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.     

Capping proteins regulate fungal development,DON-toxisome formation and virulence in Fusarium graminearum

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.     

Evaluation of Fusarium head blight in barley infected by Fusarium graminearum

Fusarium head blight, which is primarily caused by Fusarium graminearum, is a devastating disease in the barley field. A real-time PCR protocol was developed to evaluate the growth of this pathogen in the host plant tissues. All four strains harbored the gene encoding ATP-BINDING CASSETTE TRANSPORTER (FgABC; FGSG_00541) as a single copy within their genomes. Our Southern blot result was identical with the genomic data for F. graminearum strain PH-1. Based on the crossing point (CP) values obtained in our TaqMan real-time PCR analysis, two standard curves describing the relationship among the CP value, FgABC copy number, and amount of fungal DNA were constructed. Chronological enumeration of fungal growth was coincided with the symptom development.     

The ADP-ribosylation factor-like small GTPase FgArl1 participates in growth,pathogenicity and DON production in Fusarium graminearum

Fusarium graminearum is the main pathogen of Fusarium head blight (FHB) in wheat and related species, which causes serious production decreases and economic losses and produces toxins such as deoxynivalenol (DON), which endangers the health of humans and livestock. Vesicle transport is a basic physiological process required for cell survival in eukaryotes. Many regulators of vesicle transport are reported to be involved in the pathogenicity of fungi. In yeast and mammalian cells, the ADP-ribosylation factor-like small GTPase Arl1 and its orthologs are involved in regulating vesicular trafficking, cytoskeletal reorganization and other significant biological processes. However, the role of Arl1 in F. graminearum is not well understood. In this study, we characterized the Arl1-homologous protein FgArl1 in F. graminearum and showed that FgArl1 is located in the trans-Golgi apparatus. The deletion of FgARL1 resulted in a significant decrease in vegetative growth and pathogenicity. Further analyses of the ΔFgarl1 mutant revealed defects in the production of DON. Taken together, these results indicate that FgArl1 is important in the development and pathogenicity of F. graminearum.     

The FgNot3 Subunit of the Ccr4-Not Complex Regulates Vegetative Growth,Sporulation, and Virulence in Fusarium graminearum

The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of FgNOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper-branching. The ΔFgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development.     

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.     

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

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.     

Comparison of Canadian Fusarium graminearum isolates for aggressiveness,vegetative compatibility,and production of ergosterol and mycotoxins

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum

Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5′-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563-372 bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.     

Sharing a Host Plant (Wheat [Triticum aestivum]) Increases the Fitness of Fusarium graminearum and the Severity of Fusarium Head Blight but Reduces the Fitness of Grain Aphids (Sitobion avenae)

We hypothesized that interactions between fusarium head blight-causing pathogens and herbivores are likely to occur because they share wheat as a host plant. Our aim was to investigate the interactions between the grain aphid, Sitobion avenae, and Fusarium graminearum on wheat ears and the role that host volatile chemicals play in mediating interactions. Wheat ears were treated with aphids and F. graminearum inoculum, together or separately, and disease progress was monitored by visual assessment and by quantification of pathogen DNA and mycotoxins. Plants exposed to both aphids and F. graminearum inoculum showed accelerated disease progression, with a 2-fold increase in disease severity and 5-fold increase in mycotoxin accumulation over those of plants treated only with F. graminearum. Furthermore, the longer the period of aphid colonization of the host prior to inoculation with F. graminearum, the greater the amount of pathogen DNA that accumulated. Headspace samples of plant volatiles were collected for use in aphid olfactometer assays and were analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-coupled electroantennography. Disease-induced plant volatiles were repellent to aphids, and 2-pentadecanone was the key semiochemical underpinning the repellent effect. We measured aphid survival and fecundity on infected wheat ears and found that both were markedly reduced on infected ears. Thus, interactions between F. graminearum and grain aphids on wheat ears benefit the pathogen at the expense of the pest. Our findings have important consequences for disease epidemiology, because we show increased spread and development of host disease, together with greater disease severity and greater accumulation of pathogen DNA and mycotoxin, when aphids are present.     

Double-Stranded RNA Mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.