The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum–wheat interaction, as well as vital information for the general characterization of necrotroph–plant interactions.

Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins; the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.

SUMMARY Phytopathogenic fungi must feed on their hosts to propagate and cause disease. Their ability to access the rich nutrient supply offered by living plants is one of the most obvious properties that distinguish pathogens from saprophytes. Successful invasion by pathogens depends as much on their ability to utilize the available nutrient sources offered by plants as on their ability to penetrate plants and evade defensive mechanisms. Here, we review current knowledge on the nature of the nutrient supplies utilized by pathogens during infection. The available evidence is rudimentary in most cases. There is much evidence to suggest that fungal metabolism can be divided into at least two phases. The first is based on lipolysis and occurs during germination and penetration of the host. The second phase uses glycolysis and predominates during the invasion of host tissue. We also propose, mainly on theoretical grounds, that a third phase of nutrition occurs late in infection during which new spores are produced. Contrary to early assumptions, the nitrogen sources available to some pathogens appear to be abundant. The idea that nitrogen starvation is a cue that controls fungal gene expression during infection may need to be reassessed. Very little is known about the micronutrient (Fe, S, P) or vitamin supply. The knowledge gained from this research may enable the design of new antifungal strategies targeting potential weaknesses in fungal metabolism and will also impact on agronomic practices.

SummaryFungal effector–host sensitivity gene interactions play a key role in determining the outcome of septoria nodorum blotch disease (SNB) caused by Parastagonospora nodorum on wheat. The pathosystem is complex and mediated by interaction of multiple fungal necrotrophic effector–host sensitivity gene systems. Three effector sensitivity gene systems are well characterized in this pathosystem; SnToxA–Tsn1, SnTox1–Snn1 and SnTox3–Snn3. We tested a wheat mapping population that segregated for Snn1 and Snn3 with SN15, an aggressive P. nodorum isolate that produces SnToxA, SnTox1 and SnTox3, to study the inheritance of sensitivity to SnTox1 and SnTox3 and disease susceptibility. Interval quantitative trait locus (QTL) mapping showed that the SnTox1–Snn1 interaction was paramount in SNB development on both seedlings and adult plants. No effect of the SnTox3–Snn3 interaction was observed under SN15 infection. The SnTox3–Snn3 interaction was however, detected in a strain of SN15 in which SnTox1 had been deleted (tox1–6). Gene expression analysis indicates increased SnTox3 expression in tox1–6 compared with SN15. This indicates that the failure to detect the SnTox3–Snn3 interaction in SN15 is due – at least in part – to suppressed expression of SnTox3 mediated by SnTox1. Furthermore, infection of the mapping population with a strain deleted in SnToxA, SnTox1 and SnTox3 (toxa13) unmasked a significant SNB QTL on 2DS where the SnTox2 effector sensitivity gene, Snn2, is located. This QTL was not observed in SN15 and tox1–6 infections and thus suggesting that SnToxA and/or SnTox3 were epistatic. Additional QTLs responding to SNB and effectors sensitivity were detected on 2AS1 and 3AL.

SUMMARYThe fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three bestcharacterized effector-sensitivity gene systems are SnToxATsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora triticirepentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

Gna1, a gene encoding a Galpha subunit, a key component of signal transduction pathways, has been cloned and characterized in the wheat pathogen Stagonospora nodorum. Analysis of Gna1 expression during infection revealed a slight decrease in transcript levels shortly after germination, after which levels steadily increased until sporulation. Inactivation of Gna1 had a pleiotropic effect on phenotype. The Gna1 mutants were less pathogenic, attributed to coinciding with a defect in direct penetration. Also, Gna1 mutants were unable to sporulate, showed an albino phenotype, and secreted one or more brown pigments into growth media. Analysis of growth medium identified tyrosine, phenylalanine, and dihydroxyphenylalanine (L-DOPA) were excreted by the Gna1 strains but not by wild type. The presence of these compounds, and the insensitivity of melanization to tricyclazole suggest that S. nodorum synthesizes melanin via the L-DOPA pathway, the first fungal phytopathogen described to do so. Decreases in protease (and several other depolymerases) activities and sensitivity to osmotic stress were other phenotypes identified in the Gna1 mutants. Gna1 is the first signal transduction gene to be cloned and characterized from S. nodorum and its inactivation has uncovered several previously unknown facets of pathogenicity.

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).

Key message A locus on wheat chromosome 2A was found to control field resistance to both leaf and glume blotch caused by the necrotrophic fungal pathogen Parastagonospora nodorum.