HOW DO PLANTS RECOGNIZE FUNGI ?

1. HOW DO PLANTS RECOGNIZE FUNGI ? Yunzi Gou Supervisor : Martin Lipschis Prof. Georg Felix Characterization of MAMPs from Penicillium chrysogenum

2. Chitin Ergosterol … • Molecules shared by large groups of microbes but absent from the host • Highly conserved structure • Recognized by Pattern recognition receptors (PRRs , e.g. FLS2, EFR) • Induce PTI(PAMP-triggered immunity) at nanomolar concentration Flagellin(flg22) EF-Tu(elf18) … Defence signaling & responses MAMPs/PAMPs: Microbe-associated molecular patterns / Pathogen-associated molecular patterns

3. MAPKKK H+ Methionine cycle Ca2+ +++ ca. 10-30 min MAPKK MAPK signaling cascade Gene activation ca. 5 min ACC NADPH oxidase complex Cl- O2- H2O2 O2+ K+ ethylene pH assay ethylene assay PAMP induced defence reactions

4. PEN • Pen : extract from a high penicillin-producing strain of P. chrysogenum . • bulk waste product from Sandoz, Kundl, Austria: mycelium after extraction of penicillin, dried, heated for 3h at 140°C …. • penicillin or its by-product have no MAMP-activity on plants Induce Defence response B. Thuerig et al. PMPP (2006)

5. PEN • ethylene biosynthesis • extracellular alkalinisation PEN : Strong MAMP-activity in a broad range ! A. thaliana B. Thuerig et al. / Physiological and Molecular Plant Pathology (2006)

6. Approachs to purify... B. Thuerig et al. PMPP, 2006. • Ion exchange chromatography • size exclusion chromatography • C8 and C18 reversed phase chromatography • No distinct peaks • Activity was spread over a wide range of fractions  The MAMP-activity of Pen is heterogeneous in polarity, size and charge A MAMP has structural diversity ? OR A mixture of different MAMPs ?

7. Charactorization & Purification Pen • Crude extract • Aqueous solution (45mg/ml) Pen1000 • Dialysed in 1kD cut-off membrane • Get rid of small chitin fragments & other small molecule • C18 reversed phase chromatography • Eluted with Methanol gradient Penpre • Pre-purified on C18 RP pre-column with 0,1% TFA • Eluted with Methanol HPLC

8. Conductivity (mS/cm) OD(mAU) • pH assay: • 1µl from each fraction • 400µl Arabidopsis cell culture • pH measured after 25 min ΔpH in 25 min • C18 reversed phase chromatography • Eluted with Methanol gradient HPLC C D -Column : VP250/10 NUCLEOSIL 120-10 C18 -Buffer A : 0,1% TFA -Buffer B : 95% MeOH 0,1%TFA -Gradient : 0-60% B in 30 ml, 60%-100%B in 0,1s -Fraction : 1 ml Elultion Volume (ml)  No single sharp peak but 2 giant “hills„

9. Saturation experiment -Saturate PRRs for C -Add D to the same A.thaliana cells… C D • Are theythe same MAMP-activity in principle ? Extracellular pH control Time(min) • Different type of MAMP-activity, D is stronger than C • D may contain some C

10. extracellular pH extracellular pH extracellular pH • Are these 2 type of MAMP-activity sensitive to protease ? C D Prot.K Prot.K time (min) time (min) time (min) • elf18 was digested in D5, 0,3µl D5 was added. • Control : Prot.Kworked • No prot.K inhibitor presence in C & D • Protease digestion does have a small effect on both activities • But it is not a dramatic decrease of activity like it is in the case of proteinic MAMP (elf18). • C & D  unlikely protein

11. Extracellular pH Saturation experiment -Saturate PRRs for Chitin -Add C & D to the same cells… 900µg Chitin C D • Chitinasedigestionof C & D • Are theythe same MAMP-activityas Chitin ? Chitin? Chitin? 300µg Chitin / ml cell culture Control:chitinase worked C is more sensitive to chitinase than D D contains more non-chitin activity than C ! • Not the same as chitin • D seems containing more non-chitin activity than C

12. C D • Can both of them induce ethylene response ? Chitin? Chitin?  C contains major chitin-like activity  D contains a good ethylene-inducing MAMP-activity

13. Proteasedigestionof D after chitinasetreatment C D Chitin? Chitin? • Similar small effect observed again

14. D C Summary • PEN Probably overlaped ! Chitin C-type ofactiviy Chitin-like PAMP (CLP) Uninteresting! D-type ofactiviy A NOVEL PAMP ! (DP) INTERESTING ! A weak proteinic PAMP ? Proteinaceous activity or Only the peptide part linked to D ?

15. C D outlook overlapping • Find ways to purify D from Pen which is largely contaminated by Chitin & CLP • Characterize ConA-binding activity of Pen • Screen for DP-recepter mutant using ethylene bioassay Thank you for your attention!