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.

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn2Cys6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

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.

We report a fungal pan-genome study involving Parastagonospora spp., including 21 isolates of the wheat (Triticum aestivum) pathogen Parastagonospora nodorum, 10 of the grass-infecting Parastagonospora avenae, and 2 of a closely related undefined sister species. We observed substantial variation in the distribution of polymorphisms across the pan-genome, including repeat-induced point mutations, diversifying selection and gene gains and losses. We also discovered chromosome-scale inter and intraspecific presence/absence variation of some sequences, suggesting the occurrence of one or more accessory chromosomes or regions that may play a role in host–pathogen interactions. The presence of known pathogenicity effector loci SnToxA, SnTox1, and SnTox3 varied substantially among isolates. Three P. nodorum isolates lacked functional versions for all three loci, whereas three P. avenae isolates carried one or both of the SnTox1 and SnTox3 genes, indicating previously unrecognized potential for discovering additional effectors in the P. nodorum-wheat pathosystem. We utilized the pan-genomic comparative analysis to improve the prediction of pathogenicity effector candidates, recovering the three confirmed effectors among our top-ranked candidates. We propose applying this pan-genomic approach to identify the effector repertoire involved in other host–microbe interactions involving necrotrophic pathogens in the Pezizomycotina.

SummaryThree genes encoding different Ca 2+ /calmodulindependent protein kinases have been characterized in the wheat phytopathogenic fungus Stagonospora nodorum. The kinases were identified from the S. nodorum genome sequence on the basis of sequence homology to known Ca 2+ /calmodulindependent protein kinases. Expression analysis determined that each of the kinases was expressed during growth in vitro and also during infection. The onset of sporulation triggered increased transcript levels of each of the kinases, particularly CpkA where an 11-fold increase in expression was observed during sporulation in planta. The role of the kinases was further determined via a reverse genetics approach. The disruption of CpkA affected vegetative growth in vitro and also sporulation. The cpkA strains produced 20-fold less spores on complex media and were unable to sporulate on defined minimal media. Infection assays showed that CpkA was not required for lesion development but was essential for sporulation at the completion of the infection cycle. Microscopic analysis revealed that the disruption of CpkA resulted in Stagonospora nodorum being unable to differentiate the mycelial knot into immature pycnidia during sporulation. A metabolite analysis of infected leaves during sporulation excluded the possible involvement of mannitol, a compound previously shown to be involved in the sporulation of Stagonospora nodorum. The disruption of CpkB did not effect growth in vitro or pathogenicity. Stagonospora nodorum strains lacking CpkC appeared unaffected during growth in planta but showed delayed lesion development and sporulation during infection.

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.Additional keywords: necrotrophic effector (NE), septoria nodorum blotch, SnTox1, SnTox3, ToxA, wheat.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2-3weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S.nodorum.