Donald M. Gardiner

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat.

Fusarium species infect cereal crops worldwide and cause the important diseases Fusarium head blight and crown rot in wheat. Fusarium pathogens reduce yield and some species also produce trichothecene mycotoxins, such as deoxynivalenol (DON), during infection. These toxins play roles in pathogenesis on wheat and have serious health effects if present in grain consumed by humans or animals. In the present study, the response of wheat tissue to DON has been investigated. Infusion of wheat leaves with DON induced hydrogen peroxide production within 6 h followed by cell death within 24 h that was accompanied by DNA laddering, a hallmark of programmed cell death. In addition, real-time PCR analysis revealed that DON treatment rapidly induced transcription of a number of defence genes in a concentration-dependent manner. Co-treatment with DON and the antioxidant ascorbic acid reduced these responses, suggesting their induction may be at least partially mediated by reactive oxygen species (ROS), commonly known to be signalling molecules in plants. Wheat defence genes were more highly expressed in wheat stems inoculated with a DON-producing fungal strain than those inoculated with a DON-non-producing mutant, but only at a late stage of infection. Taken together, the results are consistent with a model in which DON production during infection of wheat induces ROS, which on the one hand may stimulate programmed host cell death assisting necrotrophic fungal growth, whereas, on the other hand, the ROS may contribute to the induction of antimicrobial host defences.

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two‐module non‐ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co‐regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.

On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance

The ascomycete fungal pathogen Fusarium graminearum (sexual stage: Gibberella zeae) causes the devastating head blight or scab disease on wheat and barley, and cob or ear rot disease on maize. Fusarium graminearum infection causes significant crop and quality losses. In addition to roles as virulence factors during pathogenesis, trichothecene mycotoxins (e.g. deoxynivalenol) produced by this pathogen constitute a significant threat to human and animal health if consumed in respective food or feed products. In the last few years, significant progress has been made towards a better understanding of the processes involved in F. graminearum pathogenesis, toxin biosynthesis and host resistance mechanisms through the use of high-throughput genomic and phenomic technologies. In this article, we briefly review these new advances and also discuss how future research can contribute to the development of sustainable plant protection strategies against this important plant pathogen.

Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum.

Fusarium head blight is one of the most important diseases of wheat worldwide due to crop losses and the contamination of grains with trichothecene mycotoxins. The biosynthesis of trichothecenes by Fusarium spp. is highest during infection, but relatively low levels are produced from saprophytic growth in axenic culture. A strain of Fusarium graminearum was constructed where the promoter from the TRI5 trichothecene biosynthesis gene was fused to GFP. Using this strain in large-scale nutrient profiling, a variety of amines were identified that significantly induce TRI5 expression. Analysis of trichothecene levels in the culture filtrates revealed accumulation of the toxin to over 1000ppm in response to these inducers, levels either greater than or equivalent to those observed during infection. From this work, we propose that products of the arginine-polyamine biosynthetic pathway in plants may play a role in the induction of trichothecene biosynthesis during infection.

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

BackgroundGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.ResultsPutative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.ConclusionETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.

Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts

Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.

Novel genes of Fusarium graminearum that negatively regulate deoxynivalenol production and virulence.

Fusarium head blight of wheat, caused by Fusarium graminearum, is a serious disease resulting in both reduced yields and contamination of grain with trichothecene toxins, with severe consequences for mammalian health. Recently, we have identified several related amine compounds such as agmatine and putrescine that promote the production of high levels of trichothecene toxins, such as deoxynivalenol (DON), in culture by F. graminearum and F. sporotrichioides. Here, a global analysis of fungal gene expression using the Affymetrix Fusarium GeneChip during culture under DON-inducing conditions compared with noninducing conditions is reported. Agmatine differentially regulated a large number of fungal genes, including both known and previously uncharacterized putative secondary metabolite biosynthetic gene clusters. In silico prediction of binding sites for the transcriptional regulator (TRI6) controlling TRI gene expression and gene expression analysis in a TRI6 mutant of F. graminearum showed that three of the differentially regulated genes were under the control of TRI6. Gene knock-out mutations of two of these genes resulted in mutants with massively increased production of DON and increased aggressiveness toward wheat. Our results not only identify a novel mechanism of negative regulation of DON production and virulence in F. graminearum but also point out the potential of this pathogen to evolve with an ability to produce massively increased amounts of toxins and increased virulence.

Phases of Infection and Gene Expression of Fusarium graminearum During Crown Rot Disease of Wheat

Fusarium graminearum causes head blight (FHB) and crown rot (CR) diseases in wheat. Compared with FHB, CR symptom development occurs slowly, usually taking 4 to 8 weeks to become visible. To characterize CR development, we used histological and real-time quantitative polymerase chain reaction analyses to assess fungal colonization during a timecourse of infection. Three distinct phases of infection were identified: i) initial spore germination with formation of a superficial hyphal mat at the inoculation point, ii) colonization of the adaxial epidermis of the outer leaf sheath and mycelial growth from the inoculation point to the crown, concomitant with a drop in fungal biomass, and iii) extensive colonization of the internal crown tissue. Fungal gene expression was examined during each phase using Affymetrix GeneChips. In total, 1,839 F. graminearum genes were significantly upregulated, including some known FHB virulence genes (e.g., TRI5 and TRI14), and 2,649 genes were significantly downregulated in planta compared with axenically cultured mycelia. Global comparisons of fungal gene expression with published data for FHB showed significant similarities between early stages of FHB and CR. These results indicate that CR disease development involves distinct phases of colonization, each of which is associated with a different fungal gene expression program.

A Highly Conserved Effector in Fusarium oxysporum Is Required for Full Virulence on Arabidopsis

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Δsix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Δsix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.