the pyrenophora teres f. teres secretome: role of …...the pyrenophora teres f.teres secretome:...
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The Pyrenophora teres f. teres secretome: Role of virulence-associated proteins during
net blotch disease of barley Ismail A. Ismail & Amanda J. Able
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University of Adelaide 2
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The secretome of P. teres
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Proteins NECROSIS
Aspergillomarasamines CHLOROSIS
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Resistant
Sensitivity to proteinaceous toxins is correlated with susceptibility to the pathogen
University of Adelaide 4
Susceptible
Screening tool for breeding NFNB resistance? Genetic basis for toxin sensitivity in barley?
Which individual toxins are responsible?
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The proteinaceous secretome of P. teres
• Total protein extract from culture filtrates causes symptoms BUT which individual proteins are associated with virulence?
– Compared protein profile of low and high virulent isolate (Ismail et al 2014 APP 43: 715-726) • PttSP1 and cysteine-hydrolase (PttCHFP)
• Endoxylanase (PttXyn11A) – Necrosis-inducing & cell wall degradation
• Does the proteinaceous secretome vary among a large number of isolates varying in their virulence on different barley cultivars? – Role?
University of Adelaide 5
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Culture filtrates
8 different profiles: Individual toxins
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28 isolates selected - pathotyping & genotyping
Proteomics
Further characterisation (eg. bioinformatics, qPCR & bioassay)
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260 individual proteins identified with 49 common to the most virulent isolates
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Protein families identified for 200 proteins
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16 GH families
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Predicted functions
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Predicted functions and other species?
• CWDEs – Glucanases and xylanases
• Virulence factors, effectors, fungal pathogenesis – Ceratoplatanin, Spherulins – LRR-protein – Cysteine-rich/ YxC motif – Ricin-type toxins and lectins
• Oxidation-reduction processes – Isoamyl alcohol oxidases & laccases
• Fungal development – Chitin-binding/cellulose-binding domains – 6-phosphogluconolactonase
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Predicted function, which isolates, RNA expression during the interaction, bioassay
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RNA expression patterns during the interaction
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Time 192h
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RNA expression levels
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Examples of RNA expression pattern + amount
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Resistant
Host specificity?
• Which individual toxins are responsible?
– High versus low virulence
– Susceptible versus resistant
University of Adelaide 14
Susceptible
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Host specificity? An example: ceratoplatanin
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Susc. Resistant
• For example, ceratoplatanin shown to induce cell death (Ceratocytis spp.) Linked to virulence (Magnaporthe grisea)
0
0.05
0.1
0.15
0.2
0.25
40 64 98 120 144 168 192
PttCP
Less virulent
More virulent
Ceratoplatanin
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Creating a model of the barley-Ptt interaction: toxin by toxin
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40h 64h 98h 120h 144h 168h 192h
Necrotroph Biotroph
Effectors, Virulence Factors
Cell wall degradation
Fungal development & metabolism
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Conclusions & further research
• Analysis of the secretome of P. teres f. teres identified 260 proteins associated with virulence – Using 28 isolates varying in virulence on 24 barley
varieties • Very complex
– Predicted functions include • CWDEs and metabolism • effectors/pathogenesis/virulence factors • fungal development/morphogenesis
– Timing of expression during the plant-pathogen interaction might fit with the role • Knock-out key candidates and effect?
– Host-specificity • Screening tool? • Genetic basis for resistance?
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SARDI Hugh Wallwork
ANU Celeste Linde
Adelaide Proteomics Centre
Acknowledgements
Ismail Ismail Shu Das