We are analysing the wheat pathogen Stagonospora nodorum in order to determine the molecular basi... more We are analysing the wheat pathogen Stagonospora nodorum in order to determine the molecular basis of pathogenicity. The genome sequence of the wheat pathogen Stagonospora nodorum is the first Dothideomycete to be released. This taxon includes a large number of important plant pathogens. The sequence has facilitated experiments that have defined roles for the Ca2+/calmodulin, MAP kinase and cAMP-dependant signalling pathways and for the metabolites mannitol, arabitol, xylitol, malate, trehalose and aminolevulinate. Analysis of gene expression throughout infection points to three phases of nutrition; a lipolytic phase utilising internal stores; a glycolytic phase using external low molecular weight assimilates and hydrolytic phase using external polymerised substrates. The Dothideomycete group includes most or all pathogens that produce host-specific toxins. ToxA is produced by Pyrenophora tritici-repentis, the causal agent of tan spot in wheat and is responsible for virulence of the fungus on wheat genotypes carrying the dominant susceptibility gene Tsn1. A S. nodorum predicted gene, SnToxA is nearly identical to PtrToxA. Disruption of SnToxA significantly reduced disease on wheat lines carrying Tsn1. Sequencing of ToxA genes from a world wide collection of isolates of P. tritici-repentis and S. nodorum showed that all the P. tritici-repentis sequences were identical but that the S. nodorum sequences were highly variable. This pattern is strong evidence of horizontal gene transfer from S. nodorum to P. tritici-repentis. Furthermore, comparison of the sequences showed an excess of non-synonymous codon changes over synonymous, suggesting diversifying selection. This is expected of a protein that interacts directly and significantly with the product of Tsn1. P. tritici-repentis is a recently emerged disease and we suggest this emergence is linked to the acquisition of ToxA, prior to 1941, that enabled the rapid spread around the world. SnToxA is one of several toxins produced by S. nodorum. These studies raises several important and new problems. These include the effect of toxin possession and expression on the fecundity and survival of fungal strains carrying the toxins, the history of the possession and expression of toxin receptor genes in the host wheat and the mechanisms of sequence diversification in toxin genes. Progress in these areas will be described.
ABSTRACT Genetic disease resistance is widely assumed, and occasionally proven, to cause host yie... more ABSTRACT Genetic disease resistance is widely assumed, and occasionally proven, to cause host yield or fitness penalties due to inappropriate activation of defence response mechanisms or diversion of resources to surplus preformed defences. The study of resistance gene trade-offs has so far been restricted to biotrophic pathogens. In some Pleosporales necrotrophic interactions, quantitative resistance is positively associated with insensitivity to effectors. Host lines that differ in sensitivity can easily be identified amongst current cultivars and advanced breeding lines. Large wheat cultivar trials were used to test whether lines sensitive or insensitive to three necrotrophic effectors from Pyrenophora tritici-repentis and Parastagonospora nodorum differed in yield when subjected to natural disease and stress pressures in the West Australian wheat belt. There was no significant yield penalty associated with insensitivity to the fungal effectors ToxA, SnTox1 and SnTox3. Some yield gains were associated with insensitivity and some of these gains could be attributed to increased disease resistance. It is concluded that insensitivity to these effectors does not render such plants more vulnerable to any relevant biotic or abiotic stress present in these trials. These results suggest that the elimination of sensitivity alleles for necrotrophic effectors is a safe and facile strategy for improving disease resistance whilst maintaining or improving other desirable traits.
Proteomics and transcriptomics are established functional genomics tools commonly used to study f... more Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.
Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in ... more Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in Western Australia. The pathosystem is characterised by interactions of multiple pathogen necrotrophic effectors (NE) (formerly host-specific toxins) with corresponding dominant host sensitivity loci. To date, five NE interactions have been reported in S. nodorum. Two proteinaceous NE (ToxA and SnTox3) have been cloned and expressed in microbial systems. The identification of wheat cultivars lacking sensitivity to one or more NE is a promising way to identify cultivars suitable for use in breeding for increased resistance to this economically important pathogen. The prevalence of sensitivity to the NE SnTox3 was investigated in 60 current Western Australian-adapted bread wheat (Triticum aestivum L.) cultivars. Infiltration of SnTox3 into seedling leaves caused a moderate or strong necrotic response in 52 cultivars. Six cultivars were insensitive and two cultivars exhibited a weak chloroti...
Proteomics and transcriptomics are established functional genomics tools commonly used to study f... more Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.
ABSTRACT Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particu... more ABSTRACT Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in Western Australia. The pathosystem is characterised by interactions of multiple pathogen necrotrophic effectors (NE) (formerly host-specific toxins) with corresponding dominant host sensitivity loci. To date, five NE interactions have been reported in S. nodorum. Two proteinaceous NE (ToxA and SnTox3) have been cloned and expressed in microbial systems. The identification of wheat cultivars lacking sensitivity to one or more NE is a promising way to identify cultivars suitable for use in breeding for increased resistance to this economically important pathogen.
ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tr... more ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tritici-repentis. In this study, all eight mature isoforms of the ToxA protein were purified and compared. Circular dichroism spectra indicated that all isoforms were structurally intact and had indistinguishable secondary structural features. ToxA isoforms were infiltrated into wheat lines that carry the sensitivity gene Tsn1. It was observed that different wheat lines carrying identical Tsn1 alleles varied in sensitivity to ToxA. All ToxA isoforms induced necrosis when introduced into any Tsn1 wheat line but we observed quantitative variation in effector activity, with the least-active version found in isolates of P. tritici-repentis. Pathogen sporulation increased with higher doses of ToxA. The isoforms that induced the most rapid necrosis also induced the most sporulation, indicating that pathogen fitness is affected by differences in ToxA activity. We show that differences in toxin activity encoded by a single gene can contribute to the quantitative inheritance of necrotrophic virulence. Our findings support the hypothesis that the variation at ToxA results from selection that favors increased toxin activity.
A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Mag... more A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Magnaporthe grisea was cloned and characterised from the wheat glume blotch pathogen Stagonospora nodorum. Protein sequence alignments showed the cloned gene, Mak2, is closely related to homologues from other dothideomycete fungi. Expression studies revealed Mak2 is up-regulated during in vitro growth upon nitrogen starvation but is not sensitive to carbon starvation or osmotic stress. Transcript analysis in planta showed Mak2 to be expressed throughout infection and up-regulated during the sporulation phase of the infection cycle. Fungal strains harbouring a disrupted Mak2 gene were created by homologous gene recombination. The mutant strains had a severely altered phenotype in vitro with reduced growth rate and failure to sporulate. Further phenotypic analysis revealed that the mutants had near-normal levels of secreted protease activity, were not hypersensitive to osmotic stress and appeared to have melanin synthesis intact. The mak2 strains were essentially non-pathogenic to wheat leaves. No penetration structures formed and although entry was observed through stomates, the infection rarely continued. The results within this study are discussed within the context of the differences in downstream regulation of the Mak2 MAPK pathway and the cAMP signal transduction pathway in S. nodorum; and differences are compared to mak2 mutant strains in other pathogenic fungi.
Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf a... more Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species.Taxonomy: Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria, species nodorum.Host range: Wheat, Triticum aestivum, T. durum, Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence ‘nodorum’) and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces.Useful websites: http://ocid.nacse.org/research/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), http://ohioline.osu.edu/ac-fact/0002.html (disease information), http://wwwacnfp.murdoch.edu.au/ (ACNFP homepage), http://www.broad.mit.edu/annotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), http://cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).
We are analysing the wheat pathogen Stagonospora nodorum in order to determine the molecular basi... more We are analysing the wheat pathogen Stagonospora nodorum in order to determine the molecular basis of pathogenicity. The genome sequence of the wheat pathogen Stagonospora nodorum is the first Dothideomycete to be released. This taxon includes a large number of important plant pathogens. The sequence has facilitated experiments that have defined roles for the Ca2+/calmodulin, MAP kinase and cAMP-dependant signalling pathways and for the metabolites mannitol, arabitol, xylitol, malate, trehalose and aminolevulinate. Analysis of gene expression throughout infection points to three phases of nutrition; a lipolytic phase utilising internal stores; a glycolytic phase using external low molecular weight assimilates and hydrolytic phase using external polymerised substrates. The Dothideomycete group includes most or all pathogens that produce host-specific toxins. ToxA is produced by Pyrenophora tritici-repentis, the causal agent of tan spot in wheat and is responsible for virulence of the fungus on wheat genotypes carrying the dominant susceptibility gene Tsn1. A S. nodorum predicted gene, SnToxA is nearly identical to PtrToxA. Disruption of SnToxA significantly reduced disease on wheat lines carrying Tsn1. Sequencing of ToxA genes from a world wide collection of isolates of P. tritici-repentis and S. nodorum showed that all the P. tritici-repentis sequences were identical but that the S. nodorum sequences were highly variable. This pattern is strong evidence of horizontal gene transfer from S. nodorum to P. tritici-repentis. Furthermore, comparison of the sequences showed an excess of non-synonymous codon changes over synonymous, suggesting diversifying selection. This is expected of a protein that interacts directly and significantly with the product of Tsn1. P. tritici-repentis is a recently emerged disease and we suggest this emergence is linked to the acquisition of ToxA, prior to 1941, that enabled the rapid spread around the world. SnToxA is one of several toxins produced by S. nodorum. These studies raises several important and new problems. These include the effect of toxin possession and expression on the fecundity and survival of fungal strains carrying the toxins, the history of the possession and expression of toxin receptor genes in the host wheat and the mechanisms of sequence diversification in toxin genes. Progress in these areas will be described.
ABSTRACT Genetic disease resistance is widely assumed, and occasionally proven, to cause host yie... more ABSTRACT Genetic disease resistance is widely assumed, and occasionally proven, to cause host yield or fitness penalties due to inappropriate activation of defence response mechanisms or diversion of resources to surplus preformed defences. The study of resistance gene trade-offs has so far been restricted to biotrophic pathogens. In some Pleosporales necrotrophic interactions, quantitative resistance is positively associated with insensitivity to effectors. Host lines that differ in sensitivity can easily be identified amongst current cultivars and advanced breeding lines. Large wheat cultivar trials were used to test whether lines sensitive or insensitive to three necrotrophic effectors from Pyrenophora tritici-repentis and Parastagonospora nodorum differed in yield when subjected to natural disease and stress pressures in the West Australian wheat belt. There was no significant yield penalty associated with insensitivity to the fungal effectors ToxA, SnTox1 and SnTox3. Some yield gains were associated with insensitivity and some of these gains could be attributed to increased disease resistance. It is concluded that insensitivity to these effectors does not render such plants more vulnerable to any relevant biotic or abiotic stress present in these trials. These results suggest that the elimination of sensitivity alleles for necrotrophic effectors is a safe and facile strategy for improving disease resistance whilst maintaining or improving other desirable traits.
Proteomics and transcriptomics are established functional genomics tools commonly used to study f... more Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.
Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in ... more Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in Western Australia. The pathosystem is characterised by interactions of multiple pathogen necrotrophic effectors (NE) (formerly host-specific toxins) with corresponding dominant host sensitivity loci. To date, five NE interactions have been reported in S. nodorum. Two proteinaceous NE (ToxA and SnTox3) have been cloned and expressed in microbial systems. The identification of wheat cultivars lacking sensitivity to one or more NE is a promising way to identify cultivars suitable for use in breeding for increased resistance to this economically important pathogen. The prevalence of sensitivity to the NE SnTox3 was investigated in 60 current Western Australian-adapted bread wheat (Triticum aestivum L.) cultivars. Infiltration of SnTox3 into seedling leaves caused a moderate or strong necrotic response in 52 cultivars. Six cultivars were insensitive and two cultivars exhibited a weak chloroti...
Proteomics and transcriptomics are established functional genomics tools commonly used to study f... more Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.
ABSTRACT Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particu... more ABSTRACT Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in Western Australia. The pathosystem is characterised by interactions of multiple pathogen necrotrophic effectors (NE) (formerly host-specific toxins) with corresponding dominant host sensitivity loci. To date, five NE interactions have been reported in S. nodorum. Two proteinaceous NE (ToxA and SnTox3) have been cloned and expressed in microbial systems. The identification of wheat cultivars lacking sensitivity to one or more NE is a promising way to identify cultivars suitable for use in breeding for increased resistance to this economically important pathogen.
ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tr... more ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tritici-repentis. In this study, all eight mature isoforms of the ToxA protein were purified and compared. Circular dichroism spectra indicated that all isoforms were structurally intact and had indistinguishable secondary structural features. ToxA isoforms were infiltrated into wheat lines that carry the sensitivity gene Tsn1. It was observed that different wheat lines carrying identical Tsn1 alleles varied in sensitivity to ToxA. All ToxA isoforms induced necrosis when introduced into any Tsn1 wheat line but we observed quantitative variation in effector activity, with the least-active version found in isolates of P. tritici-repentis. Pathogen sporulation increased with higher doses of ToxA. The isoforms that induced the most rapid necrosis also induced the most sporulation, indicating that pathogen fitness is affected by differences in ToxA activity. We show that differences in toxin activity encoded by a single gene can contribute to the quantitative inheritance of necrotrophic virulence. Our findings support the hypothesis that the variation at ToxA results from selection that favors increased toxin activity.
A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Mag... more A gene encoding a mitogen-activated protein kinase (MAPK) putatively orthologous to Pmk1 from Magnaporthe grisea was cloned and characterised from the wheat glume blotch pathogen Stagonospora nodorum. Protein sequence alignments showed the cloned gene, Mak2, is closely related to homologues from other dothideomycete fungi. Expression studies revealed Mak2 is up-regulated during in vitro growth upon nitrogen starvation but is not sensitive to carbon starvation or osmotic stress. Transcript analysis in planta showed Mak2 to be expressed throughout infection and up-regulated during the sporulation phase of the infection cycle. Fungal strains harbouring a disrupted Mak2 gene were created by homologous gene recombination. The mutant strains had a severely altered phenotype in vitro with reduced growth rate and failure to sporulate. Further phenotypic analysis revealed that the mutants had near-normal levels of secreted protease activity, were not hypersensitive to osmotic stress and appeared to have melanin synthesis intact. The mak2 strains were essentially non-pathogenic to wheat leaves. No penetration structures formed and although entry was observed through stomates, the infection rarely continued. The results within this study are discussed within the context of the differences in downstream regulation of the Mak2 MAPK pathway and the cAMP signal transduction pathway in S. nodorum; and differences are compared to mak2 mutant strains in other pathogenic fungi.
Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf a... more Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species.Taxonomy: Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria, species nodorum.Host range: Wheat, Triticum aestivum, T. durum, Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence ‘nodorum’) and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces.Useful websites: http://ocid.nacse.org/research/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), http://ohioline.osu.edu/ac-fact/0002.html (disease information), http://wwwacnfp.murdoch.edu.au/ (ACNFP homepage), http://www.broad.mit.edu/annotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), http://cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).
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