nikita da camara supervisor: dr. l.a. piater co-supervisor ...the proteomic analysis of inap-treated...

Nikita da Camara

Supervisor: Dr. L.A. Piater Co-supervisor: Prof. I.A. Dubery

Figure 1: The various levels of defence in plant systems.

Figure 2: The primary innate immunity is either localized or systemic.

(Van der Ent et al., 2009; Barilli et al., 2012; Henry et al., 2012 ;Pastor, 2012; Slaughter et al., 2012)

Figure 3: The chemical structure of INAP.

(Dubery et al., 1999; Pritchard and Birch, 2011; Madala et al., 2012; Ramptisch and Bykova, 2012; Rodrigues et al., 2012; Tanou et al., 2012; Maake, 2013)

• Isonitrosoacetophenone. • ‘priming’ agent??? • 2 3� ‘omics’ completed.

• Plant-pathogen language = PROTEINS.

• Bridges the Gap. • Complexes + PTMs = priming. • Physiological state of an organism.

The proteomic analysis of INAP-treated A. thaliana plants could provide a greater understanding of the physiological state of the plants and, in conjunction with the identification of the protein profiles, will determine the validity of INAP as a priming agent.

SDS-PAGE IEF

2D Gels

Urea/CHAPS Buffer SDS Sample Buffer

Western Blotting

Amido Black (BSA Standards)

Broad Range (pH 3 – 10)

Narrow Range (pH 4 – 7)

Anti-Active MAPK Anti-Pho-Tyr

(Wang et al., 2006)

Protein extraction (TCA-Phenol-Acetone)

INAP [1 mM] elicitation (10 mM MgCl2 Control) over 0, 8, 16, and 24 hours

Cultivation and growth of A. thaliana plants.

± 2 g plant material

kDa M C T C T C T C T 0 h 8 h 16 h 24 h

Time Study Sample

260 140 100 70 50 40 35 25 15 10

PageRuler Broad Range

Protein Ladder

1 2 3 4 5 6 7 8 9

Lane Number

Figure 4: A representative 10% SDS-PAGE gel of induction confirmation in A. thaliana.

260 140 100 70 50 40 35 25 15 10

kDa M


Protein Ladder

pH 3 – 10

0h C 260 140 100 70 50 40 35 25 15 10

kDa M


Protein Ladder

pH 3 – 10

8h C

260 140 100 70 50 40 35 25 15 10

kDa M


Protein Ladder

pH 3 – 10

16h C 260 140 100 70 50 40 35 25 15 10

kDa M


Protein Ladder

pH 3 – 10

24h C

Figure 5: Representative 2D gels for broad range IEF protein analysis in A. thaliana.

0h T 8h T

16h T 24h T

Figure 6: Representative 2D gels for narrow range IEF protein analysis in A. thaliana.

0h C 260 140 100 70 50 40 35 25

kDa M


Protein Ladder

pH 4 – 7

8h C 260 140 100 70 50 40 35 25 15

kDa M


Protein Ladder

pH 4 – 7

16h C 24h C 260 140 100 70 50 40 35 25 15

kDa M


Protein Ladder

pH 4 – 7

260 140 100 70 50 40 35 25 15

kDa M


Protein Ladder

pH 3 – 10

0h T 8h T

16h T 24h T

kDa M C T C T C T C T 0 h 8 h 16 h 24 h

Time Study Sample

260 140 100 70 50 40 35 25 15 10


Protein Ladder

1 2 3 4 5 6 7 8 9

Lane Number Figure 7: Western Blot for Anti-Active MAP Kinase in A. thaliana.

(Schaefer et al., 2009; MacPhee, 2010; Mahmood and Yang, 2012)

CPGR

8-Plex iTRAQ/nano LC/MS

Extraction Buffer [6 M Guanidine, 25 mM HEPES,

5 mM DTT and 5% (w/w) PVP pH 7.5]

Resuspension Buffer [50 mM NH4HCO3 1% RapiGest

and 2.5 mM DTT pH 8.5]

Induction with 1 mM INAP in acetone (0 h, 8 h, 16 h, 24 h) + all controls

0.5 g of cells ground to powder

Amido Black (BSA Standards)

Pellets

SDS-PAGE

Lysozyme Spike [30 μg/mL]

Silver Staining

(Personal Communication, Mare Vlok, CPGR)

Data Analysis

0h T

8h T

8h U

T

16h

T

16h

UT

24h

T

24h

UT

Biosynthetic 9%

Transport 14%

DNA/Transcription 6%

Metabolism/Energy 14%

Translation 13%

Unclassified 19%

Response 10%

Growth 4%

Signal Transduction 8%

Defence 3%

Figure 8: A Pie Chart indicative of the various proteins obtained and their biological classification.

Down-Regulated 53%

Up-Regulated 44%

Up/Down-Regulation

3%

Figure 9: A Pie Chart displaying the split between the up/down-regulated proteins.

Figure 10: The classification of the down-regulated proteins.

Biosynthetic 7%

Transport 21%



Translation 13%

Unclassified 16%

Response 7%

Growth 4%


Defence 3%

Biosynthesis Plasma membrane ATPase 4 ATP generation Transport Pleiotropic drug resistance protein 1 Secretion of antimicrobial terpenes Reticulon-like protein Membrane, Intracellular trafficking Clathrin heavy chain Receptor–Mediated Endocytosis Small GTPase Rab5b Vesicle transport DNA/Transcription Histone H2B DNA compaction and Gene access Transcription elongation factor SPT5 Transcription Endonuclease 1 DNA Replication/Repair Metabolism/Energy OrfB protein Encodes protein involved in ATP generation Cytochrome c oxidase subunit 2 Electron transport chain, ATP generation Progesterone 5beta reductase-A Cardenolide formation Proton pump interactor 1 ATP generation Translation 40S ribosomal protein S24 Protein translation 60S ribosomal protein L23a Protein translation 60S ribosomal protein L36 Protein translation Putative translation initiation factor 2B beta subunit Protein translation DnaJ-like protein Chaperone, involved in protein folding Uncharacterised CYP73A47v3 ?????? Response Mitochondrial small heat shock protein Heat shock protein/Chaperone increase stress tolerance Putative methyltransferase family protein Normal growth, pectin/lignin biosynthesis, antimicrobial compounds from

phenylpropanoids ASR (Fragment) Regulation of ABA response gene expression Signal Transduction Superoxide dismutase [Cu-Zn] Detoxify O2•-- Inorganic pyrophosphatase * Signalling Ras-related GTP-binding protein MAPK activation Calcium-dependent protein kinase Early signal transduction Phospholipase D * Production of Phosphatidic Acid Defence Polygalacturonase inhibiting protein Inhibition of pathogen polygalacturonase

Table 1: Identifiable proteins displaying Down-regulation

(Blackbourn and Jackson, 1996; Liscovitch et al., 2000; Morsomme and Boutry, 2000; Gardiner et al., 2001; Çakir et al., 2003; D’Ovidio et al., 2004; He et al., 2004; Ludwig et al., 2005; Wang 2005; Bargmann and Munnik 2006; Di Matteo et al., 2006; Federici et al., 2006; Frankel et al., 2006; Juge, 2006; Klimecka and Muszyńska, 2007; Kobayashi et al., 2007; Ahuja et al., 2010; Dodd et al., 2010; Nodd, 2010; Maake, 2013)

Biosynthetic 10%

Transport 5%



Translation 14%

Unclassified 22%

Response 12%

Growth 3%


Defence 3%

Figure 11: The classification of the up-regulated proteins

Biosynthesis Phosphomannomutase ABA biosynthesis pathway L-galactose dehydrogenase ABA biosynthesis pathway UDP-glucose:glucosyltransferase Glycosylation Dimethylallyltransferase Phenylpropanoid pathway DNA/Transcription Histone H3 DNA compaction and Gene access Histone H1D DNA compaction and Gene access Metabolism/Energy NADH-Ubiquinone oxidoreductase subunit 7 ATP generation Tobacco pre-pro-cysteine proteinase Protein turnover Glycylpeptide N-tetradecanoyltransferase Myristoylation Translation 40S ribosomal protein S5 Protein translation 40S ribosomal protein S12 Protein translation Response Glycine-rich RNA-binding protein Response to abiotic stress Probable phospholipid hydroperoxide glutathione peroxidase Reduce lipid hydroperoxides

Glutathionine S-transferase (Fragment) Detoxify herbicides Probable glutathione S-transferase MSR-1 Detoxify herbicides Signal Transduction Superoxide dismutase (Fragment) Detoxify O2•-- Inorganic pyrophosphatase * Signalling SKP1 protein Response to auxin and jasmonate Protein phosphatase 2A Negative regulator of signalling e.g. ABA SUMO Protein modification Phospholipase D * Linked to production of Phosphatidic Acid Defence Thioredoxin ROS

Table 2: Identifiable proteins displaying Up-regulation

(Hérouart et al., 1993; Showalter, 1993; Rea, 2000; Conklin, 2001; Alscher et al., 2002; Zhou et al., 2004; Gabaldόn et al., 2005; Wang 2005; Azevedo et al., 2006; Iqbal et al., 2006; Mukhopadhyay and Dasso, 2007; Selvakumar et al., 2006; Kim et al., 2008; Linster and Clarke, 2008; Manzano et al., 2008; Gill and Tuteja, 2010; Tarrago and Gladyshev, 2012; Gupta et al., 2012; Musrap et al., 2014)

Thus far differential proteome profiles have been prompted for A. thaliana leaves when treated with the chemical inducer, INAP. However, further insight shall be obtained from the conduction of 8-plex iTRAQ/nanoLC/MS for the A. thaliana leaves with respect to the up/down-regulated states of the proteins, as has been done with the N. tabacum cell suspensions which provided a more detailed insight into the proteomic activity within the cells.

Barilli, E., Rubiales, D. and Castillejo, M.A. (2012) Comparative Proteomic Analysis of BTH and BABA-Induced Resistance in Pea (Pisum sativum) toward Infection with Pea Rust (Uromyces pisi), Journal of Proteomics, 75: 5189 – 5205. Conrath, U. (2009) Priming of Induced Plant Defence Response, Advances in Botanical Research, 51: 362 – 395. Dubery, I.A., Louw, A.E. and van Heerden, F.R. (1999) Synthesis and Evaluation of 4-(3-methyl-2-butenoxy)isonitrosoacetophenone, a Radiation Induced Stress Metabolite in Citrus, Phytochemistry, 50: 983 – 989. Gong, Y., Liquin, R. and Yu, D. (2013) Abiotic Stress in Plants, Agricultural Chemistry, 10: 114 – 152. Henry, G., Thonart, P. and Ongena, M. (2012) PAMPs, MAMPs, DAMPs and others: an Update on the Diversity of Plant Immunity Elicitors, Biotechnol. Agron. Soc Environ., 16: 257 – 268. Iliuk, A. B., Martin, V. A., Alicie, B. M., Geahlen, R. L., and 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 – 2172 . Jones, J.D.G. and Dangl, J.L. (2006) The Plant Immune System, Nature, 444: 323 – 329. Jorrín, J.V., Maldonado, A.M. and Castillejo, M.A. (2007) Plant Proteome Analysis: A 2006 Update, Proteomics, 7: 2947 – 2962. Maake, M.P. (2013) Differential Gene Expression in Nicotiana Tabacum Cells in response to Isonitrosoacetophenone, MSc Dissertation, University of Johannesburg, Submitted. Madala, N.E., Steenkamp, P.A., Piater, L.A. and Dubery, I.A. (2012) Biotransformation of Isonitrosoacetophenone (2-keto-2-phenyl-acetaldoxime) in tobacco cell suspensions, Biotechnol. Lett, 34: 1351 – 1356. Pastor, V., Luna, E., Mauch-Mani, B., Ton, J. and Flors, V. (2012) Primed Plants do not Forget, Environmental and Experimental Botany, in press. Peters, E.C. (2005) A Polymeric Solution for Enriching the Phosphoproteome, Nature Methods, 2(8): 579 – 580. Pritchard, L. and Birch, P. (2011) A Systems Biology Perspective on Plant-microbe Interactions: Biochemical and Structural Targets of Pathogen Effectors, Plant Science, 180: 584 – 603. Quirino, B.F., Candido, E.S., Campos, P.F., Franco, O.L. and Krüger, R.H. (2010) Proteomic Approaches to Study Plant-Pathogen Interactions, Phytochemistry, 71: 351 – 362. Rodrigues, P.M., Silva, T.S, Dias, J. and Jessen, F. (2012) Proteomics in Aquaculture: Applications and Trends, Journal of Proteomics, 75: 4325 – 4345. Ramptisch, C. and Bykova, N.V. (2012) Proteomics and Plant Disease: Advances in Combating a Major Threat to the Global Food Supply, Proteomics, 12: 673 – 690. Slaughter, A., Daniel, X., Flors, V., Luna, E., Hohn, B. and Mauch-Mani, B. (2012) Descendants of Primed Arabidopsis Plants Exhibit Resistance to Biotic Stress, Plant Physiology, 158: 835 – 843. Tao, W.A., Wollscheid, B., O’Brien, R., Eng, J.K., Li, X, Bodenmiller, B., Watts, J.D., Hood, L. and Aebersold, R. (2005) Quantitative Phosphoproteome Analysis using Dendrimer Conjugation Chemistry and Tandem Mass Spectometry, Nature Methods, 2(8): 591 – 598. Tanou, G., Fotopolous, V. and Molassiotis, A. (2012) Priming Against Environmental Challenges and Proteomics in Plants: Update and Agricultural Perspectives, Frontiers in Science, 3(216): 1 – 5. Van der Ent, S., Koornneef, A., Ton, J. and Pieterse, C.M.J. (2009) Chapter 11: Induced Resistance-Orchestrating Defence Mechanisms Through Crosstalk and Priming, Annual Plant Reviews, 34: 334 – 370. 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. Wang, W., Vignani, R., Scali, M. and Cresti, M. (2006) A Universal and Rapid Protocol for Protein Extraction from Recalcitrant Plant Tissues for Proteomic Analysis, Electrophoresis, 27: 2782 – 2786.

Dr. Piater Prof. Dubery Dr. Madala Dr. James UBUNTU lab CPGR NRF Molecular Plant Pathogen Interactions Group Department of Biochemistry UJ

INAP 4’-hexopyranosyloxy-3’-methyloxyisonitrosoacetophenone

o Hydroxylation, methoxylation and glucosylation. o Phenylpropanoid pathway.

(Madala et al., 2012)

• Thiols

• [Detergents] • Active Proteases

• Ammonium Acetate • Ammonium Bicarbonate • Ammonium Citrate • Ammonium Tartrate • AMPD • Aminoguandine Bicarbonate Salt • AMP • Ethanolamine • Gly-gly • Tris Buffers

Cysteine Blocking Trypsin Inactivation iTRAQ labelling

250 140 100 70 50 40 35 25 15 10

PageRuler Broad Range Pre-stained

Protein Ladder

kDa M 8h T 8h Tp 8h UT 8h UTp 8h(R) Pilot Study Sample

1 2 3 4 5 6 Lane No.

Figure 6: Pilot 10% SDS-PAGE gel of effective iTRAQ protein extraction.

(Madala et al., 2012)

Proteome Analysis of Isonitrosoacetophenone-treated Plant SystemsBackgroundSlide Number 3Slide Number 4HypothesisSlide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19ReferencesSlide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28

nikita da camara supervisor: dr. l.a. piater co-supervisor ...the proteomic analysis of inap-treated...

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