The Pyrenophora teres f. teres secretome: Role of virulence-associated proteins during
net blotch disease of barley Ismail A. Ismail & Amanda J. Able
University of Adelaide 2
The secretome of P. teres
University of Adelaide 3
Proteins NECROSIS
Aspergillomarasamines CHLOROSIS
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?
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
Culture filtrates
8 different profiles: Individual toxins
University of Adelaide 6
28 isolates selected - pathotyping & genotyping
Proteomics
Further characterisation (eg. bioinformatics, qPCR & bioassay)
260 individual proteins identified with 49 common to the most virulent isolates
University of Adelaide 7
Protein families identified for 200 proteins
University of Adelaide 8
16 GH families
Predicted functions
University of Adelaide 9
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
University of Adelaide 10
Predicted function, which isolates, RNA expression during the interaction, bioassay
RNA expression patterns during the interaction
University of Adelaide 11
Time 192h
RNA expression levels
University of Adelaide 12
Examples of RNA expression pattern + amount
University of Adelaide 13
Resistant
Host specificity?
• Which individual toxins are responsible?
– High versus low virulence
– Susceptible versus resistant
University of Adelaide 14
Susceptible
Host specificity? An example: ceratoplatanin
University of Adelaide 15
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
Creating a model of the barley-Ptt interaction: toxin by toxin
University of Adelaide 16
40h 64h 98h 120h 144h 168h 192h
Necrotroph Biotroph
Effectors, Virulence Factors
Cell wall degradation
Fungal development & metabolism
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?
University of Adelaide 17
SARDI Hugh Wallwork
ANU Celeste Linde
Adelaide Proteomics Centre
Acknowledgements
Ismail Ismail Shu Das