Erik Lysøe

One fungus, one name

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

ABSTRACT Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

The Transcriptome of Fusarium graminearum During the Infection of Wheat

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.

The Transcription Factor FgStuAp Influences Spore Development, Pathogenicity, and Secondary Metabolism in Fusarium graminearum

Fusarium graminearum is an important plant-pathogenic fungus and the major cause of cereal head blight. Here, we report the functional analysis of FgStuA, the gene for a transcription factor with homology to key developmental regulators in fungi. The deletion mutant was greatly reduced in pathogenicity on wheat heads and in production of secondary metabolites. Spore production was significantly impaired in ΔFgStuA, which did not develop perithecia and sexual ascospores, and lacked conidiophores and phialides, leading to delayed production of aberrant macroconidia. FgStuAp appears to act as a global regulator that may affect many diverse aspects of the life cycle of F. graminearum. Transcriptome analysis shows that thousands of genes are differentially expressed in the mutant during asexual sporulation and infection of wheat heads and under conditions that induce secondary metabolites, including many that could account for the mutant phenotypes observed. The primary regulatory targets of FgStuAp are likely genes involved in cell-cycle control, and the predicted FgStuAp sequence has an APSES domain, with homology to helix-loop-helix proteins involved in cell-cycle regulation. The Aspergillus StuAp response element (A/TCGCGT/ANA/C) was found highly enriched in the promoter sequences of cell-cycle genes, which was upregulated in the ΔFgStuA deletion mutant.

An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium

Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

The Genome of the Generalist Plant Pathogen Fusarium avenaceum Is Enriched with Genes Involved in Redox, Signaling and Secondary Metabolism

Fusarium avenaceum is a fungus commonly isolated from soil and associated with a wide range of host plants. We present here three genome sequences of F. avenaceum, one isolated from barley in Finland and two from spring and winter wheat in Canada. The sizes of the three genomes range from 41.6–43.1 MB, with 13217–13445 predicted protein-coding genes. Whole-genome analysis showed that the three genomes are highly syntenic, and share>95% gene orthologs. Comparative analysis to other sequenced Fusaria shows that F. avenaceum has a very large potential for producing secondary metabolites, with between 75 and 80 key enzymes belonging to the polyketide, non-ribosomal peptide, terpene, alkaloid and indole-diterpene synthase classes. In addition to known metabolites from F. avenaceum, fuscofusarin and JM-47 were detected for the first time in this species. Many protein families are expanded in F. avenaceum, such as transcription factors, and proteins involved in redox reactions and signal transduction, suggesting evolutionary adaptation to a diverse and cosmopolitan ecology. We found that 20% of all predicted proteins were considered to be secreted, supporting a life in the extracellular space during interaction with plant hosts.

Real-Time Quantitative Expression Studies of the Zearalenone Biosynthetic Gene Cluster in Fusarium graminearum

The estrogenic mycotoxin zearalenone (ZON) produced by some Fusarium spp. causes reproductive problems and hyperestrogenic syndromes in mammals. In an effort to elucidate the molecular pathways of ZON production, we present a comparative real-time quantitative polymerase chain reaction expression study of seven contiguous genes in the ZON biosynthetic cluster on sterile rice and during wheat and oat infection. Under ZON production on rice, the polyketide synthase (PKS) genes PKS4 and PKS13, alcohol oxidase FG12056 gene, and transcriptional regulator FG02398 gene showed similarly upregulated patterns, whereas the nonribosomal peptide synthetase (NPS) FG02394, the K(+) channel beta subunit FG12015, and the protein kinase FG02399 displayed a variant pattern. During the same time period under wheat infection when no ZON was produced, the PKS genes and the NPS were downregulated relative to rice whereas the K(+) channel beta subunit gene FG12015 was markedly upregulated, suggesting that it may play a role in the infection process. This is the first expression study of ZON biosynthetic genes in planta. The results give insight into the regulation and activities of the ZON gene cluster under different experimental systems and suggest a connection between ZON and a K(+) channel that could reveal a novel function for ZON in Fusarium spp.

Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis

The available genome sequences show that the number of secondary metabolite genes in filamentous fungi vastly exceeds the number of known products. This is also true for the global plant pathogenic fungus Fusarium graminearum, which contains 15 polyketide synthase (PKS) genes, of which only 6 have been linked to products. To help remedy this, we focused on PKS14, which has only been shown to be expressed during plant infections or when cultivated on rice or corn meal (RM) based media. To enhance the production of the resulting product we introduced a constitutive promoter in front of PKS14 and cultivated two of the resulting mutants on RM medium. This led to the production of two compounds, which were only detected in the PKS14 overexpressing mutants and not in the wild type or PKS14 deletion mutants. The two compounds were tentatively identified as orsellinic acid and orcinol by comparing spectroscopic data (mass spectroscopy and chromatography) to authentic standards. NMR analysis of putative orcinol isolated from the PKS14 overexpressing mutant supported our identification. Orcinol and orsellinic acid, not previously detected in Fusarium, have primarily been detected in lichen fungi. Orsellinic acid is hypothesized to be the PKS release product which is transformed to orcinol through decarboxylation. Phylogenetic analyses of PKSs placed PKS14 in a subclade of known OA synthases. Expression analysis by microarray of 55 experiments identified seven genes near PKS14 that were expressed in a similar manner. One of the seven genes encodes a predicted carboxylase, which could be responsible for transforming orsellinic acid to orcinol.

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.