1 / 15

HOW DO PLANTS RECOGNIZE FUNGI ?

HOW DO PLANTS RECOGNIZE FUNGI ?. Yunzi Gou Supervisor : Martin Lipschis Prof. Georg Felix. Characterization of MAMPs from Penicillium chrysogenum. Chitin Ergosterol …. Molecules shared by large groups of microbes but absent from the host Highly conserved structure

miach
Download Presentation

HOW DO PLANTS RECOGNIZE FUNGI ?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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!

More Related