Ormonde D. C. Waters

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.

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.

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.

Prevalence and importance of sensitivity to Stagonospora nodorum necrotrophic effector SnTox3 in current Western Australian wheat cultivars.

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 chlorotic response. Five of the cultivars that were insensitive or weakly sensitive to SnTox3 were noticeably more resistant to the disease. The 60 cultivars gave a very similar reaction to SnTox3 and to the crude S. nodorum SN15 culture filtrate demonstrating that SnTox3 is the dominant NE in this isolate. We conclude that a simple screen using both SnTox3 and ToxA effectors combined with simple greenhouse disease evaluation, will allow breeders to select cultivars that are more resistant to the disease, allowing them to concentrate resources on other still intractable breeding objectives.

Sensitivity to three Parastagonospora nodorum necrotrophic effectors in current Australian wheat cultivars and the presence of further fungal effectors

Abstract. Parastagonospora nodorum is a major fungal pathogen of wheat in Australia, causing septoria nodorum blotch (SNB). Virulence of P. nodorum is quantitative and depends largely on multiple effector–host sensitivity gene interactions. The pathogen utilises a series of proteinaceous, necrotrophic effectors to facilitate disease development on wheat cultivars that possess appropriate dominant sensitivity loci. Thus far, three necrotrophic effector genes have been cloned. Proteins derived from these genes were used to identify wheat cultivars that confer effector sensitivity. The goal of this study was to determine whether effector sensitivity could be used to enhance breeding for SNB resistance. We have demonstrated that SnTox1 effector sensitivity is common in current commercial Western Australian wheat cultivars. Thirty-three of 46 cultivars showed evidence of sensitivity to SnTox1. Of these, 19 showed moderate or strong chlorotic/necrotic responses to SnTox1. Thirteen were completely insensitive to SnTox1. Disease susceptibility was most closely associated with SnTox3 sensitivity. We have also identified biochemical evidence of a novel chlorosis-inducing protein or proteins in P. nodorum culture filtrates unmasked in strains that lack expression of ToxA, SnTox1 and SnTox3 activities.

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.