Barley powdery mildew control in Western Australia and beyond

Australia is one of the largest barley exporters in the world, with Western Australia accounting for some 40% of national production. The crop is predominantly grown in the south and south-west of the state in winter and spring, where temperate conditions and higher rainfall levels are more suited to barley than northern and eastern regions. Between 2007 and 2013, prolonged outbreaks of barley powdery mildew (BPM) occurred. This was brought about by a combination of the extensive use of susceptible cultivars and an over-reliance on a small number of single mode-of-action demethylation inhibitor fungicides, which select for mutations in the C14α-demethylase (Cyp51A) gene. This review highlights the steps taken to reduce losses to BPM, breeding efforts to introduce resistance into cultivars and the success of pre-breeding research to find new and durable resistance genes. We also draw comparisons with powdery mildew in Australian wheat, where similar factors are leading to substantial outbreaks

Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, yet relatively few virulence-specific regulators are characterised in detail and their evolutionary trajectories are not well understood. Hence, this study compared the full range of TFs across taxonomically-diverse fungal proteomes and classified their lineages through an orthology analysis. The primary aims were to characterise differences in the range and profile of TF lineages broadly linked to plant-host association or pathogenic lifestyles, and to better characterise the evolutionary origin and trajectory of experimentally-validated virulence regulators. We observed significantly fewer TFs among obligate, host-associated pathogens, largely attributed to contractions in several Zn2Cys6 TF-orthogroup lineages. We also present novel insight into the key virulence-regulating TFs Ste12, Pf2 and EBR1, providing evidence for their ancestral origins, expansion and/or loss. Ultimately, the analysis presented here provides both primary evidence for TF evolution in fungal phytopathogenicity, as well as a practical phylogenetic resource to guide further detailed investigation on the regulation of virulence within key pathogen lineages

Comparative sub-cellular proteome analyses reveals metabolic differentiation and production of effector-like molecules in the dieback phytopathogen Phytophthora cinnamomi.

Phytopathogenic oomycetes pose a significant threat to global biodiversity and food security. The proteomes of these oomycetes likely contain important factors that contribute to their pathogenic success, making their discovery crucial for elucidating pathogenicity. Phytophthora cinnamomi is a root pathogen that causes dieback in a wide variety of crops and native vegetation world-wide. Virulence proteins produced by P. cinnamomi are not well defined and a large-scale approach to understand the biochemistry of this pathogen has not been documented. Soluble mycelial, zoospore and secreted proteomes were obtained and label-free quantitative proteomics was used to compare the composition of the three sub-proteomes. A total of 4635 proteins were identified, validating 17.7% of the predicted gene set. The mycelia were abundant in transporters for nutrient acquisition, metabolism and cellular proliferation. The zoospores had less metabolic related ontologies but were abundant in energy generating, motility and signalling associated proteins. Virulence-associated proteins were identified in the secretome such as candidate effector and effector-like proteins, which interfere with the host immune system. These include hydrolases, cell wall degrading enzymes, putative necrosis-inducing proteins and elicitins. The secretome elicited a hypersensitive response on the roots of a model host and thus suggests evidence of effector activity

Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BACKGROUND: Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained. RESULTS: In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellularassociated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome. CONCLUSION: We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies

Proteomic identification of extracellular proteins regulated by the Gna1 Gα subunit in Stagonospora nodorum

The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn.leaf and glume blotch) disease of wheat. The Gna1-encoded Ga protein is an important signaltransduction component in the fungus, which is required for full pathogenicity, sporulationand extracellular depolymerase production. In this study, we sought to gaina better understanding of defects associated with the gna1 mutant by using twodimensionalgel electrophoresis to analyse the extracellular proteome for differences tothe wildtype. Mass spectrometry analysis of altered abundant protein spots and peptidematching to the Stagonospora nodorum genome database have led to the identification ofgenes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compoundmetabolism. In addition, quantitative RT-PCR has demonstrated that some of theencoding genes showed differential expression throughout host infection. Implicationsof these proteins and their corresponding genes in fungal virulence are discussed.The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn. leaf and glume blotch) disease of wheat. The Gna1-encoded Gα protein is an important signal transduction component in the fungus, which is required for full pathogenicity, sporulation and extracellular depolymerase production. In this study, we sought to gain a better understanding of defects associated with the gna1 mutant by using two-dimensional gel electrophoresis to analyse the extracellular proteome for differences to the wildtype. Mass spectrometry analysis of altered abundant protein spots and peptide matching to the Stagonospora nodorum genome database have led to the identification of genes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compound metabolism. In addition, quantitative RT-PCR has demonstrated that some of the encoding genes showed differential expression throughout host infection. Implications of these proteins and their corresponding genes in fungal virulence are discussed

GWAS analysis reveals distinct pathogenicity profiles of Australian Parastagonospora nodorum isolates and identification of marker-trait-associations to septoria nodorum blotch

The fungus Parastagonospora nodorum is the causal agent of septoria nodorum leaf blotch (SNB) and glume blotch which are common in many wheat growing regions in the world. The disease is complex and could be explained by multiple interactions between necrotrophic effectors secreted by the pathogen and matching susceptibility genes in wheat. An Australian P. nodorum population was clustered into five groups with contrasting properties. This study was set to identify their pathogenicity profiles using a diverse wheat panel of 134 accessions which are insensitive to SnToxA and SnTox1 in both in vitro and in vivo conditions. SNB seedling resistance/susceptibility to five representative isolates from the five clusters, responses to crude culture-filtrates (CFs) of three isolates and sensitivity to SnTox3 semi-purified effector together with 11,455 SNP markers have been used for linkage disequilibrium (LD) and association analyses. While quantitative trait loci (QTL) on 1D, 2A, 2B, 4B, 5B, 6A, 6B, 7A, 7D chromosomes were consistently detected across isolates and conditions, distinct patterns and isolate specific QTL were also observed among these isolates. In this study, SnTox3–Snn3-B1 interaction for the first time in Australia and SnTox3–Snn3-D1 interaction for the first time in bread wheat were found active using wild-type isolates. These findings could be due to new SnTox3 haplotype/isoform and exotic CIMMYT/ICARDA and Vavilov germplasm used, respectively. This study could provide useful information for dissecting novel and different SNB disease components, helping to prioritise research targets and contributing valuable information on genetic loci/markers for marker-assisted selection in SNB resistance wheat breeding programme

Chromosome-level genome assembly and manually-curated proteome of model necrotroph Parastagonospora nodorum Sn15 reveals a genome-wide trove of candidate effector homologs, and redundancy of virulence-related functions within an accessory chromosome

Background: The fungus Parastagonospora nodorum causes septoria nodorum blotch (SNB) of wheat (Triticum aestivum) and is a model species for necrotrophic plant pathogens. The genome assembly of reference isolate Sn15 was first reported in 2007. P. nodorum infection is promoted by its production of proteinaceous necrotrophic effectors, three of which are characterised – ToxA, Tox1 and Tox3. Results: A chromosome-scale genome assembly of P. nodorum Australian reference isolate Sn15, which combined long read sequencing, optical mapping and manual curation, produced 23 chromosomes with 21 chromosomes possessing both telomeres. New transcriptome data were combined with fungal-specific gene prediction techniques and manual curation to produce a high-quality predicted gene annotation dataset, which comprises 13,869 high confidence genes, and an additional 2534 lower confidence genes retained to assist pathogenicity effector discovery. Comparison to a panel of 31 internationally-sourced isolates identified multiple hotspots within the Sn15 genome for mutation or presence-absence variation, which was used to enhance subsequent effector prediction. Effector prediction resulted in 257 candidates, of which 98 higher-ranked candidates were selected for in-depth analysis and revealed a wealth of functions related to pathogenicity. Additionally, 11 out of the 98 candidates also exhibited orthology conservation patterns that suggested lateral gene transfer with other cereal-pathogenic fungal species. Analysis of the pan-genome indicated the smallest chromosome of 0.4 Mbp length to be an accessory chromosome (AC23). AC23 was notably absent from an avirulent isolate and is predominated by mutation hotspots with an increase in non-synonymous mutations relative to other chromosomes. Surprisingly, AC23 was deficient in effector candidates, but contained several predicted genes with redundant pathogenicity-related functions. Conclusions: We present an updated series of genomic resources for P. nodorum Sn15 – an important reference isolate and model necrotroph – with a comprehensive survey of its predicted pathogenicity content

Dissecting the role of histidine kinase and Hog1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat.

The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity

Proteomic analysis revealed that the oomyceticide phosphite exhibits multi-modal action in an oomycete pathosystem

Phytopathogenic oomycetes constitute some of the most devastating plant pathogens and cause significant crop and horticultural yield and economic losses. The phytopathogen Phytophthora cinnamomi causes dieback disease in native vegetation and several crops. The most commonly used chemical to control P. cinnamomi is the oomyceticide phosphite. Despite its widespread use, the mode of action of phosphite is not well understood and it is unclear whether it targets the pathogen, the host, or both. Resistance to phosphite is emerging in P. cinnamomi isolates and other oomycete phytopathogens. The mode of action of phosphite on phosphite-sensitive and resistant isolates of the pathogen and through a model host was investigated using label-free quantitative proteomics. In vitro treatment of sensitive P. cinnamomi isolates with phosphite hinders growth by interfering with metabolism, signalling and gene expression; traits that are not observed in the resistant isolate. When the model host Lupinus angustifolius was treated with phosphite, proteins associated with photosynthesis, carbon fixation and lipid metabolism in the host were enriched. Increased production of defence-related proteins was also observed in the plant. We hypothesise the multi-modal action of phosphite and present two models constructed using comparative proteomics that demonstrate mechanisms of pathogen and host responses to phosphite. Significance: Phytophthora cinnamomi is a significant phytopathogenic oomycete that causes root rot (dieback) in a number of horticultural crops and a vast range of native vegetation. Historically, areas infected with phosphite have been treated with the oomyceticide phosphite despite its unknown mode of action. Additionally, overuse of phosphite has driven the emergence of phosphite-resistant isolates of the pathogen. We conducted a comparative proteomic study of a sensitive and resistant isolate of P. cinnamomi in response to treatment with phosphite, and the response of a model host, Lupinus angustifolius, to phosphite and its implications on infection. The present study has allowed for a deeper understanding of the bimodal action of phosphite, suggested potential biochemical factors contributing to chemical resistance in P. cinnamomi, and unveiled possible drivers of phosphite-induced host plant immunity to the pathogen

Differential effector gene expression underpins epistasis in a plant fungal disease.

Fungal 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 characterised 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 to SN15. This indicates that the failure to detect the SnTox3-Snn3 interaction in SN15 is due - at last 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