Quantitative variation in effector activity of ToxA isoforms from Stagonospora nodorum and Pyrenophora tritici-repentis.

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.

Metabolomics protocols for filamentous fungi.

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.

Decoding the mannitol enigma in filamentous fungi.

Mannitol is a 6-carbon polyol that is among the most abundant biochemical compounds in the biosphere. Mannitol has been ascribed a multitude of roles in filamentous fungi including carbohydrate storage, reservoir of reducing power, stress tolerance and spore dislodgement and/or dispersal. The advancement of genetic manipulation techniques in filamentous fungi has rapidly accelerated our understanding of the roles and metabolism of mannitol. The targeted deletion of genes encoding proteins of mannitol metabolism in several fungi, including phytopathogens, has proven that the metabolism of mannitol does not exist as a cycle and that many of the postulated roles are unsupported. These recent studies have provided a much needed focus on this mysterious metabolite and make this a fitting time to review the roles and metabolism of mannitol in filamentous fungi.

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch).

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.

Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat.

UNLABELLED: SUMMARY 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).

The Mak2 MAP kinase signal transduction pathway is required for pathogenicity in Stagonospora nodorum.

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.