Comparative enrichment of Phosphopeptides from ergosterol-treated A.thaliana leaves

1. Comparative enrichment of Phosphopeptides from ergosterol-treated A.thaliana leaves Robyn Klemptner University of Johannesburg MSc supervisors: Dr. L.A. Piater Prof. I.A. Dubery Prof. R. Meijboom

2. 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)

3. 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. Figure 1: A figure that clearly indicates the two mechanisms of pathogen detection and induction of corresponding immune responses. (Klemptner et al., 2014)

4. 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. A B C D E F 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. (Avrovaet al., 2004; Wang , 2004; Rossard et al., 2010; Weeteet al., 2010; Klemptner et al., 2014)

5. 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.

6. Proteomics VS Genomics and Metabolomics

7. Phosphorylation = Post-translational modification = structural change = functional change Serine, Threonine and Tyrosine residues of proteins = kinases = signal transduction activation. KinasesvsPhosphatases= regulation. (Schulze, 2010)

8. Phosphoproteins & signal transduction Figure 3: An overview of signal transduction pathways in defense responses in plants. (Yang et al, 1997; Thurston et al., 2005)

9. Enriching phosphoproteins • Important 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. (Meimounet al., 2007; Iliuk et al., 2010)

10. Dendrimers Figure 4: Dendrimer nanopolymers of varying generations. (Holisteret al., 2003)

11. Dendrimer isolation mechanism Add dendrimer to tryptic digest Filter through spin-column to isolate dendrimer + bound peptides Phosphorylated groups bind to surface amino groups Cleave peptides by acid hydrolysis Figure 5: The fundamental dendrimer-based phosphopeptide isolation mechanism. (Peters, 2005)

12. PolyMAC and PAMAM • Dendrimers with modified terminal groups on the surface. • Specific affinity for phosphorylated amino acid residues. A B Figure 6A & B: The PolyMAC dendrimer and its 2 types of side-chain moieties; the traditional PAMAM dendrimer with amine surface groups. (Iliuk et al., 2010; Mandeville & Tajmir-Raihi, 2010)

13. Hypothesis

14. Objectives • Elicitation 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 .

15. Methodology • PAMP treatment of A.thaliana plants • Untreated control • 250 nM ergosterol • EtOH control • 0, 6, 12, 24, 48, 72 hr and 7 days • SDS sample buffer • SDS-PAGE gels (1D) • Western blotting • Total protein extraction • Liquid N2 • TCA/acetone/phenol • Ammonium acetate/meOH precipitation • Buffers for downstream protocols • 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)

16. Methodology • SDS-PAGE (1D) • 10 ug total/lane • 10% gel • Fairbanks/silver staining • Western Blotting • 1° Ab • = Anti-active MAPK • = Anti-phosphoTyr • 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

17. SDS-PAGE: total protein kDA 260 140 100 70 50 40 35 25 15 10 EtOH EtOH EtOH Erg EtOH EtOH Erg Erg EtOH EtOH Erg Erg Erg Erg M UT M ~27 kDa 0hr 6hr 12hr 24hr 48 hr 72hr 7 days 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.

18. Table 1: Protein identities following Mass Spectrometry of gel slices

19. A B pH 4 - 7 pH 4 - 7 C D pH 4 - 7 pH 4 - 7 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.

20. Western Blotting – Anti phosphotyrosine ~40 kDa ~27 kDa UT 0hr 6hr 12hr 24hr 48 hr 72hr 7 days 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.

21. Western blotting – Anti active MAPK ~ 40 - 45 kDa ~ 15 - 25 kDa UT 0hr 6hr 12hr 24hr Figure 11: Autoradiography film showing the presence of MAPKs at 42 – 45 kDa.

22. MALDI-TOF mass spectrometry • Preliminary analysis of phosphopeptide enrichment. • DHB and CHCA matrices. • α-casein/BSA standard + samples + calibration peptides. • BrukerDaltonicsAutoFlex at the CSIR, Biosciences. • Nitrogen laser/ positive ion mode.

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

24. 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)

25. 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.

26. Acknowledgements • 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.

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29. Thank you