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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 22

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code HH3216SFV

2. Project title

Novel proteomic and biosensor based strategies for the detection of downy mildew infection.

3. Contractororganisation(s)

Centre for Research in Plant Sciences, University of the West of England, Coldharbour Lane, Bristol. BS16 1QY

54. Total Defra project costs £ 364,283(agreed fixed price)

5. Project: start date................ 03 March 2003

end date................. 02 March 2006


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

1. Project aimTo identify protein biomarkers that are indicative of pea downy mildew infection and investigate rapid detection technologies for this pathogen.

2. Objectives1. To research and identify cellular biomarkers that are specifically over or under-expressed in pea

plants infected by downy mildew.2. To produce high affinity monoclonal antibodies (MAbs) to the specific biomarker(s) identified3. To write and present a coherent constructive scientific report of the research undertaken.

3. Summary of methods and main findings

Proteomics is a relatively recent technique that enables all the proteins with a cell system (e.g. plant leaves infected by a pathogen such as Peronospora viciae, which causes downy mildew of pea) to be extracted and analysed in order to generate what is termed a proteome. We have compared the proteomes of healthy pea leaves with the proteomes of leaves infected by the pathogenic microorganisms P. viciae, Erysiphe pisi (powdery mildew), Botrytis cinerea (grey mould) and Pseudomonas syringae pv. pisi (bacterial blight) and leaves subjected to water deficit and mechanical wounding stresses. This has enabled the identification of proteins that change their abundance following specific infections or stresses, and allowed selection of proteins that can be used as markers for these stresses. A gene for one of these proteins, abscisic acid response protein 17 (ABR17) has been cloned, and this has enabled production of the protein in sufficient quantities to allow antibodies to be raised. Following screening for specificity, one antibody has been selected and is available for incorporation into disposable diagnostic kits, such as the lateral flow device developed by the UK Government’s Central Science Laboratory (CSL).

The main conclusions and areas for further work are listed below.

1) This final report demonstrates that the project has met the original objectives. Work on the ABR17 protein provides proof-of-principle that the proteomics approach can be used to identify potential marker proteins, which can be cloned, expressed in vitro and used to raise monoclonal antibodies (MAbs) suitable

2) The comparative proteomic analysis of the different abiotic and biotic stresses has indicated that ABR17 has potential as a general marker for fungal and oomycete infection of pea leaves (at least for


those pathogens and biotic stresses assessed).

3) Two pea-derived proteins (chloroplastic GAPDH and photosystem I reaction centre subunit) were identified as only being increased in abundance for P. viciae at 4 days post-inoculation (dpi), and have potential as markers of pre-symptomatic downy mildew infection. They merit further investigation (proteomic, gene cloning, in vitro expression, MAb production) as for ABR17.

4) Several other proteins identified show differential changes in abundance (either increase, decrease or no change) depending on the particular cause of stress.

5) Of particular significance is one unidentified protein that increased in abundance only following B. cinerea infection. This has particular potential as an early warning marker for grey mould in the cut flower and soft fruit market, and merits further investigation.

6) The abundance of proteins was different at different stages of P. viciae infection (4 vs 7 vs 10 dpi). Therefore future work on this and other biotic and biotic stresses should include time-course studies.

7) The additional work completed (i.e. work not in the original contract) has started to build proteome maps of pea leaves and P. viciae conidiospores. These provide the basis for further proteomic analysis in order to identify specific markers for the major biotic and biotic stresses that reduce yield in UK pea crops.

8) Comparison of the potential protein biomarkers for P. viciae in other downy mildew pathogens (especially Peronospora species) and their host plants merits investigation. This may lead to common markers and to a generic detection system for a range of downy mildew diseases of important UK crops.

9) Further work should extend this analysis to root infections (e.g. by P. viciae, Pythium and Aphanomyces oospores, and nematodes) because infections of roots may induce proteome changes in leaves and provide opportunities for developing diagnostics for root diseases based on leaf sampling.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

1. Project aimTo identify protein biomarkers that are indicative of pea downy mildew infection and investigate rapid detection technologies for this pathogen.

2. Objectives4. To research and identify cellular biomarkers that are specifically over or under-expressed in pea plants

infected by downy mildew.5. To produce high affinity monoclonal antibodies (MAbs) to the specific biomarker(s) identified6. To write and present a coherent constructive scientific report of the research undertaken.

3. Methods, results and conclusionsThese are listed sequentially within objectives 1-3 and in milestone order within each objective. Secondary milestones are marked (S).


3.1 Objective 1: To research and identify cellular biomarkers that are specifically over or under-expressed in pea plants infected by downy mildew.

Milestone 01/01: QA of the proteomic study. SOP for the identification and characterisation of biomarkers.Milestone S01/02: Review of the proteomic studies. Critical review of the outcomes of the proteomic studies

to date identifying the strategy for further studies.Milestone 01/03: 2nd Review of proteomic studies. The positioning of all biomarker targets will have been

recorded and a selection made for further analysis.Milestone 01/04: Characterised and expressed biomarker(s). The relevance and appropriateness of each

biomarker will have been critically assessed.Milestone 01/05: Functional studies report. Outcomes of strategic importance to infection control strategies

and further research will have been identified and assessed.

Assessment of P. viciae-infected pea leaves at 4 days post-inoculation.

The aim of this work was to identify cellular biomarkers indicative of early-stage, pre-symptomatic infection of pea by the downy mildew pathogen Peronospora viciae.

METHODSInoculation of pea with Peronospora viciaePeronospora viciae isolate Nitouche (kindly provided by Dr David Kenyon, NIAB, Cambridge) was maintained on a mixture of seven pea cultivars (Pisum sativum L. cvs Livioletta, Kelvedon Wonder, Maro, Krupp Pelushka, Early Onward, Solara and Progreta). For protein extractions, plants of P. sativum cv Livioletta were cultivated from seed in compost (Levington F2S) in growth chambers (Sanyo; 16 h light at 20oC, 8 h dark at 14oC). At ten days after sowing, the surfaces of fully developed leaflets were rubbed gently to flatten waxes before being inoculated with P. viciae conidia by the method of El-Gariani and Spencer-Phillips (2004). Control plants were inoculated with sterile distilled water only. Protein extraction and preparationProteins were extracted from fully developed leaflets of healthy pea plants and from P. viciae-infected plants at four days post-inoculation (dpi) according to the method of Giavilisco et al. (2003). The method results in three fractions, comprising (I) cytosolic proteins, (II) membrane bound proteins and (III) nucleic acid-associated proteins. For the present investigation the protein fractions II and III were pooled for analysis. The proteins were prepared for two-dimensional gel electrophoresis (2DE) using the 2D Clean-up kit (GE Healthcare, Little Chalfont, UK) re-suspended in lysis buffer (30 mM Tris pH 8.5; 4% w/v 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulphonate) (CHAPS); 7 M urea; 2 M thiourea) and quantified using the 2D Quant kit (GE Healthcare) according to the manufacturer’s instructions. Protein samples were stored at -80oC until further analysis.

CyDye labellingSamples were labelled using fluorescent cyanine dyes (GE Healthcare) according to the manufacturer’s protocols. The cyanine dyes were reconstituted in fresh 99.8% anhydrous dimethyl formamide. Aliquots of 50 μg of protein were labelled with 400 pmol of amine reactive CyDye for 30 min on ice in the dark, then 1 μl of 10 μM lysine was added to the tube and incubated on ice in the dark to halt the reaction. The samples were made up to 100 μl with rehydration solution (8 M urea; 2% w/v CHAPS; 0.002% w/v Bromophenol Blue; 0.2% w/v 1,4-dithiothreitol (DTT); 2% w/v immobilised pH gradient (IPG) buffer (pH 3-10, GE Healthcare).

Two-dimensional gel electrophoresisSamples were subjected to isoelectric focusing (IEF) using IPG strips (24 cm, GE Healthcare) in the pH 3-10 non-linear range, with rehydration loading to separate proteins in the first dimension according to isoelectric point. The IPG strips were rehydrated overnight at room temperature in the protein sample made up to 450 μl with rehydration solution and covered with mineral oil. The strips were transferred to an Ettan IPGphor II (GE Healthcare) and IEF was performed with a 50 μA limit/IPG strip. IEF voltage conditions were 300 V step and hold for 3 h, 1000 V gradient for 6 h, 8000 V gradient for 3 h and 8000 V step for 4 h 40 min.

SDS-PAGE was used to separate proteins in the second dimension according to molecular weight. Following focusing in the first dimension, each strip was removed from the IEF unit and equilibrated in 15 ml equilibration buffer (50 mM Tris pH 8.8; 6 M urea; 30% v/v glycerol; 2% w/v sodium dodecyl sulphate (SDS); 0.002% w/v bromophenol blue) amended with 150 mg DTT with gentle shaking for 15 min at room temperature. The strips were further equilibrated in 15 ml equilibration buffer amended with 375 mg iodoacetamide with gentle shaking for 15 min at room temperature in the dark. The strips were equilibrated finally in 10 ml equilibration buffer alone for 5 min at room temperature. The strips were loaded onto a 12.5% acrylamide gel (dimensions 24 cm × 20 cm × 1 mm) and overlaid with 1% agarose in SDS running buffer (25 mM Tris pH 8.3; 192 mM glycine; 0.1% SDS) amended with 0.002% (w/v) bromophenol blue. The gels were electrophoresed in an Ettan DALTsix gel system (GE Healthcare) in SDS buffer at 2.5 W per gel for 30 min, followed by 100 W until the bromophenol blue dye


front had run off the bottom of the gels. Two replicate gels were produced for fraction I and the combined fractions II plus III of the extracted proteins. A minimum of three additional, non-CyDye-labelled gels were run for each sample for protein spot picking.Image analysisGels were scanned on a Typhoon 9400 imager (GE Healthcare) to visualise CyDye-labelled proteins. Cy3 scans were obtained using a 532 nm laser and emission filter of 580 nm BP30. Cy5 scans were obtained using a 633 nm laser and a 670 nm BP30 emission filter. Scans were performed at 100 μm resolution with the photomultiplier tube voltage set for a maximum pixel intensity of 60 to 80000 pixels. All images were cropped using ImageQuant V5.2 software prior to analysis to remove areas outside the gel. Analysis of each of the gels was performed with DeCyder Differential In-gel Analysis module software (V5.0) (GE Healthcare) using the double detection setting and an estimated protein spot number of 2500. Parameters for an exclusion filter were determined and applied according to the manufacturer’s instructions, with resulting spots confirmed individually by visual inspection. Protein spots increasing in abundance consistently on all gels were selected for analysis.

MALDI-TOF and ESI Q-TOF MS analysisProtein spots were excised from the gels using an Ettan Spot Picker (GE Healthcare) and subsequently digested using an Ettan Digester (GE Healthcare) with 10 μl trypsin (20 ng μl -1; Sequencing Grade Porcine Modified, Promega, Southampton, UK) in 20 mM ammonium bicarbonate (Sigma, Poole, UK) overnight at room temperature. Following tryptic digestion, the peptides were extracted in 50% acetonitrile/0.1% trifluoroacetic acid to a clean microtitre plate and transferred to an Ettan Spotter (GE Healthcare). The peptides were mixed with matrix (10 mg ml-1 α-cyano-4-hydroxycinnamic acid (Sigma) in 50:50 v/v methanol/acetonitrile) for spotting onto Micromass target plates for analysis in a MALDI-TOF mass spectrometer (Waters-Micromass, UK). Peptide mixtures were analysed using a nitrogen UV laser (337nm). MS data was acquired in the MALDI reflector positive ion mode in the mass range 800-3500 Da. Identification of proteins from the mass fingerprints generated was performed using Proteinlynx Global Server software (V2.0.5, Waters-Micromass). Search parameters included one missed cleavage per peptide, fixed carbamidomethylation of cysteine and variable oxidation of methionine modifications.

Nanoelectrospray ionisation tandem mass spectra were acquired using a Q-TOF Micro mass spectrometer (Waters-Micromass) coupled to a LC Packings capillary liquid chromatography system. Aliquots (15 μl) of peptide solutions prepared as before were injected using an auxiliary solvent flow of 30 μl/min and desalted on a C18

PepMap Nano-Precolumn (5 x 0.3 mm internal diameter (i.d.), 5 μm particle size; Dionex, Amsterdam, The Netherlands) for 4 min. Peptides were eluted and separated using a C18 PepMap100 nano column (15 cm x 75 μm i.d., 3 μm particle size) with a gradient flow of 200 nl/min and solvent system of: auxiliary solvent, 0.1% HCOOH; solvent A, 5% v/v CH3CN/95% v/v 0.1% v/v aqueous HCOOH; solvent B, 80% v/v CH3CN/20% v/v 0.1% v/v aqueous HCOOH. The solvent gradient was 4 min at 5% aqueous solvent B, 5% to 55% B over 40 min, 55% to 80% B over 1 min, maintained at 80% B for 5 min, then reduced to 5% B in 0.1 min and the column washed with solvent A for 9.9 min before the next sample injection. The column was connected to the nanosprayer of the Z-spray ion source using a short length of 75 μm i.d. capillary. Voltages used were 3500 V for the capillary, 45 V for the sample cone and 2.5 V for the extraction cone. MS spectra were acquired throughout the chromatographic run while MS/MS spectra were acquired in data-dependent mode on the most abundant ions having charge states of 2+, 3+ and 4+ between m/z 400-2000. The collision cell was pressurised with 1.38 bar ultra-pure argon (99.999%, BOC) and collision voltages depended on the m/z and charge states of the parent ions. The mass spectrometer was calibrated daily using MS/MS fragment ions from [Glu1]-fibrinopeptide B (Sigma). Processed data were submitted to ProteinLynx Global Server (V2.0.5, Waters-Micromass) and also to MASCOT (Matrix Science) for searching against SwissProt databases. Search criteria were: peptide tolerance of 100 ppm; fragment tolerance of 0.1 Da; 2 missed cleavages per peptide; fixed carbamidomethylation of cysteine and variable oxidation of methionine modifications.

ABR17 gene cloning and expressionSense (ABR17L 5’-CACCATGGGTGTCTTTGTTTTTGAT-3’) and antisense (ABR17R 5’-TTAGTAACCAGGATTTGCCAAAACG-3’) primers designed to amplify the entire ABR17 gene sequence from pea were synthesised (Operon, Germany). ABR17L includes an additional four bases (CACC) at the 5’ end to allow directional cloning into the Gateway pENTR-D/TOPO vector (Invitrogen, Paisley, UK). RNA isolated from P. viciae-infected pea leaves at 4 dpi, using Tri-Reagent according to the manufacturer’s protocol, was used as a template for cDNA synthesis using Superscript III (Invitrogen). Polymerase chain reaction (PCR) amplification (95oC 5 min; 35 cycles of 95oC 30 s, 60oC 30 s, 68oC 30 s; 68oC 4 min) was performed using the proof-reading Pfu polymerase mix (Invitrogen) according to the manufacturer’s instructions. PCR products were purified (Wizard SV gel and PCR clean-up system; Promega) and cloned into the pENTR/D-TOPO entry vector according to the manufacturer’s instructions. The resulting vector was transformed into competent TOP10TM Escherichia coli cells (Invitrogen), plated on Luria Bertani (LB) + kanamycin selective media and colonies screened using PCR to identify those containing the ABR17 gene. The PCR screening used identical conditions to those described above, except that the PCR Mastermix (Promega) was used in place of a proof-reading polymerase, and a colony stab was used as template. Plasmids from three positive colonies were purified (Wizard Plus SV Minipreps Purification System; Promega) and sequenced (Cogenics™, Essex, UK). The plasmids were also digested using


EcoRI (Promega) to release the insert, with agarose gel electrophoresis used to separate the plasmid fragments and confirm insert size. One of the plasmids was used in the LR recombination reaction to transfer the ABR17 gene from the entry vector into the destination vector pDEST17 (Invitrogen), following the manufacturer’s instructions. Fifty μl of the recombination reaction was transformed into chemically competent DH5α cells, plated onto LB + ampicillin and colonies screened by PCR to confirm presence of the ABR17 gene. Plasmids were prepared from two positive colonies and sequenced as before. One of the plasmids containing the entire ABR17 gene inserted in the correct orientation was selected and transformed into competent E. coli BL21-A One-shot cells (Invitrogen) and induced to express the recombinant ABR17 protein according to the manufacturer’s instructions. The cells were harvested, lysed and resulting proteins analysed by 12.5% SDS-PAGE. Bands of interest were excised from the gel using a clean razor blade and subjected to MALDI-TOF mass spectrometry as described above.

RESULTSThe proteins from pea plants inoculated with P. viciae and sterile distilled water controls were visualised using 2D-DIGE, with each gel comprising two experimental samples labelled with Cy3 (control) and Cy5 (P. viciae-infected). Following scanning, DeCyder software detected between 977 and 1337 protein spots on the gel images (Table 1). The total number of spots detected on the gels varied by 19.7% between the two replicates of fraction I (cytosolic soluble proteins), and by 26% between the replicates of combined fractions II plus III (membrane-associated and nucleic acid-associated proteins). The proportion of proteins with decreased abundance following P. viciae infection was 1.7 and 3.4% for the replicates of fraction I, and 0.38 and 0.92% for the replicates of fractions II plus III. The proportion of proteins with increased abundance was 5.2 and 9.2% for fraction I, and 2.3 and 4.5% for fraction II plus III. Therefore the proteins for matrix assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) and quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS) were selected on the basis that their abundance altered significantly and reproducibly on all gel replicates (two CyDye labelled replicates plus a minimum of three additional unlabelled replicate gels) thus removing potential gel artefacts from the analysis. This resulted in 12 proteins that increased in abundance during P. viciae infection. These are indicated in Fig. 1, a representative gel from this experiment. An additional protein (spot number 6) is indicated on the gel as an example of a protein that does not alter in abundance upon infection by P. viciae. The relative fold abundance of protein spots determined by DeCyder software, and selected individual spot images, are illustrated in Figs 2 and 3. The molecular weight, pI, matched peptides and sequence coverage of each protein are given in Table 2.

Table 1. The relative abundance of proteins detected in 2D gels for fractions I (cytosolic proteins) and fractions II + III (membrane and nucleic acid-associated proteins) from pea leaves four days after inoculation with conidia of Peronospora viciae compared to sterile distilled water in two separate experiments (a and b).

Relative abundance of proteins (no. of spots detected)Fraction Decreased Similar Increased TotalIa 22 1245 70 1337Ib 37 960 77 1074II + IIIa 5 1285 30 1320II + IIIb 9 924 44 977

Protein 1 (Figs 1 and 2) was identified as the disease resistance response protein PI176 from pea (accession number P13239). Compared to control plants, its abundance increased by 3.4 and 6.6 fold in fractions I and fractions II plus III respectively. The protein has similar predicted and observed molecular weights and iso-electric points. The three peptides matching published sequences of PI176 represented 16.4% of its amino acid sequence. Protein 2 was identified as abscisic acid responsive protein ABR17 from pea (accession number Q06931). The abundance of this protein resembled that of PI176, with increases of 2.9 and 5.8 for fractions I and fractions II plus III respectively. Theoretical and observed values for molecular weight and pI are in accord, and the seven matched peptides provided 51% coverage of the amino acid sequence. Protein 3 matched to a glycine-rich RNA binding protein from Sinapis alba (accession number P49311) and, compared to controls, had increased abundances of 6.4 and 6.5 for fraction I and fraction II plus III respectively. The theoretical and observed molecular weight and pI values agreed, and the single peptide covered 4.7% of the amino acid sequence. Protein 4 matched to cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from pea (accession number P34922). In comparison to control plants, the abundance of the protein increased by 6.1 in fraction I and by 6.3 in fractions II plus III. Predicted and observed molecular weight and pI values were similar, with the nine matched peptides covering 34.6% of the amino acid sequence. Protein 5 was identified as a chloroplastic precursor of GAPDH A from pea (accession number P12858). Compared to control samples, the protein increased in abundance by 7.7 for fraction I and 8.6 for fractions II plus III. The predicted and observed molecular weight and pI values were in accord with 15 peptides matched, covering 44.7% of the amino acid sequence. Protein 6 was identified tentatively (only one peptide matched) as a thioredoxin M-type chloroplast precursor from pea (accession number P48384) and was selected as a protein that differed by less than two-fold compared to control plants. Indeed, in some gels (e.g. Fig. 1), its abundance appeared unchanged following P. viciae infection.


4 10 pI

7

8

9

10

11

12

13

1

2

3

4

5

6

MW

(kD

)

15

35

50

Table 2. Proteins identified using MALDI-TOF and ESI Q-TOF mass spectrometry that differ in abundance in response to infection by P. viciae at 4 days post-inoculation. All proteins differed in abundance by more than two-fold, except protein 6 which was essentially unchanged between treatments. Spot numbers relate to Fig. 1. * denotes protein matched by de novo sequencing using ESI Q-TOF MS/MS

Spot number

Matching protein

Protein accession no.

Predicted mW (Da)

Predicted pI

Matched peptides

Sequence coverage

1 PI176 P13239 16909 5.113 3 16.42 ABR17 Q06931 16618 5.114 7 51.03 Glycine-rich

RNA binding protein

P49311 16360 5.54 1* 4.7*

4 Cytosolic GAPDH

P34922 36586 6.997 9 34.6

5 Chloroplastic GAPDH

P12858 43311 9.002 15 44.7

6 Thioredoxin M-type precursor

P48384 12501 5.420 1 5.2

11 Photosystem I reaction centre subunit II

Q9S7H1 23102 9.76 3 10.1

12 ATP synthase epsilon chain

P05039 15212 6.59 3 15.3

13 Photosystem I iron sulphur centre

P10793 9220 7.5 3 17.3

Protein 11 had the highest peptide match to the photosystem I reaction centre subunit II precursor from Arabidopsis thaliana (accession number Q9S7H1), and also matched to a partial sequence obtained for the same protein in pea (accession number P20117). The predicted molecular weight and pI values were slightly different to those observed, both being larger than the values observed on the gel. The greatest increase in abundance was observed for this protein (Fig. 2), which increased by 21.7-fold in fraction I compared to an increase of 3.3 for fractions II plus III. Three peptides from protein 12 matched to an ATP synthase epsilon chain from pea (accession number P05039), covering 15.3% of the amino acid sequence. The pI and molecular weight for the protein observed on the gel matched those predicted. Protein 13 matched to the photosystem I iron sulphur centre from pea (accession number P10793). Three peptides were matched, covering 17.3% of the amino acid sequence, with predicted pI and molecular weight values matching those observed.

The abundances of proteins 7, 10, 12 and 13 (Figs 1 and 2) increased similarly in both fractions. The abundance of protein 8 was greater in the soluble fraction I than the membrane and nucleic acid associated proteins of

fraction II plus III, having a 3.3- and 2.5- fold increase in abundance compared to control samples, respectively. In

fraction II plus III, protein 9 increased in abundance by more than twice that

observed in fraction I (9.3 and 4.3 respectively). Thus differences between the relative abundance of proteins isolated from the two fractions were apparent for six of the thirteen proteins (Fig. 2).


0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12 13

Protein

Relat

ive ab

unda

nce (

inoc

ulat

ed/co

ntro

l)

Figure 1. A typical 2D DIGE gel obtained from analysis of the pea leaf proteome during early stage infection (4 days post-inoculation) by Peronospora viciae, compared to proteins from control leaves treated with sterile distilled water, for the cytosolic fraction I. Spot colour indicates the effect of P. viciae on protein abundance: red = increased; green = decreased; yellow = no change. Proteins that increased in abundance reproducibly on all gels are indicated by the numbers 1-13, except for spot 6 which represents a protein showing no change in abundance during infection by P. viciae (see Fig. 2).

Figure 2. Relative abundance of proteins 1-13 that increase in pea leaves at 4 dpi after inoculation with P. viciae, compared to proteins from control leaves treated with sterile distilled water . Protein abundances for fraction I ( cytosolic proteins) and fractions II plus III ( membrane and nucleic acid-associated proteins) were calculated using DeCyder Software (GE Healthcare) from 2 replicate experiments. Standard errors are indicated.

Protein Experiment Fraction I Fractions II plus IIIControl P. viciae 4

dpiControl P. viciae 4

dpi


Protein 2

Q06931ABA-responsive protein ABR17

a

b

Protein 11

Q9S7H1Photo-system I reaction centre subunit II

a

b

Protein 6

P48384Thioredoxin M-type, chloroplast precursor

a

b

Figure 3. Images of representative protein spots on 2D gels, from two replicate experiments (a and b). Protein 2 (ABR17) increases in abundance during P. viciae infection of pea leaves in fractions I and fractions II plus III; protein 11 (photosystem I reaction centre subunit II) increases to a greater extent in fraction I than fractions II plus III; protein 6 (thioredoxin M-type, chloroplast precursor) does not differ in abundance between control (sterile distilled water) and infected pea leaves.

An amplicon of approximately 480 bp was amplified by PCR using ABR17-specific primers from pea cDNA and cloned into the GatewayTM entry vector pENTR-D/TOPO (Invitrogen). Sequencing of the insert revealed that it encodes the entire ABR17 protein along with a 5’ His-tag sequence. The gene was mobilised into the GatewayTM

destination vector pDEST17 (Invitrogen) via a recombination reaction. The resulting vector, designed for expression in bacterial systems, was transformed into competent Escherichia coli cells where expression of ABR17 was induced using L-arabinose. SDS-PAGE of induced cells illustrated that an additional 19.6 kD protein band was present compared to controls (data not shown). The protein was excised from the gel and analysed using MALDI-TOF MS resulting in the amino acid sequence coverage maps shown in Fig. 4, alongside the original map for ABR17 obtained from protein spot 2 (Fig. 1). The protein was identified as the ABR17 protein from pea (17 kD). A difference between the size of the expressed ABR17 band on the gel (19.6 kD) and the expected size of ABR17 (16.6 kD) was observed, and is due to the N-terminal histidine tag.

a) Protein from 2D gel

SID 5 (Rev. 3/06) Page 10 of 22

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b) Protein from recombinant E. coli cells

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Figure 4. Amino acid sequence coverage map for abscisic acid response protein 17 (ABR17) excised from (a) a 2D gel of proteins isolated from pea infected by P. viciae (spot 2 on Fig. 1) and from (b) the ABR17 protein expressed in recombinant Escherichia coli cells. Both maps matched to ABR17 when analysed using ProteinLynx Global Server software (V2.0.5). Key: matched to a peptide ; matched to a partial peptide ; overlapping peptides .

Comparison of the pea proteome during drought and wounding stress, and upon infection by Peronospora viciae, Botrytis cinerea, Pseudomonas syringae and Erysiphe pisi.

The aim of this work was to determine the specificity of ABR17 as a potential marker protein for P. viciae infection, and to identify additional proteins that specifically increase in abundance during the various stress conditions.

METHODSInoculation of pea with Peronospora viciae, Botrytis cinerea, Erysiphe pisi and Pseudomonas syringaePlants of P. sativum cv. Livioletta were used throughout these experiments at 10 days after sowing seeds. Pea plants were inoculated with P. viciae spores and proteins extracted at 4, 7 and 10 dpi as described above. For Botrytis cinerea, spores were washed from 14 day old agar cultures and the spore density amended to 10 6

spores/ml. The spores were then sprayed on to pea plants as for P. viciae and leaves were harvested at 4 dpi for protein extraction. For Erysiphe pisi, infected plants with sporulating colonies were shaken over the plants to be inoculated, with the resulting spore cloud settling on to the leaf surfaces. Leaves were harvested at 4 dpi for protein extraction. To inoculate peas with the bacterium Pseudomonas syringae pv. pisi, leaves of pea plants were punctured at ten evenly distributed places using a sterile mounted needle, and 10 μl of P. syringae cell suspension was added to each puncture hole. Plants were harvested at 4 dpi.

Application of wounding and drought stress to pea plants.Fully developed leaves from 10 day old pea plants (cv. Livioletta) were wounded by pushing a sterile, mounted needle through each pea leaf at ten evenly distributed places. Four days post-wounding the leaves were excised from the plants for protein extractions. For drought stress, ten day old pea plants (cv. Livioletta) were grown as described above but watering was limited to once at the initial sowing stage. Leaves were excised from the

SID 5 (Rev. 3/06) Page 11 of 22

drought stressed plants at 14 days to match the age of the leaves for other treatments, thus negating time-related differences in the pea proteome.

Protein extractionsAll protein extractions were performed as described above.

CyDye labellingGels were scanned as described above, except scans on Cy2-labelled proteins were also performed using a 488 nm laser and emission filter of 520 nm BP40. Additionally, analysis of each of the gels was performed with DeCyder Differential Biological Variance Analysis module software (V5.0) (GE Healthcare) using the triple detection setting and an estimated protein spot number of 2500.

Two-dimensional gel electrophoresisLarge-format 2D gels were prepared as described above.

Image analysisImage analysis was performed as described above

MALDI-TOF and ESI Q-TOF MS analysisMass spectrometry analysis was performed as described above.

RESULTSFor P. viciae infections, proteins were harvested from fully developed pea leaflets at 4, 7 and 10 dpi, which represented asymptomatic, early sporulation and heavy sporulating stages of infection respectively. Additionally, proteins from pea leaves infected by B. cinerea, P. syringae and E. pisi at 4 dpi were analysed along with proteins from drought- and wound-stressed leaves. The proteins varying in abundance in pea leaflets during infection and during abiotic stress were examined using two dimensional difference gel electrophoresis (2D-DIGE). Table 3 indicates the proteins identified by mass spectrometry and whether they altered in abundance following the varyious treatments. The relative proportions are illustrated in Fig. 5.

ABR17 levels appeared to increase during all biotic stresses except P. syringae infection, but not during the abiotic stress conditions. A further unidentified protein consistently increased in abundance specifically following B. cinerea infection.

Table 3. Identified pea proteins and their abundance relative to controls (sterile distilled water inoculated for pathogens; untreated for biotic stresses) following treatment with various stress conditions.Accession Number

Protein Molecular

Weight

pI Response

O04300 Alpha-1,4-glucan protein synthase

41572 5.73 No change

O49169 Elongation factor 1 alpha 49339 9.5 Increased abundance on E. pisi infection

O82043 Ketol acid reductoisomerase

39404 8.9 No change

O98997 RuBisCO activase chloroplast precursor

47871 8.1 No change

P04717 RuBisCO large chain precursor large subunit

52729 7.0 Increased abundance on E. pisi and P. syringae infection

P05037 ATP synthase beta chain 53089 5.2 No change P05039 ATP synthase epsilon chain 15212 6.6 No change P07689 RuBisCO small chain 3A 20213 9.4 Increased abundance on E.

pisi infection, P. viciae infection at 4, 7 and 10 dpi and B. cinerea infection

P08215 ATP synthase alpha chain 54591 5.9 No change P08926 RuBisCO large subunit

binding protein subunit alpha chloroplast precursor 60 kDa chaperonin subunit

61941 5.2 No change

P08927 RuBisCO subunit binding protein beta subunit chloroplast precursor

62945 5.99 No change

P10793 Photosystem 1 iron sulphur centre

9220 7.5 Decreased abundance for all stresses except P. viciae

SID 5 (Rev. 3/06) Page 12 of 22

infectionP11964 Superoxide dismutase

CuZn chloroplast precursor20613 6.3 No change

P12858 Chloroplastic GAPDH Increased abundance on P. viciae infection at 4 dpi

P13239 Pathogen induced 176 protein

16909 5.1 Increased abundance on E. pisi infection, P. viciae infection at 4, 7 and 10 dpi and B. cinerea infection

P14226 Oxygen evolving enhancer protein 1 chloroplast precursor 33 kDa subunit

34871 6.6 Increased abundance on P. viciae infection at 4 dpi, decreased abundance during P. viciae infection at 7 and 10 dpi

P14710 Disease resistance response protein Pi49

16742 4.94 Increased abundance on E. pisi infection, P. viciae infection at 4, 7 and 10 dpi and B. cinerea infection

P16048 Glycine cleavage system H protein mitochondrial precursor

17687 5.2 No change


28030 8.5 Increased abundance on E. pisi and B. cinerea infection and wounding stress


28030 8.5 Increased abundance on E. pisi infection (different spot to previous)

P17067 Carbonic anhydrase 35355 7.6 Decreased abundance on P. viciae infection at 7 and 10 dpi, E. pisi infection, wounding and drought stress

P25079 RuBisCO large chain precursor large subunit

52910 7.0 No change

P26969 Glycine dehydrogenase decarboxylating mitochondrial precursor. Glycine decarboxylase glycine cleavage system P protein


P34899 Serine hydroxymethyl transferase

57256 8.9 No change

P34922 Cytosolic GAPDH Increased abundance on P. viciae infection at 4 and 10 dpi and E. pisi infection

P35100 ATP dependent Clp protease ATP binding subunit clpC homolog chloroplast precursor


P46256 Fructose bisphosphate aldolase cytoplasmic isozyme 1

38467 7.3 No change

P46257 Fructose bisphosphate aldolase cytoplasmic isozyme 2

38467 7.3 No change

P47922 Nucleoside diphosphate kinase

16498 8.8 No change

P48384 Thioredoxin M-type precursor

12501 5.4 Decreased abundance on E. pisi, P. syringae and B. cinerea infection and wounding stress, increased abundance for drought stress

P48534 L ascorbate peroxidase cytosolic

27044 5.7 No change

P49311 Glycine rich RNA binding 16360 5.5 Increased abundance on E.

SID 5 (Rev. 3/06) Page 13 of 22

Number of proteins

24

3

5

3

1

2

1

11

11 1 1

No change in abundance in response to any stresscondition

Increased abundance during E. pisi infection

Increased abundance during E. pisi and P. syringaeinfection

Increased abundance during E. pisi, B. cinerea andP. viciae infection at 4, 7 and 10dpi

Decreased abundance for all stresses except P.viciae infection

Increased abundance during P. viciae infection at4dpi

Increased abundance during P. viciae infection at4dpi, decreased abundance during P. viciaeinfection at 7 and 10dpiIncreased abundance during E. pisi, B. cinerea andwounding

Decreased abundance during P. viciae infection at7 and 10dpi, E. pisi infection, wounding and drought

Increased abundance during P. viciae infection at 4and 10dpi and during E. pisi infection

Increased abundance during drought stress,decreased abundance during infection by E. pisi, P.syringae, B. cinerea and woundingIncreased abundance during P. viciae infection at4dpi and E. pisi infection

Increased abundance during P. viciae infection at 4and 7dpi, E. pisi infection and B. cinerea infection

protein pisi infection and P. viciae infection at 4 dpi

P49364 Aminomethyl transferase (mitochondrial precursor)

44256 9.0 Increased abundance during all stress conditions

P50218 Isocitrate dehydrogenase NADP


P52576 Isoflavone reductase 2 hydroxy IFR NADPH IFR oxidoreductase

35407 5.5 No change

Q01517 Fructose bisphosphate aldolase 2 chloroplast

37803 5.6 No change

Q02028 Stromal 70 kDa heat shock related protein chloroplast precursor

75469 5.3 No change

Q06931 Abscisic acid response protein 17

16618 5.1 Increased abundance on P. viciae infection at 4 and 7 dpi, E. pisi and B. cinerea infection

Q40977 Monodehydroascorbate reductase


Q42450 RuBisCO activase B, chloroplast precursor

47228 7.6 No change

Q42961 Phosphoglycerate kinase chloroplast precursor

50145 8.8 Increased abundance on E. pisi infection

Q7X9A0 RuBisCO 1 chloroplast precursor activase 1 alpha form

52107 6.8 No change

Q96551 S-adenosylmethionine synthetase 1

43022 5.8 No change

Q9BBU0 ATP synthase beta chain 53779 5.5 No change Q9S7H1 Photosystem I reaction

centre subunit23102 9.76 Increased abundance on P.

viciae infection at 4 dpi

Figure 5. The abundance of the 45 identified proteins (Table 3) following the various treatments, relative to control leaves (numbers of proteins indicated).

The pea proteome

SID 5 (Rev. 3/06) Page 14 of 22

In addition to identifying several proteins that alter in abundance during various plant stresses, many additional proteins were identified using mass spectrometry in order to build a map of the pea proteome (Fig. 6). This is an additional output not in the original contract but which will be useful for further studies of the pea proteome.

Figure 6. Map of the pea proteome. Protein spots were excised from 2D gels and identified using MALDI-TOF mass spectrometry and Q-TOF mass spectrometry.

Identification of proteins from the spores of Peronospora viciae

This proof-of-principle study was undertaken to determine whether proteins isolated from P. viciae spores and pre-invasion infection processes could be characterised using a 2D gel approach. Proteins isolated from spores (and subsequently from germlings) may reveal potential novel biomarkers for early stage downy mildew infection. This is an additional output not in the original contract. METHODSProtein extractionFor P. viciae protein extraction, 100 μg of spores were collected by washing conidia from pea leaves at 10 dpi. The spores were pelleted by centrifugation (14,000 rpm, 4 oC, 30 min), the supernatant discarded and the remaining pellet snap frozen in liquid nitrogen (LN). The pellets were then ground to a fine powder under LN using a mortar and pestle. The powder was transferred to a pre-cooled 1.5 ml Eppendorf tube and 1 ml of lysis buffer was added and incubated on ice for 1 h. The solution was centrifuged (14,000 rpm, 4 oC, 30 min) and the supernatant containing soluble proteins snap frozen and stored at -80 oC until required.

Two-dimensional gel electrophoresisLarge-format 2D gels were prepared as described above. Gels were stained using Brilliant Blue G Colloidal stain (Sigma) and scanned using an Imagescanner (GE Healthcare)

MALDI-TOF and ESI Q-TOF MS analysisMass spectrometry analysis was performed as described above.

RESULTSOver 250 protein spots could be identified following separation of a protein extraction of P. viciae spores using large format 2D electrophoresis (Fig. 8). Of those proteins which showed sufficient abundance to be excised and sequenced, 22 showed matches to plant, bacterial and yeast proteins whilst 53 showed no homologies to any proteins in the databases searched. A further 12 proteins were identified which showed homologies to proteins from other oomycetes. These proteins could be clustered into functional groups including those involved in

SID 5 (Rev. 3/06) Page 15 of 22

The Pea Proteome

Q9BBU0

O98997

P08215

P14226

P16059

P48534

P52576

Q01517

P05037

P08926

Q02028

P04717

P26969

Q42961P46257

P35100

Q7X9A0

Q96551

O49169

P25079

Q40977

P49311

Q06931

P13239

P48384

Q9S7H1

P05039

P10793

P47922

P34922

P17067

P49364

P07689

P08927

P04717

O04300

O82043

P46256Q42450

P48534

P50218

P34899

P16048

P11964

P14710

P12858

glycolysis/metabolism/energy pathways, cytoskeleton/shape/form/organisation, protein and amino acid synthesis, and protein binding/transport/folding.

.

Figure 8. Image of the P viciae conidiospore proteome.

Objective 1 Conclusions One pea-derived protein (ABR17) was identified as having potential as a generic marker of E. pisi, P.

viciae and B. cinerea infection. Two pea-derived proteins (choloplastic GAPDH and photosystem I reaction centre subunit) were

identified as only being increased in abundance for P. viciae at 4 dpi, and have potential as markers of pre-symtomatic downy mildew infection.

A further 12 novel proteins, identified directly from the P. viciae spore proteome, have potential for use as P. viciae specific, or downy mildew generic, markers.

One unidentified protein increased in abundance specifically to the necrotrophic pathogen Botrytis cinerea, having potential as a marker in the cut flower and soft fruit markets.

3.2 Objective 2: To produce high affinity monoclonal antibodies (MAbs) to the specific biomarker(s) identified

Milestone S02/01 Review of MAb production. Outcomes of the MAbs production to date, reviewing progress and identifying the strategy for the MAb production in the coming 12 months.

Milestone 02/02: MAbs to all biomarkers. Production of fully characterised antibodies with recommendations on the most appropriate biomarker/antibody combination.

The aim of this work was to generate monoclonal antibodies to the proteins identified in the work of objective 1.

METHODSImmunisationExtracts of downy mildew infected pea material were provided by UWE for CSL to raise MAbs. Three, 10 week old Balb/c mice were immunised with 0.1 ml of the pea material. The first injections were emulsified 1:1 in Freunds complete adjuvant and administered subcutaneously. Three subsequent immunisations were given at 2 weekly intervals, 1:1 in Freunds incomplete adjuvant, by intra-peritoneal injection.

Tail bleedsBlood was taken from the tail and screened by ELISA at week 8 using the immunogen to capture the antibodies. The mouse producing the highest polyclonal response was given a final boost of 0.1 ml antigen in sterile phosphate buffered saline (PBS) 4 days prior to fusion.

Fusion

SID 5 (Rev. 3/06) Page 16 of 22

3 7

50

35

25

15

150

The boosted mouse was killed and the spleen removed aseptically. The fusion and subsequent cell culture were carried out according to standard procedures (Kohler and Milstein, 1975: Galfre and Milstein, 1982). Splenocytes were fused at a ratio of 3:1 with SP2/0-Ag14 mouse myeloma cells (ECACC). Hybrids were plated out in 96 well tissue culture plates and screened on day 14.

Hybridoma screeningAll hybridomas were screened in a plate trapped antigen format ELISA, using a polyclonal anti-mouse IgG alkaline phosphatase conjugate with p-nitrophenyl phosphate powder (pNPP) as the substrate. Hybridomas were selected on the basis of good positive results (OD >1.0 at 405nm) using ABR17 coated onto plates at 1 µg/ml to screen the antibodies.

Inhibition ELISAAntibodies selected for further evaluation were screened using an inhibition ELISA. In this format, free antigen is used to compete with plate bound antigen for antibody binding. Plates were coated with ABR17 at 1 µg/ml, and antigen added at a range of dilutions when the antibody is added. Antibody was screened using both ABR17 and pea leaf extract as the inhibiting antigens.

Detection of ABR17 in plant materialThis was approached in 2 different ways. Firstly, plant material was coated directly onto the plate and the anti-ABR17 monoclonal antibody was used to detect ABR17 protein in the downy mildew infected pea material. Secondly, infected pea leaf was used to inhibit binding of antibody to ABR17 (as described above).

PurificationAntibodies isotyped as IgG were purified using a protein G affinity HiTrap column (GE Healthcare).

ConjugationAntibodies were conjugated to horse radish peroxidase (HRP) using a LinkIt kit (PerBio, Cramlington, UK).

RESULTSTail bleedsTail bleeds were screened in a plate trapped antigen format ELISA, using purified ABR17 to capture the antibodies. The results are shown in Figure 8.

Figure 8. ELISA data for tail bleed blood of mice immunised with ABR17.

Hybridoma screeningThree fusions wee performed, and given the identification codes F366, F375 and F377. All hybridomas were screened using standard protocols, selecting positive hybridomas based on ELISA screening results. Table 4 shows a summary of the antibodies generated.

SID 5 (Rev. 3/06) Page 17 of 22

Table 4. Summary of anti-ABR17 antibodies generated.

Fusion number Fusion date % of wells containing hybridomas Antibodies produced Isotype

366 07/06/2005 96 366/9.D2.E6.H1 IgM375 20/09/2005 94 375/7.G3.B12 IgM

375/10.G9.D11 IgM377 20/09/2005 69 377/1.H12.D11.E11 IgG

Preliminary inhibition dataInhibition was seen with all antibodies, but 377/1.H12.D11 showed the greatest reduction (Fig. 9). Western blot data confirmed that antibody 377/1.H12.D11 showed the greatest promise.

This antibody was allocated the identification code Y144. Monoclonal antibodies which are IgG isotype are usually easier to incorporate into a lateral flow device format, and so this was an added advantage of selecting this MAb.

Inhibition data using purified antibodyY144 was collected in bulk and purified. Figure 10 shows inhibition of purified Y144 antibody using free ABR17.

Purification of the IgM antibodies proved problematic, and once purified they did not perform well in ELISA, so to maximise chances of success, all effort was focussed on the Y144 IgG.

Figure 9. Preliminary ELISA inhibition data of anti-ABR17 monoclonal antibodies.

SID 5 (Rev. 3/06) Page 18 of 22

Figure 10: Inhibition of anti-ABR17 antibody (Y144) using free ABR17

Inhibition at 50% was achieved with free ABR17 at a concentration of 3.23 µg/ml (= 3.23 ppm; Fig. 10).

Antibody Y144 was conjugated to horseradish peroxidase (HRP) to see if assay sensitivity could be improved. Figure 11 shows a comparison of the 2 different plate trapped antigen assays, using either a secondary alkaline phosphatase conjugated polyclonal to detect unconjugated Y144, or a directly linked HRP conjugated Y144. Antibody concentration was standardised to 1 mg/ml.

Figure 11. Comparison of indirect vs direct ELISA (AP = alkaline phosphatase; HRP = horseradish peroxidase).

Sensitivity was not improved by using the HRP conjugated Y144 instead of the indirect format in a plate trapped antigen ELISA (Fig. 11). The HRP conjugated Y144 was also used to detect ABR17 in a double antibody sandwich ELISA, using Y144 to trap ABR17, and using HRP conjugated Y144 as the detecting antibody. This did not improve assay sensitivity.

SID 5 (Rev. 3/06) Page 19 of 22

Indirect ELISA using AP

Direct ELISA using HRP

Detection of ABR17 in plant materialFigure 12 shows an increased OD when detecting ABR17 in infected sap compared to healthy sap, but these data could not be replicated in an inhibition ELISA (Fig. 13). This could be for one of 2 reasons: either there is an error in the ELISA system we have been unable to detect, or the ABR17 protein is not accessible to the antibody in the plant sap. The latter is most likely, and would merit further investigation if ABR17 was to be used as a marker protein.

For example, ABR17 may be located on an internal structural surface within plant cells and would not be accessible in plant material ground using the standard method used to detect bacterial, fungal and viral antigens. The standard method uses a grinding wheel with PBS/0.02% Tween with 2% PVP.

Objective 2 ConclusionsFigure 10 confirms that the proteomics approach is able to identify potential marker proteins, which can be cloned, expressed in vitro and used to raise MAbs.

Figure 12. Detection of ABR17 in infected vs healthy pea leaf material in a simple plate trapped-antigen format.

Figure 13. Inhibition of Y144 using downy mildew infected plant sap. Plate coated with ABR17 at 1 µg/ml.

SID 5 (Rev. 3/06) Page 20 of 22

3.3 Objective 3: To write and present a coherent constructive scientific report of the research undertaken.

Milestone 03/01: Submission of final report. A clear and logical evaluation of the outcomes of the research will be presented together with recommendation for further research opportunities (if applicable), technology transfer and commercialisation.

This report completes objective 3, with the key conclusions and recommendations for further research listed below.

1) This report demonstrates that the project has met the original objectives. The work on the ABR17 protein provides proof-of-principle that the proteomics approach can be used to identify potential marker proteins, which can be cloned, expressed in vitro and used to raise MAbs suitable for rapid detection devices (such as the CSL lateral flow format).

2) The comparative proteomic analysis of the different abiotic and biotic stresses has indicated that ABR17 has potential as a general marker for fungal and oomycete infection of pea leaves (at least for those pathogens and biotic stresses assessed).

3) Two pea-derived proteins (chloroplastic GAPDH and photosystem I reaction centre subunit) were identified as only being increased in abundance for P. viciae at 4 dpi, and have potential as markers of pre-symptomatic downy mildew infection. They merit further investigation (proteomic, gene cloning, in vitro expression, MAbs) as for ABR17.

4) Several other proteins identified show differential changes in abundance (either increase, decrease or no change) depending on the particular cause of stress.

5) Of particular significance is the one unidentified protein that increased in abundance only following B. cinerea infection. This has particular potential as an early warning marker for grey mould in the cut flower and soft fruit market, and merits further investigation.

6) The abundance of proteins was different at different stages of P. viciae infection (4 vs 7 vs 10 dpi). Therefore future work on this and other biotic and biotic stresses should include time-course studies.

7) The additional work completed (i.e. work not in the original contract) has started to build proteome maps of pea leaves and P. viciae conidiospores. These provide the basis for further proteomic analysis in order to identify specific markers for the major biotic and biotic stresses that reduce yield in UK pea crops.

8) Comparison of the potential protein biomarkers for P. viciae in other downy mildew pathogens (especially Peronospora species) and their host plants merits investigation. This may lead to common markers and to a generic detection system for a range of downy mildew diseases of important UK crops.

9) Further work should extend this analysis to root infections (e.g. by P. viciae, Pythium and Aphanomyces oospores, and nematodes) because infections of roots may induce proteome changes in leaves and provide opportunities for developing diagnostics for root diseases based on leaf sampling.

References

El-Gariani, N.K. and Spencer-Phillips, P.T.N. (2004). Isolation of viable Pernospora viciae hyphae from infected Pisum sativum leaves and accumulation of nutrients in vitro. In:Advances in Downy Mildew Research. Vol 2. (P. Spencer-Phillips and M. Jeger, eds), pp. 249-264. Kluwer Academic Publishers, Dordrecht.

Galfre, G. and Milstein C. (1982). Chemical typing of human kappa light chain subgroups expressed by human hybrid myelomas. Immunology 45: 125-128.

Giavalisco P, Nordhoff E, Lehrach H, Gobom J, and Klose J. (2003). Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis. Electrophoresis 24:207-216.

Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.

SID 5 (Rev. 3/06) Page 21 of 22

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Amey, R., Schleicher, T., Danks, C., Macdonald, H., Neill, S. and Spencer-Phillips, P. (2003). Novel proteomic and biosensor based strategies for detection of downy mildew infection. In: Abstracts, Plant GEMs/GARNet, York, United Kingdom, p. 192.

Amey, R., Schleicher, T., Danks, C., Macdonald, H., Neill, S. and Spencer-Phillips, P. (2003). Novel proteomic and biosensor based strategies for detection of downy mildew infection. In: Abstracts, Proteomics of Plant Proteins, Rothamsted Research, UK.

Amey, R., Schleicher, T., Danks, C., Macdonald, H., Neill, S. and Spencer-Phillips, P. (2003). Novel proteomic and biosensor based strategies for detection of downy mildew infection. Molecular Biology of Fungal Pathogens XIV, Ambleside, UK.

Amey, R., Schleicher, T., Macdonald, H., Neill, S. and Spencer-Phillips, P (2004). Proteomic analysis of pea downy mildew infections. 7th Conference of the European Foundation for Plant Pathology & British Society for Plant Pathology Presidential Meeting 2004, University of Aberdeen, UK, p11.

Amey, R., Taylor, L., Schleicher, T., Macdonald, H., Neill, S. and Spencer-Phillips, P. (2004). A proteomic approach to identify proteins differentially regulated during Peronospora viciae infection in susceptible and resistant pea cultivars. 7th Conference of the European Foundation for Plant Pathology & British Society for Plant Pathology Presidential Meeting 2004, University of Aberdeen, UK, p35.

Amey, R., Taylor, L., Schleicher, T., Macdonald, H., Neill, S. and Spencer-Phillips, P. (2005). Proteomic analysis of pea downy mildew infections. British Society for Plant Pathology SW Regional Plant Pathology Meeting, University of Bath.

Amey, R., Schleicher, T., Tomkies, V., Danks, C., Macdonald, H., Neill, S. and Spencer-Phillips, P. Heather Macdonald, Steve Neill & Peter Spencer-Phillips (2005). Novel proteomic and biosensor strategies for detection of downy mildew infection in pea. SET for BRITAIN, House of Commons, UK.

Amey, R.C. and Spencer-Phillips, P.T.N. (2006). Towards developing diagnostics for downy mildew diseases. Outlooks on Pest Management 17, 4-8.

Chuisseu Wandji, J.L., Amey, R.C., Butt, E., Harrison, J., Macdonald, H. and Spencer-Phillips, P.T.N. (2007). Towards proteomic analysis of Peronospora viciae conidiospores. In: Advances in Downy Mildew Research, Volume 3 (A. Lebeda and P.T.N. Spencer-Phillips, eds), pp. 95-100. Palacky University in Olomouc and JOLA, v.o.s., Kostelec na Hane (Czech Republic).

Amey, R.C., Schleicher, T., Slinn, J., Lewis, M., Macdonald, H., Neill, S. & Spencer-Phillips, P.T.N. (2007). Proteomic analysis of a compatible interaction between Pisum sativum (pea) and the downy mildew pathogen Peronospora viciae. European Journal of Plant Pathology, submitted.

SID 5 (Rev. 3/06) Page 22 of 22

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