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