Characterization of MAMPs from Penicillium chrysogenum

Yunzi Gou

Supervisor :

Martin Lipschis

Prof. Georg Felix

HOW DO PLANTS RECOGNIZE FUNGI ?

MAMPs/PAMPs:Microbe-associated molecular patterns / Pathogen-associated molecular patterns

Defence signaling & responses

Flagellin(flg22)

EF-Tu(elf18)…

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

ChitinErgosterol

…

signaling cascade +++

K+

H+

Cl-

Ca2+

NADPHoxidasecomplex

O2+ O2

- H2O2

ethylene

Methioninecycle

ACC

ca. 5 min

ca. 10-30 min

pH

assay

eth

yle

ne a

ssay

PAMP induced defence reactions

MAPKKK

MAPKK MAPK

Gene activation

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

InduceDefence response

B. Thuerig et al. PMPP (2006)

A. thaliana

extracellular alkalinisation ethylene

biosynthesis

PENPEN

B. Thuerig et al. / Physiological and Molecular Plant Pathology (2006)

PEN : Strong MAMP-activityPEN : Strong MAMP-activity

in a broad range !in a broad range !

Ion exchange chromatography

size exclusion chromatography

C8 and C18 reversed phase chromatography

B. Thuerig et al. PMPP, 2006.

No distinct peaks Activity was spread

over a wide range of fractions

APPROACHS TO PURIFY...

The MAMP-activity of Pen is The MAMP-activity of Pen is heterogeneousheterogeneous in polarity, in polarity, size and size and chargecharge

A MAMP has structural diversity ?

ORA mixture of different

MAMPs ?

Pen• Crude extract• Aqueous solution (45mg/ml)

Pen1000

• Dialysed in 1kD cut-off membrane• Get rid of small chitin fragments &

other small molecule

Penpre

• Pre-purified on C18 RP pre-column with 0,1% TFA

• Eluted with Methanol

CHARACTORIZATION & PURIFICATION

• C18 reversed phase chromatography

• Eluted with Methanol gradientHPLC

HPLC

• C18 reversed phase chromatography• Eluted with Methanol gradient

-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 mlElultion Volume (ml)

pH assay:

• 1µl from each fraction• 400µl Arabidopsis cell culture• pH measured after 25 min

No single sharp peak but 2 giant “hills„ No single sharp peak but 2 giant “hills„

Are they the same MAMP-activity in principle ?

Saturation experiment -Saturate PRRs for C -Add D to the same A.thaliana cells…

Different type of MAMP-activity, D is stronger than C D may contain some C

Time(min)

control

Are these 2 type of MAMP-activity sensitive to protease ?

-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

time (min)time (min)

elf18 was digested in D5, 0,3µl D5 was added.

time (min)

• Control : Prot.K worked

Are they the same MAMP-activity as Chitin ? Saturation experiment -Saturate PRRs for Chitin -Add C & D to the same cells…

300µg Chitin / ml cell culture

900µg Chitin

Not the same as chitin D seems containing more non-chitin activity than C

Chitinase digestion of C & D

C is more sensitive to chitinase than DD contains more non-chitin activity than C !

Control:chitinase worked

Can both of them induce ethylene response ?

C contains major chitin-like activity D contains a good ethylene-inducing MAMP-activity

Protease digestion of D after chitinase treatment

-Similar small effect observed again

SUMMARY

Proteinaceous activity

A weak proteinic PAMP ?

or

Only the peptide part linked to D ?

Probably overlaped !

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

OUTLOOK

overlapping