Kasia Rybak

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

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.

Investigating the role of calcium/calmodulin-dependent protein kinases in Stagonospora nodorum

Three genes encoding different Ca2+/calmodulin‐dependent 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 Ca2+/calmodulin‐dependent 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.

Ubiquity of ToxA and absence of ToxB in Australian populations of Pyrenophora tritici-repentis.

Pyrenophora tritici-repentis, the causal organism of the necrotrophic foliar wheat disease tan spot [also known as yellow (leaf) spot in Australia] is an important disease in Australia and in many parts of the world. North American isolates of the pathogen have been shown to produce combinations of three host-specific toxins, ToxA, ToxB and ToxC. Each toxin interacts with a host sensitivity locus, respectively Tsn1, Tsc2 and Tsc1. The virulence of an isolate is partially correlated with the presence of these toxins and resistance in the host is associated with absence of the sensitivity loci. Breeding for resistance to tan spot can, therefore, be aided by knowledge of the prevalence of the toxin-encoding genes in local pathogen populations. Two of the toxins, A and B, are encoded by known genes and molecular tests for the genes have been developed. We screened a diverse collection of 119 tan spot isolates collected between 1984 and 2008 and from all affected regions of Australia (Queensland, New South Wales, Victoria and Western Australia). In all cases, the gene for ToxA was present and the gene for ToxB was absent. The implications for resistance breeding and epidemiology of the disease are discussed. We also define a diagnostic molecular marker for P. tritici-repentis.

A metabolomic approach to dissecting osmotic stress in the wheat pathogen Stagonospora nodorum

A non-targeted metabolomics approach was used to identify significant changes in metabolism upon exposure of the wheat pathogen Stagonospora nodorum to 0.5M NaCl. The polyol arabitol, and to a lesser extent glycerol, was found to accumulate in response to the osmotic stress treatment. Amino acid synthesis was strongly down-regulated whilst mannitol levels were unaffected. A reverse genetic approach was undertaken to dissect the role of arabitol metabolism during salt stress. Strains of S. nodorum lacking a gene encoding an l-arabitol dehydrogenase (abd1), a xylitol dehydrogenase (xdh1) and a double-mutant lacking both genes (abd1xdh1) were exposed to salt and the intracellular metabolites analysed. Arabitol levels were significantly up-regulated upon salt stress in the xdh1 strains but were significantly lower than the wild-type. Arabitol was not significantly different in either the abd1 or the abd1xdh1 strains during osmotic stress but the concentration of glycerol was significantly higher indicating a compensatory mechanism in operation. Genome sequence analysis identified a second possible enzyme capable of synthesizing arabitol explaining the basal level of arabitol present in the abd1xdh1 strains. This study identified that arabitol is the primary compatible solute in S. nodorum but in-built levels of redundancy are present allowing the fungus to tolerate osmotic stress.

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.

Emergence of tan spot disease caused by toxigenic Pyrenophora tritici-repentis in Australia is not associated with increased deployment of toxin-sensitive cultivars.

The wheat disease tan (or yellow leaf) spot, caused by Pyrenophora tritici-repentis, was first described in the period 1934 to 1941 in Canada, India, and the United States. It was first noted in Australia in 1953 and only became a serious disease in the 1970s. The emergence of this disease has recently been linked to the acquisition by P. tritici-repentis of the ToxA gene from the wheat leaf and glume blotch pathogen, Stagonospora nodorum. ToxA encodes a host-specific toxin that interacts with the product of the wheat gene Tsn1. Interaction of ToxA with the dominant allele of Tsn1 causes host necrosis. P. tritici-repentis races lacking ToxA give minor indistinct lesions on wheat lines, whereas wheat lines expressing the recessive tsn1 are significantly less susceptible to the disease. Although the emergence and spread of tan spot had been attributed to the adoption of minimum tillage practices, we wished to test the alternative idea that the planting of Tsn1 wheat lines may have contributed to the establishment of the pathogen in Australia. To do this, wheat cultivars released in Australia from 1911 to 1986 were tested for their sensitivity to ToxA. Prior to 1941, 16% of wheat cultivars were ToxA-insensitive and hence, all other factors being equal, would be more resistant to the disease. Surprisingly, only one of the cultivars released since 1940 was ToxA insensitive, and the area planted to ToxA-insensitive cultivars varied from 0 to a maximum of only 14% in New South Wales. Thus, the majority of the cultivars were ToxA-sensitive both before and during the period of emergence and spread of the disease. We therefore conclude that the spread of P. tritici-repentis in Australia cannot be causally linked to the deployment of ToxA-sensitive cultivars.