Recent advances in genes involved in secondary metabolite synthesis,hyphal development,energy metabolism and pathogenicity in Fusarium graminearum (teleomorph Gibberella zeae)

The ascomycete fungus, Fusarium graminearum (teleomorph Gibberella zeae), is the most common causal agent of Fusarium head blight (FHB), a devastating disease for cereal crops worldwide. F. graminearum produces ascospores (sexual spores) and conidia (asexual spores), which can serve as disease inocula of FHB. Meanwhile, Fusarium-infected grains are often contaminated with mycotoxins such as trichothecenes (TRIs), fumonisins, and zearalenones, among which TRIs are related to the pathogenicity of F. graminearum, and these toxins are hazardous to humans and livestock. In recent years, with the complete genome sequencing of F. graminearum, an increasing number of functional genes involved in the production of secondary metabolites, hyphal differentiation, sexual and asexual reproduction, virulence and pathogenicity have been identified from F. graminearum. In this review, the secondary metabolite synthesis, hyphal development and pathogenicity related genes in F. graminearum were thoroughly summarized, and the genes associated with secondary metabolites, sexual reproduction, energy metabolism, and pathogenicity were highlighted.     

Significance of the class II hydrophobin FgHyd5p for the life cycle of Fusarium graminearum

Hydrophobins are small secreted proteins ubiquitously found in filamentous fungi. Some hydrophobins were shown to have functions in fungal development, while others lack known function. Class II hydrophobins from Fusarium graminearum and Fusarium culmorum are characterized by formation of low stability aggregates and their solubility in organic solvents. They are economically relevant to the brewing industry because they can induce beer gushing. Since cellular functions of Hyd5p's are still unknown, we analyzed the influence of FgHyd5p on growth and morphology of F. graminearum using FgΔhyd5 knock-out mutants expressing sGFP under the control of the hyd5 promoter and compared them with the performance of the parent wild type strain. Results demonstrate that FgHyd5p does not affect the colony and hyphal morphology. FgHyd5p affects the hydrophobicity of aerial mycelia but had no obvious function in penetration of hyphae through the water air interface. The hydrophobin affects the morphology of conidia, but not their fitness. Different sources of carbon and nitrogen as well as different pH have no effect on the expression of the hyd5 gene, which was demonstrated to be expressed upon growth of F. graminearum on hydrophobic surfaces.     

Analysis of simple sequence repeats (SSRs)dynamics in fungus Fusarium graminearum

The abundance and inherent potential for variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. We describe the organization and abundance of SSRs in fungus Fusarium graminearum (causative agent for Fusarium head blight or head scab of wheat). We identified 1705 SSRs of various nucleotide repeat motifs in the sequence database of F. graminearum. It is observed that mononucleotide repeats (62%) were most abundant followed by di- (20%) and trinucleotide repeats (14%). It is noted that tetra-, penta- and hexanucleotide repeats accounted for only 4% of SSRs. The estimated frequency of Class I SSRs (perfect repeats ≥20 nucleotides) was one SSR per 124.5 kb, whereas the frequency of Class II (perfect repeats >10 nucleotides and ≫20 nucleotides) was one SSR per 25.6 kb. The dynamics of SSRs will be a powerful tool for taxonomic, phylogenetic, genome mapping and population genetic studies as SSR based markers show high levels of allelic variation, codominant inheritance and ease of analysis.     

Relocation of genes generates non-conserved chromosomal segments in Fusarium graminearum that show distinct and co-regulated gene expression patterns

Background

Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of Fusarium graminearum to elucidate the significance of non-conserved regions.

Results

The genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster.

Conclusions

Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-191) contains supplementary material, which is available to authorized users.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

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.     

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.     

Viability of Heterodera glycines Exposed to Fungal Filtrates

Filtrates from nematode-parasitic fungi have been reported to be toxic to plant-parasitic nematodes. Our objective was to determine the effects of fungal filtrates on second-stage juveniles and eggs of Heterodera glycines. Eleven fungal species that were isolated from cysts extracted from a soybean field in Florida were tested on J2, and five species were tested on eggs in vitro. Each fungal species was grown in Czapek-Dox broth and malt extract broth. No toxic activity was observed for fungi grown in Czapek-Dox broth. Filtrates from Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, and Fusarium solani grown in malt extract broth were toxic to J2, whereas filtrates from Exophiala pisciphila, Fusarium oxysporum, Gliocladium catenulatum, Pyrenochaeta terrestris, Verticillium chlamydosporium, and sterile fungi 1 and 2 were not toxic to J2. Filtrates of P. lilacinus, S. heteroderae, and N. vasinfecta grown in malt extract broth reduced egg viability, whereas F. oxysporum and P. terrestris filtrates had no effect on egg viability.     

The Fusarium Graminearum virulence factor FGL targets an FKBP12 immunophilin of wheat

Wheat scab, caused by the fungal pathogen Fusarium graminearum is a devastating disease worldwide. Despite an extensive and coordinated effort to investigate this pathosystem, little progress has been made to understand the molecular basis of host–pathogen interactions, for example how the pathogen causes disease in plant. Recently, a secreted lipase (FGL1) has been identified from the fungus and shown to be an important virulence factor; however, the intrinsic function of FGL1 in plant is unknown. Here, we report the identification of the molecular components that may possibly be involved in the FGL virulence pathway using yeast two hybrid system. FGL gene was amplified from a local virulent strain (F15) and shown to be 99.5% identical to the original published FGL at the amino acid level. We showed that transient expression of this FGL gene by Agroinfiltration in tobacco leaves causes cell death further implicating the role of FGL in virulence. To identify FGL initial physical target in plant, we screened two wheat cDNA libraries using the FGL protein as the bait. From both libraries, a small FKBP-type immunophilin protein, designated wFKBP12, was found to physically interact with FGL. The direct interaction of FGL with wFKBP12 was confirmed in living onion epidermal cells by biomolecular fluorescence complementation (BiFC) assay. To investigate further, we then used wFKBP12 protein as bait and identified an elicitor-responsive protein that contains a potential Ca^2 + binding domain. Semi-quantitative PCR showed that this elicitor-responsive gene is down-regulated during the F. graminearum infection suggesting that this protein may be an important component in FGL virulence pathway. This work serves as an initial step to reveal how fungal lipases act as a general virulence factor.     

Suppression of Alfalfa Growth by Concommitant Populations of Pratylenchus penetrans and two Fusarium species

Growth of alfalfa (Medicago sativa cv. Vernal) seedlings was compared after inoculation with combinations of either Pratylenchus penetrans and Fusarium soloni or P. penetrans and F. oxysporum f. sp. medicaginis. A synergistic disease interaction occurred in alfalfa when F. oxysporum and P. penetrans were added simultaneously to the soil. Alfalfa growth was suppressed at all inoculum levels of P. penetrans and F. oxysporum, but not with F. solani. Seedlings inoculated with the nematode alone gave lower yields than when inoculated with either Fusarium species alone. Fusarium oxysporum, but not F. solani, was pathogenic to alfalfa under similar experimental conditions. Fusarium oxysporum did not alter the populations of P. penetrans in alfalfa roots, whereas the presence of F. solani was associated with a diminished number of P. penetrans in the roots.     

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.     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.     

Interaction of Rotylenchulus reniformis with Seedling Disease Pathogens of Cotton

The impact of 10 Fusarium species in concomitant association with Rotylenchulus reniformis on cotton seedling disease was examined under greenhouse conditions. In experiment 1, fungal treatments consisted of Fusarium chlamydosporum, F. equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. oxysporum f.sp. vasinfectum, F. proliferatum, F. semitectum, F. solani, and F. sporotrichioides; Rhizoctonia solani; and Thielaviopsis basicola. The experimental design was a 2 × 14 factorial consisting of the presence or absence of R. reniformis and the 12 fungal treatments plus two controls in autoclaved field soil. In experiment 2, the same fungal and nematode treatments were examined in autoclaved or non-autoclaved soil. This experimental design was a 2 × 2 × 14 factorial consisting of field or autoclaved soil, presence or absence of R. reniformis, and the 12 fungal treatments plus two controls. In both tests, Fusarium oxysporum f. sp. vasinfectum, F. solani, R. solani, and T. basicola consistently displayed extensive root and hypocotyl necrosis that was more severe (P ≤ 0.05) in the presence of R. reniformis. Soil treatment (autoclaved vs. non-autoclaved) influenced the impact of the Fusarium species on cotton seedling disease, with disease being more severe in the autoclaved soil. Rotylenchulus reniformis reproduction on cotton seedlings was greater in field soil compared to autoclaved soil (P ≤ 0.05). This study suggests the importance of Fusarium species and R. reniformis in cotton seedling disease.     

Cladistic and phenetic analyses of relationships among Fusarium spp. in Dongtan wetland by morphology and isozymes

Variations of 12 morphological characters and 78 isozymic bands among 78 isolates of five Fusarium spp. from Dongtan wetland were described and analysed with cladistic parsimony and phenetic UPGMA methods. Hierarchical cluster analysis of 12 morphological characters grouped 78 strains into five defined species with a high overlap between isolates. Hierarchical cluster analysis of isozyme patterns showed a higher degree of relationship among five Fusarium spp., in which Fusarium nivale, Fusarium semitectum and Fusarium oxysporum clustered as one group, and F. semitectum was closer to F. nivale than to F. oxysporum; Fusarium graminearum and Fusarium moniliforme formed one group and showed clearly distinct from the first group. Groups of individual isolates indicated by a plot of principal component analysis were consistent with these findings. The comparison of two different data sets revealed that isozyme patterns showed higher variations between species and among individual isolates than morphological characters. Parsimony analysis of morphological characters yielded unresolved cladograms. Parsimony analysis of isozymes as presence/absence characters revealed the same five species in general as the results indicated by phenetic analysis, differing in the relative position of species in subclusters.     

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.     

Functional evaluation of a homologue of plant rapid alkalinisation factor (RALF) peptides in Fusarium graminearum

The cereal infecting fungus Fusarium graminearum is predicted to possess a single homologue of plant RALF (rapid alkalinisation factor) peptides. Fusarium mutant strains lacking FgRALF were generated and found to exhibit wildtype virulence on wheat and Arabidopsis floral tissue. Arabidopsis lines constitutively overexpressing FgRALF exhibited no obvious change in susceptibility to F. graminearum leaf infection. In contrast transient virus-mediated over-expression (VOX) of FgRALF in wheat prior to F. graminearum infection, slightly increased the rate of fungal colonisation of floral tissue. Ten putative Feronia (FER) receptors of RALF peptide were identified bioinformatically in hexaploid wheat (Triticum aestivum). Transient silencing of two wheat FER homoeologous genes prior to F. graminearum inoculation did not alter the subsequent interaction outcome. Collectively, our VOX results show that the fungal RALF peptide may be a minor contributor in F. graminearum virulence but results from fungal gene deletion experiments indicate potential functional redundancy within the F. graminearum genome. We demonstrate that virus-mediated over-expression is a useful tool to provide novel information about gene/protein function when results from gene deletion/disruption experimentation were uninformative.