Comparative enrichment of Phosphopeptides from ergosterol-

treated A.thaliana leaves

Robyn KlemptnerUniversity of Johannesburg

MSc supervisors:Dr. L.A. Piater

Prof. I.A. DuberyProf. R. Meijboom

Background

BIGGEST 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)

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. Signal transduction. WRKYs. MAMP/PAMP-triggered

immunity (M/PTI).

Effectors Against specific host. Suppress M/PTI. Effector-triggered

immunity (ETI). Recognized by

intracellular receptors. ROS, HR, SAR.

Ergosterol – an “orphan” MAMP Ergosterol = 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)

What we know…. Calcium-dependent

protein kinases – Ca2+

influx.

Phospholipase Kinase C.

MAPKs.

WRKY transcription factors.

Phenylpropanoid pathway – metabolites.

H2O2 generation.

Ergosterol perception is specific.

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???

= Post-translational modification= structural change = functional change

Serine, Threonine and Tyrosine residues of proteins = kinases = signal transduction activation.

Kinases vs Phosphatases = regulation.

(Schulze, 2010)

Phosphorylation

(Yang et al, 1997; Thurston et al., 2005)

Figure 3: An overview of signal transduction pathways in defense responses in plants.

Phosphoproteins & signal transduction

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 chromatography Antibody-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)

Dendrimers

(Holister et al., 2003)Figure 4: Dendrimer nanopolymers of varying generations.

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.

PolyMAC and PAMAM

(Iliuk et al., 2010; Mandeville & Tajmir-Raihi, 2010)

Figure 6A & 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.

Hypothesis

Dendrimer-based technologies provide enhanced phosphopeptide enrichment from A.thaliana following ergosterol elicitation.

Objectives

1. Elicitation of A.thaliana with ergosterol and total protein expression profiles.

2. Enrich plant phosphopeptides using dendrimer technologies.

3. Compare efficiencies of PAMAM vs. PolyMAC dendrimer enrichment techniques.

4. Successful identification of differentially expressed phosphorylated proteins by Mass spectrometry.

5. Possibly elucidate ergosterol-induced signal transduction pathway of A. thaliana .

MethodologyPAMP treatment of A.thaliana plants Untreated control 250 nM ergosterol EtOH control 0, 6, 12, 24, 48, 72 hr and 7 days

Total protein extraction Liquid N2 TCA/acetone/phenol Ammonium acetate/meOH

precipitation Buffers 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)

Methodology

Western Blotting 1° Ab= Anti-active MAPK= Anti-phosphoTyr

SDS-PAGE (1D) 10 ug total/lane 10% 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 = MASCOT

SDS-PAGE: total protein

Figure 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.

kDA260140 10070

50

40

35

25

15

10

0hr 6hr 12hr 24hr 48 hr 72hr 7 days

Erg EtOH EtO

H

EtOH

EtOH

EtOH

EtOH

M Erg Erg Erg Erg

Erg ErgEtOH

MUT

~27 kDa

Accession Description MW [kDa] calc. pI

P94072 Germin-like protein subfamily 3 member 21.8 6.76

Q9ZUU4 Ribonucleoprotein At2g37220, chloroplastic 30.7 5.16

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 23.5 6.23

P19366 ATP synthase subunit beta, chloroplastic 53.9 5.50

Table 1: Protein identities following Mass Spectrometry of gel slices

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 hr and 12 hr ergosterol treatment respectively.

A B

C D

pH 4 - 7

pH 4 - 7

pH 4 - 7

pH 4 - 7

Western Blotting – Anti phosphotyrosine

Figure 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 days

Western blotting – Anti active MAPK

Figure 11: Autoradiography film showing the presence of MAPKs at 42 – 45 kDa.

~ 40 - 45 kDa

~ 15 - 25 kDa

UT 0hr 6hr 12hr 24hr

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.

MALDI-TOF

Figure 12: MALDI-TOF spectra of phosphopeptide standard (α-casein/BSA) and PolyMAC enriched sample.

Conclusions Preliminary 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)

Further studies and research outcomes

Final 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.

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.

Acknowledgements

ReferencesAvrova, A.O., Taleb, N., Rokka, V-M., Heilbronn, J., Campbell, E., Hein, I., Gilroy, E.M., Cardle, L., Bradshaw, J.E., Stewart, H.E., Fakim, Y.J., Loake, G. and Birch, P.R.J. (2004) Potato oxysterol binding protein and cathepsin B are rapidly up-regulated in independent defense pathways that distinguish R-gene-mediated and field resistance to Phytophthora infestans. Molecular Plant Pathology, 5: 45-56.

Boller T. and He Y.S, (2009) Innate Immunity in Plants: An Arms Race Between Pattern Recognition Receptors in Plants and Effectors in Microbial Pathogens. Journal of Science, 324: 742-744.

Dodds, P. N., & Rathjen, J. P. (2010). Plant immunity: towards an integrated view of plant-pathogen interactions. Nature reviews. Genetics, 11 (8), 539-48.

Fairbanks G, Steck TL, W. D. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10 (13), 2606-17.

Godfray, H. C. J., Beddington, J. R., Crute, I. R., Haddad, L., Lawrence, D., Muir, J. F., Pretty, J., et al. (2010). Food security: the challenge of feeding 9 billion people. Science , 327 (5967), 812-8. Goldring, J. P., & Ravaioli, L. (1996). Solubilization of protein-dye complexes on nitrocellulose to quantify proteins spectrophotometrically. Analytical biochemistry, 242 (2), 197-201.

Holister, P., Vas, C.R., Harper, T., (2003) Dendrimers. Clientifica, New York, pg 2-15.

Iliuk, A. B., Martin, V. A, Alicie, B. M., Geahlen, R. L., & Tao, W. A. (2010). In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Molecular & Cellular Proteomics , 9 (10): 2162-72.

Klajnert, B., & Bryszewska, M. (2001). Dendrimers: properties and applications. Acta biochimica Polonica, 48 (1), 199-208.

Klemptner, R.L., Sherwood, J. S., Tugizimana, F., Piater, L. A., & Dubery, I. A. (2014). Ergosterol, an orphan fungal microbe-associated molecular pattern (MAMP). Molecular Plant Pathology

Lochman J. and Mikes V., (2006) Ergosterol treatment leads to the expression of a specific set of defence-related genes in tobacco. Journal of Plant Molecular Biology, 62:43–51.

Mandeville, J. S., & Tajmir-Riahi, H. A. (2010). Complexes of dendrimers with bovine serum albumin. Biomacromolecules, 11 (2): 465-72.

Meimoun, P., Ambard-Bretteville, F., Colas-des Francs-Small, C., Valot, B., & Vidal, J. (2007). Analysis of plant phosphoproteins. Analytical biochemistry, 371 (2): 238-46.

Peters, E. C. (2005). A polymeric solution for enriching the phosphoproteome Insect transgenesis by site-specific. Nature Methods, 2 (8): 579-580.

Rossard S., Roblin G. and Atanassova R., (2010) Ergosterol triggers characteristic elicitation steps in Beta vulgaris leaf tissues. Journal of Experimental Botany, 61: 1807–1816.

Schulze, W. X. (2010). Proteomics approaches to understand protein phosphorylation in pathway modulation. Current opinion in plant biology, 13 (3): 280-87.

Tao, W. A., Wollscheid, B., Brien, R. O., Eng, J. K., Li, X.-jun, Bodenmiller, B., Watts, J. D., et al. (2005). Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nature Methods, 2 (8): 591-598.

Thurston G., Regan S., Rampitsch C., Xing T., (2005) Proteomic and phosphoproteomic approaches to understand plant–pathogen interactions. Journal of Physiological and Molecular Plant Pathology, 66: 3–11.

Wang, W., Vignani, R., Scali, M., & Cresti, M. (2006). A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis, 27 (13): 2782-6.

Weete J.D., Abril M., Blackwel M. (2010) Phylogenetic Distribution of Fungal Sterols. PLoS One. 5: 1-6.

Yang Y., Shah J., and Klessig D.F. (1997) Signal perception and transduction in plant defence response. Journal of Genes and development, 12: 1621-1628.

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