHB) is a disease of the floral tissues of wheat and barley for which highly resistant varieties are not available. Thus, there is a need to identify genes/mechanisms that can be targeted for the control of this devastating disease. Fusarium graminearum is the primary causal agent of FHB in North America. In addition, it also causes Fusarium seedling blight. Fusarium graminearum can also cause disease in the model plant Arabidopsis thaliana. The Arabidopsis–F. graminearum pathosystem has facilitated the identification of targets for the control of disease caused by this fungus. Here, we show that resistance against F. graminearum can be enhanced by flg22, a bacterial microbe-associated molecular pattern (MAMP). flg22-induced resistance in Arabidopsis requires its cognate pattern recognition receptor (PRR) FLS2, and is accompanied by the up-regulation of WRKY29. The expression of WRKY29, which is associated with pattern-triggered immunity (PTI), is also induced in response to F. graminearum infection. Furthermore, WRKY29 is required for basal resistance as well as flg22-induced resistance to F. graminearum. Moreover, constitutive expression of WRKY29 in Arabidopsis enhances disease resistance. The PTI pathway is also activated in response to F. graminearum infection of wheat. Furthermore, flg22 application and ectopic expression of WRKY29 enhance FHB resistance in wheat. Thus, we conclude that the PTI pathway provides a target for the control of FHB in wheat. We further show that the ectopic expression of WRKY29 in wheat results in shorter stature and early heading time, traits that are important to wheat breeding.     

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

Cystathionine gamma-synthase is essential for methionine biosynthesis in Fusarium graminearum

Methionine (Met) plays an important role in various cellular processes in both eukaryotes and prokaryotes. Cystathionine gamma-synthase encoded by STR2 gene is a key enzyme in Met biosynthesis in Saccharomyces cerevisiae. In this study, we identified FgMETB, a homologue of S. cerevisiae STR2, from Fusarium graminearum using the Protein Basic Local Alignment Search Tool (BLASTP) program. The FgMETB deletion mutants were unable to grow on fructose gelatin agar (FGA) medium containing SO₄²⁻ as sole sulphur source. In addition, more than 90 % conidia of the mutants were not able to germinate in 2 % sucrose solution within 6 or 12 h of incubation. Supplementation of 1 mM Met or 0.5 mg ml⁻¹ homocysteine, but not 1 mM cysteine or 0.5 mg ml⁻¹ glutathione, rescued the defect of mycelial growth and spore germination of FgMETB deletion mutants. These results indicated that the enzyme encoded by FgMETB is involved in conversion of cysteine into homocysteine. Inoculation tests showed that the FgMETB deletion mutant exhibited decreased virulence significantly on wheat heads, which is consistent with a low level of deoxynivalenol (DON) production of the mutant in wheat kernels. Fungicide sensitivity assays revealed FgMETB deletion mutants showed increased sensitivity to the sterol demethylation inhibitor tebuconazole, but did not change their sensitivities to other fungicides. Taken together, results of this study indicated that FgMETB plays a critical role in the regulation of various cellular processes in F. graminearum.     

Time‐resolved dissection of the molecular crosstalk driving Fusarium head blight in wheat provides new insights into host susceptibility determinism

Fungal plant diseases are controlled by a complex molecular dialogue that involves pathogen effectors able to manipulate plant susceptibility factors at the earliest stages of the interaction. By probing the wheat–Fusarium graminearum pathosystem, we profiled the coregulations of the fungal and plant proteins shaping the molecular responses of a 96‐hr‐long infection's dynamics. Although no symptoms were yet detectable, fungal biomass swiftly increased along with an extremely diverse set of secreted proteins and candidate effectors supposed to target key plant organelles. Some showed to be early accumulated during the interaction or already present in spores, otherwise stored in germinating spores and detectable in an in vitro F. graminearum exudate. Wheat responses were swiftly set up and were evidenced before any visible symptom. Significant wheat protein abundance changes co‐occurred along with the accumulation of putative secreted fungal proteins and predicted effectors. Regulated wheat proteins were closely connected to basal cellular processes occurring during spikelet ontogeny, and particular coregulation patterns were evidenced between chloroplast proteins and fungal proteins harbouring a predicted chloroplast transit peptide. The described plant and fungal coordinated responses provide a resourceful set of data and expand our understanding of the wheat–F. graminearum interaction.     

The Secreted Antifungal Protein Thionin 2.4 in Arabidopsis thaliana Suppresses the Toxicity of a Fungal Fruit Body Lectin from Fusarium graminearum

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.     

Identification and characterization of a fusarium head blight resistance gene TaACT in wheat QTL‐2DL

Fusarium head blight (FHB) resistance in wheat is considered to be polygenic in nature. Cell wall fortification is one of the best resistance mechanisms in wheat against Fusarium graminearum which causes FHB. Metabolomics approach in our study led to the identification of a wide array of resistance‐related (RR) metabolites, among which hydroxycinnamic acid amides (HCAAs), such as coumaroylagmatine and coumaroylputrescine, were the highest fold change RR metabolites in the rachis of a resistant near‐isogenic line (NIL‐R) upon F. graminearum infection. Placement of these metabolites in the secondary metabolic pathway led to the identification of a gene encoding agmatine coumaroyl transferase, herein referred to as TaACT, as a candidate gene. Based on wheat survey sequence, TaACT was located within a FHB quantitative trait loci on chromosome 2DL (FHB QTL‐2DL) between the flanking markers WMC245 and GWM608. Phylogenetic analysis suggested that TaACT shared closest phylogenetic relationship with an ACT ortholog in barley. Sequence analysis of TaACT in resistant and susceptible NILs, with contrasting levels of resistance to FHB, led to the identification of several single nucleotide polymorphisms (SNPs) and two inversions that may be important for gene function. Further, a role for TaACT in FHB resistance was functionally validated by virus‐induced gene silencing (VIGS) in wheat NIL‐R and based on complementation studies in Arabidopsis with act mutant background. The disease severity, fungal biomass and RR metabolite analysis confirmed TaACT as an important gene in wheat FHB QTL‐2DL, conferring resistance to F. graminearum.     

In Planta Stage-Specific Fungal Gene Profiling Elucidates the Molecular Strategies of Fusarium graminearum Growing inside Wheat Coleoptiles

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall–degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)–related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection.     

Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

Transducin Beta-Like Gene FTL1 Is Essential for Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley. In a previous study, we identified several mutants with reduced virulence by insertional mutagenesis. A transducin beta-like gene named FTL1 was disrupted in one of these nonpathogenic mutants. FTL1 is homologous to Saccharomyces cerevisiae SIF2, which is a component of the Set3 complex involved in late stages of ascospore formation. The Δftl1 mutant was significantly reduced in conidiation and failed to cause typical disease symptoms. It failed to colonize the vascular tissues of rachis or cause necrosis on the rachis of inoculated wheat heads. The Δftl1 mutant also was defective in spreading from infected anthers to ovaries and more sensitive than the wild type to plant defensins MsDef1 and osmotin. However, the activation of two mitogen-activated protein kinases, Mgv1 and Gpmk1, production of deoxynivalenol, and expression of genes known to be important for plant infection in F. graminearum were not affected, indicating that the defect of the Δftl1 mutant in plant infection is unrelated to known virulence factors in this pathogen and may involve novel mechanisms. The Δftl1 deletion mutant was significantly reduced in histone deacetylation, and many members of the yeast Set3 complex are conserved in F. graminearum. FTL1 appears to be a component of this well-conserved protein complex that plays a critical role in the penetration and colonization of wheat tissues.The filamentous ascomycete Fusarium graminearum (teleomorph Gibberella zeae) is the main causal agent of Fusarium head blight (FHB), or scab, which is an important disease on wheat and barley throughout the world (18). It also causes stalk and ear rots of maize and infects other small grains. In addition to causing yield losses, this pathogen often contaminates infested grains with trichothecene and estrogenic mycotoxins, such as deoxynivalenol (DON) and zearalenone. Unfortunately, complete resistance to F. graminearum is lacking in wheat, and fungicide application is not cost-effective for FHB control in wheat and barley.F. graminearum overwinters in infected plant debris and produces ascospores in the spring. Ascospores are forcibly discharged from mature perithecia (52) and function as the primary inoculum for FHB. The multicellular conidia or macroconidia are important for spreading the disease in the field and colonizing plant vegetative tissues. Wheat spikes are most susceptible to FHB at anthesis (34a). Although F. graminearum can colonize glumes, anthers are the main site of primary infection on flowering wheat heads (3, 38). Earlier studies indicated that wheat anther extracts stimulate F. graminearum virulence on wheat. Choline and glycine betaine were identified as two major components in anthers that stimulate fungal growth and predispose wheat to F. graminearum infection (50, 51). Under conducive conditions, the fungus can spread from the infected floret along the rachis and cause severe damage. The production of DON, the first virulence factor identified in F. graminearum (11, 42), is not necessary for the initial infection but is important for the spread of FHB on infected wheat heads (2).In the past few years, genetic and genomic studies of F. graminearum have advanced significantly. The genome of F. graminearum has been sequenced (10) and a whole-genome microarray of this haploid homothallic fungus is commercially available (21). A number of pathogenicity or virulence factors have been identified by insertional mutagenesis or targeted gene deletion approaches. Two mitogen-activated protein (MAP) kinase genes, MGV1 and GPMK1, are essential for pathogenicity in F. graminearum (23, 24). Genes that are important for full virulence in F. graminearum on wheat include FGL1 (54), GzCPS1 (31), FBP1 (22), FSR1 (48), SID1 (19), NPS6 (37), RAS2 (5), GzGPA2 and GzGPB1 (56), and HMR1 (47). These virulence-associated genes encode proteins with various biochemical activities, such as lipase, nonribosomal peptide synthase, Ras protein, and 3-hydroxy 3-methylglutaryl coenzyme A reductase. Several genes involved in the primary metabolism, such as the CBL1, RSY1, GzHIS7, ADE5, and ARG2 genes (29, 44, 46) that are required for methionine, histidine, and arginine syntheses, also have been implicated in plant infection in F. graminearum. Overall, molecular mechanisms underlying F. graminearum pathogenesis appear to be complex and remain to be fully understood.In a previous study, we identified 11 restriction enzyme-mediated integration (REMI) mutants that are defective in plant infection (46). In one of these mutants, the transforming vector was inserted in a predicted gene named FTL1 (for Fusarium transducin beta-like gene 1). FTL1 is homologous to the mammalian TBL1 or TBLR1 genes (40, 55) and the Saccharomyces cerevisiae SIF2 gene (8). The products of these genes are components of protein complexes involving histone deacetylases (HDACs). In mammalian cells, TBL1 and TBLR1 are parts of the N-CoR/SMRT/HDAC complexes (40). In yeast, SIF2 is a part of the Set3 complex regulating ascospore formation. In F. graminearum, the Δftl1 gene replacement mutant was significantly reduced in conidiation and failed to cause typical head blight symptoms on flowering wheat heads. It failed to colonize vascular tissues or cause necrosis on the rachis of inoculated wheat heads. The Δftl1 mutant also was defective in spreading from infected anthers to ovaries and was more sensitive than the wild type to plant defensins MsDef1 and osmotin. Although it was normal in the production of deoxynivalenol and the expression of known virulence factors, the Δftl1 mutant was significantly reduced in HDAC activities. FTL1 appears to be a component of this well-conserved HDAC complex that plays a critical role in the penetration and colonization of wheat tissues.     

Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides

Plant peptide hormones are important players that control various aspects of the lives of plants. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies have increased our understanding of the evolutionary processes that shaped the RALF family of peptides. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analyzed the phylogeny of the proposed multigenic RALF peptide family in the model plant Arabidopsis (Arabidopsis thaliana), ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a revision to the consensus AtRALF peptide family annotation. We show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate reactive oxygen species (ROS) production in Arabidopsis. Moreover, our findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Additionally, we show that for the majority of the peptides, these responses are genetically dependent on FERONIA, suggesting a pivotal role for this receptor kinase in the perception of multiple RALF peptides.

Synthetic RAPID ALKALINIZATION FACTOR peptides induced a variety of physiological responses, many of which depend on FERONIA, a receptor kinase with functions in development and defense