Athaliana leaves Robyn Klemptner University of Johannesburg MSc supervisors Dr LA Piater Prof IA Dubery Prof R Meijboom Background BIGGEST CHALLENGE 9 BILLION people by 2050 ID: 275196
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Slide1
Comparative enrichment of Phosphopeptides from ergosterol-treated A.thaliana leaves
Robyn KlemptnerUniversity of JohannesburgMSc supervisors:Dr. L.A. PiaterProf. I.A. DuberyProf. R. MeijboomSlide2
BackgroundBIGGEST CHALLENGE
: 9 BILLION people by 2050!!!Food security – global importance.Plant exposed to multiple pathogens.Price hikes – plant diseases.
Preformed defenses.
Innate immunity = overcome pathogens.
PAMP-Triggered Immunity (PTI) + Effector-triggered immunity (ETI).
(Lochman & Mikes, 2006 ; Godfray, 2009)Slide3
Figure 1: A figure that clearly indicates the two mechanisms of pathogen detection and induction of corresponding immune responses.(Klemptner
et al., 2014)Innate immune responses
MAMPs/PAMPs
Preformed defenses compromised.
Bind PRR at cell membrane.S
ignal transduction.WRKYs.MAMP/PAMP-triggered immunity (M/PTI).
Effectors
A
gainst specific host.
S
uppress M/PTI.
E
ffector-triggered immunity (ETI).
Recognized by intracellular receptors.
ROS, HR, SAR.Slide4
Ergosterol – an “orphan” MAMPErgosterol
= Fungal sterol, fungal cell membrane component.Implicated in major crop losses world wide.Receptor/signal transduction pathway not yet elucidated.
Trigger immune response in sugar beet, grape, tomato and tobacco plants.
Reactive oxygen species, ion fluxes,
PR proteins, LTPs
.
Figure 2
: 3D models of various sterol compounds that have been used to study receptor interactions in plant-pathogen interactions. A: Ergosterol; B:
Brassicasterol
;
C: Sitosterol
;
D: Stigmasterol; E: Campesterol; F: Cholesterol.
A B C D E F
(
Avrova
et al
.,
2004;
Wang
,
2004;
Rossard
et al
.,
2010
;
Weete
et al.,
2010; Klemptner
et al.,
2014
)Slide5
What we know….
Calcium-dependent protein kinases – Ca2+ influx.Phospholipase Kinase C.MAPKs.
WRKY transcription factors.
Phenylpropanoid pathway – metabolites.
H
2O2 generation.
Ergosterol perception is specific.Slide6
Proteomics VS Genomics and Metabolomics
Genomics= genetic level = mRNA….
But
mRNA = protein? NOT ALWAYS!
“Lost in Translation”
Proteomics
= key players in
signaling.
= receptors, kinases,
PR-proteins.
Metabolomics
= metabolites:
jasmonates
etc
.
= overlapping/
intersecting.
= “end products”
= pathways???Slide7
= Post-translational modification= structural change = functional changeSerine
, Threonine and Tyrosine residues of proteins = kinases = signal transduction activation.Kinases
vs
Phosphatases = regulation
.(Schulze, 2010)
Phosphorylation
Slide8
(Yang et al, 1997; Thurston et al.,
2005)Figure 3: An overview of signal transduction pathways in defense responses in plants. Phosphoproteins & signal transductionSlide9
Enriching phosphoproteinsImportant players in signal transduction
BUT occur in low abundance! < only transiently phosphorylated!Provide a greater knowledge of defense-related signal transduction networks.Methods of enrichment include:Affinity chromatographyAntibody-based affinity capture
Chemical derivatization
Metal ion-based affinity capture
Thus, more sensitive and reliable method required = DENDRIMERS
!Novel proteome investigation in plants since dendrimer-based enrichment techniques have yet to be applied to plant studies.
(
Meimoun
et al.,
2007; Iliuk
et al.,
2010)Slide10
Dendrimers
(Holister
et al
.,
2003)Figure 4:
Dendrimer nanopolymers of varying generations.Slide11
Dendrimer isolation mechanism
(Peters, 2005)
Add dendrimer to
tryptic
digest
Phosphorylated groups bind to surface amino groups
Filter through spin-column to isolate dendrimer + bound peptides
Cleave peptides by acid hydrolysis
Figure 5
:
The fundamental dendrimer-based phosphopeptide isolation mechanism. Slide12
PolyMAC and PAMAM
(Iliuk et al., 2010; Mandeville & Tajmir-Raihi, 2010)
Figure
6
A & B
: The PolyMAC dendrimer and its 2 types of side-chain moieties; the traditional PAMAM dendrimer with amine surface groups.
A
B
Dendrimers with modified terminal groups on the surface.
Specific affinity for phosphorylated amino acid residues.Slide13
HypothesisSlide14
ObjectivesElicitation of
A.thaliana with ergosterol and total protein expression profiles.Enrich plant phosphopeptides using dendrimer technologies.Compare efficiencies of PAMAM vs. PolyMAC dendrimer enrichment techniques.
Successful identification
of differentially expressed phosphorylated proteins by Mass spectrometry.
Possibly elucidate ergosterol-induced signal transduction pathway
of A. thaliana .Slide15
Methodology
PAMP treatment of A.thaliana plantsUntreated control 250 nM ergosterol EtOH control0, 6, 12, 24, 48, 72 hr and 7 days
Total protein extraction
Liquid N
2
TCA/acetone/phenol
Ammonium acetate/meOH precipitationBuffers for downstream protocols
SDS sample buffer
SDS-PAGE gels (1D)
Western blotting
Urea sample buffer
PolyMAC and PAMAM enrichment
IEF sample buffer
Isoelectric focusing (2D)
Protein concentration quantification
Amido black assay
BSA standards (0.625, 1.25, 2.5, 5 and 10
ug
/
uL
)
Samples and standards – nitrocellulose membrane
Absorbance at 600 nm
(Granado, 1995; Lochman and Mikes, 2004; Wang
et al.,
2006)Slide16
Methodology
Western Blotting1° Ab= Anti-active MAPK= Anti-phosphoTyr
SDS-PAGE (1D)
10
ug total/lane10% gel
Fairbanks/silver staining
IEF (2D-PAGE)
pH 3-10 and pH 4-7
Fairbanks/silver staining
Dendrimer enrichment
Trypsin digest
C-18 peptide clean up
Enrichments
= PAMAM
=PolyMAC
Mass spectrometry analysis
MALDI-TOF
=DHB/CHCA
LC-MS/MS
Peptide sequences
Protein ID = MASCOTSlide17
SDS-PAGE: total proteinFigure 8: SDS-PAGE separation of all protein samples. Despite there being a large number of bands that are common to all the samples, there is a protein that shows differential expression and has an approximate size of 27 kDa.
kDA
260
140
100
70
50
40
35
25
15
10
0hr 6hr 12hr 24hr 48 hr 72hr 7 days
Erg
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
M
Erg
Erg
Erg
Erg
Erg
Erg
EtOH
M
UT
~27 kDaSlide18
AccessionDescription
MW [kDa]calc. pIP94072Germin-like protein subfamily 3 member 21.8
6.76
Q9ZUU4
Ribonucleoprotein At2g37220, chloroplastic
30.75.16
Q9FN48
Calcium sensing receptor,
chloroplastic
41.3
9.39
Q05431
L-ascorbate peroxidase 1, cytosolic
27.5
6.13
O65282
20 kDa chaperonin, chloroplastic
26.8
8.88
Q9SIU8-2
Isoform 2 of Probable protein phosphatase 2C 20
30.5
6.14
Q41951
Aquaporin TIP2-1
25.0
5.64
Q0WP12-2
Isoform 2 of Thiocyanate methyltransferase 1
25.3
4.82
O24456
Guanine nucleotide-binding protein subunit beta-like protein A
35.7
7.71
Q41963
Aquaporin TIP1-2
25.8
5.06
Q8LAA6
Probable aquaporin PIP1-5
30.6
8.82
Q96291
2-Cys peroxiredoxin BAS1, chloroplastic
29.1
7.44
P42742
Proteasome subunit beta type-1
24.6
7.40
Q9LS02
Allene oxide cyclase 2, chloroplastic
27.6
7.43
P42758
Dehydrin Xero 2
20.9
9.38
O23016
Probable voltage-gated potassium channel subunit beta
36.5
7.42
P46422
Glutathione S-transferase F2
24.1
6.35
Q9SRH5
Mitochondrial outer membrane protein porin 1
29.4
8.73
Q9LHA7
Peroxidase 31
35.3
9.06
Q9ZRW8
Glutathione S-transferase U19
25.6
6.04
O04834
GTP-binding protein SAR1A
22.0
7.53
P43297
Cysteine proteinase RD21a
50.9
5.41
Q9ZTW3
Vesicle-associated membrane protein 721
24.7
8.75
P28186
Ras-related protein RABE1c
23.8
7.83
P41916
GTP-binding nuclear protein Ran-1
25.3
6.86
P41088
Chalcone--flavonone isomerase 1
26.6
5.50
Q39258
V-type proton ATPase subunit E1
26.0
6.40
P94040
Germin-like protein subfamily 3 member
21.5
9.20
O64518
Metacaspase-5
44.8
6.61
Q84W80-2
Isoform 2 of F-box/LRR-repeat protein
22.8
8.22
O81147
Proteasome subunit alpha type-6-B
27.3
6.09
Q42592
L-ascorbate peroxidase S, chloroplastic/mitochondrial
40.4
8.28
Q8LE52
Glutathione S-transferase DHAR3, chloroplastic
28.5
7.74
P43286
Aquaporin PIP2-1
30.5
8.40
P42760
Glutathione S-transferase F6
23.56.23P19366ATP synthase subunit beta, chloroplastic53.95.50
Table 1: Protein identities following Mass Spectrometry of gel slicesSlide19
Figure
9A, B, C & D
:
2D-PAGE gels (11.25%) of ergosterol-treated samples following
IEF,on
a pH 4-7 IPG strip.
Figure A shows spots resulting from the untreated control and those in figure B show those resulting from a 0 hour ergosterol
treatment. Figures C and D show spots resulting from a 6
h
r and 12 hr ergosterol treatment respectively.
A B
C D
pH 4 - 7
pH 4 - 7
pH 4 - 7
pH 4 - 7Slide20
Western Blotting –
Anti phosphotyrosineFigure 10: Autoradiography films showing Tyrosine-phosphorylated proteins following Western blotting. The dotted yellow boxes indicate a ~27 kDa protein that exhibits a strong binding signal to the anti-active phosphotyrosine antibody.
~27 kDa
~40 kDa
UT
0hr 6hr 12hr 24hr 48 hr 72hr 7 daysSlide21
Western blotting – Anti active MAPKFigure 11: Autoradiography film showing the presence of MAPKs at 42 – 45 kDa.
~ 40 - 45 kDa
~ 15 - 25 kDa
UT
0hr 6hr 12hr 24hr Slide22
MALDI-TOF mass spectrometry
Preliminary analysis of phosphopeptide enrichment.DHB and CHCA matrices.α-casein/BSA standard + samples + calibration peptides.Bruker Daltonics
AutoFlex
at the CSIR, Biosciences.Nitrogen laser/ positive ion mode.Slide23
MALDI-TOFFigure 12: MALDI-TOF spectra of phosphopeptide standard (
α-casein/BSA) and PolyMAC enriched sample.Slide24
ConclusionsPreliminary MALDI analysis indicates successful phosphopeptide enrichment.
Anti-PhosphoTyr = specific phosphoproteins.~27 kDa protein across samples = phosphorylated protein. Confirm identity.
Ergosterol-specific proteins = germin-like protein.
Defense and stress-related proteins are evident =
aquaporins
, LRR, calcium binding, Ras
-related protein.
(Klemptner
et al.,
2014)Slide25
Further studies and research outcomesFinal LC-MS/MS analysis = CSIR (Pretoria)/CPGR (Cape Town).
Identify total differentially expressed proteins.Compare to western blots, SDS-PAGE and 2D.Compare enrichment of in-gel digested proteins to proteins in solution – efficiency of dendrimer-based enrichments.Compare genomic, proteomic and metabolomic data.Slide26
Dr. L. Piater, Prof. Dubery, Prof. R. Meijboom.Prof. A.W. Tao – Tymora Analytical/ Purdue University – Indiana, USA.
National Research Foundation.Dr. Stoyan Stoychev – CSIR Biosciences, Pretoria.Dr. Salome Snyman – Stellenbosch University.
AcknowledgementsSlide27
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Thank you