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Proteomics and its applications in phytopathology

  2. Proteomics and its applications in phytopathology Abhijeet shankar kashyap PhD - 10333 plant pathology
  4. Proteins - key to Biology Proteins are large biological molecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins are the work horses of biological systems. – They play key roles in constructing and maintaining living cells Our genes code for proteins Proteins are polymers of amino acids Roles played by proteins include  Enzymes (biological catalysts)  Hormones  Storage proteins  Transport proteins  Structural proteins  Protective protein  Toxic proteins
  5. Genomics DNA (Gene) Transcriptomics RNA Proteomics PROTEIN Metabolomics METABOLITE Transcription Translation Enzymatic reaction What’s “proteomics” ? The term ‘proteome’ was first coined in 1994 by an Australian post doctoral fellow named Marc wilkins. Proteome refers to the total set of proteins expressed in a given cell at a given time. To study the dynamic protein products of the genome and their interaction. A large-scale characterization and functional analysis of the proteins expressed by a genome
  6. Why Proteomics? • The behavior of gene products is difficult or impossible to predict from gene sequence. • Even if a gene is transcribed, its expression may be regulated at the level of translation. • Genome sequence tells us about the sequence of proteins but there are many post translational modification taking place in cells. Genomics fails to explain these modifications. • Proteomic is the field that bridge the gap between genome sequencing and cellular behavior. DNA Transcription RNA processing mRNA Translation Protein Transcriptional Regulation Alternative splicing mRNA editing Translational Regulation proteolysis Post- translational modification Compartmentalizations
  7. General classification for proteomics Structural Proteomics -Goal is to map the 3D structure of protein and protein X-ray crystallography and NMR spectroscopy Functional Proteomics To study protein protein interaction, 3D structure cellular localization and PTMS in order to understand the physiological function of the whole set of proteome. Expression proteomics - Quantitative study of protein expression between samples that differ by some variable
  8. • Functional proteomics is the analysis of protein interactions at larger scale. • The characterization of protein-protein interactions are useful to determine the protein functions • It also explains the way proteins assemble in bigger complexes. • Technologies such as affinity purification, mass spectrometry, and the yeast two-hybrid system are particularly useful in interaction proteomics Functional Proteomics
  9. • Expression proteomics includes the analysis of protein expression at larger scale. • It helps identify main proteins in a particular sample, and those proteins differentially expressed in related samples—such as diseased vs. healthy tissue. • Major Techniques in Expression Proteomics Expression proteomics • 1D- and 2D-SDS PAGE • MALDI-TOF • Liquid chromatography • MS-MS • SELDI • Protein microarray
  10. Protein Mixture Individual Proteins PeptidesPeptide Mass Protein Identification Separation 2D-SDS-PAGE Digestion Trypsin Mass Spectroscopy MALDI-TOF Database Search Spot Cutting Overview of steps involved in proteomic analysis
  11. Goal : To separate and display all gene products present in sample. Two-dimensional Gel Electrophoresis • gel with an immobilised pH gradient • electric current causes charged proteins to move until it reaches the isoelectric point (pH gradient makes the net charge 0). The first dimension of 2DE is isoelectricfocusing(IEF) Isoelectric point pI and molecular mass of protein are used to separate particular protein from the mixture of proteins.
  12. 2nd dimension pH 3 pH 10 The strip is loaded onto a SDS gel Mw pI Staining ! Proteins that were separated on IEF gel are next separated in the second dimension based on their molecular weights. -SDS denatures and linearises the protein (to make movement solely dependent on mass, not shape) Silver staining Coomassie blue staining Sypro Ruby staining
  13. Pros and Cons of 2D PAGE • Good resolution of proteins • Detection of posttranslational modifications • Not for hydrophobic proteins • Limited by pH range • Not easy for low abundant proteins • Analysis and quantification are difficult
  14. Methods for protein identification
  15. Mass Spectrometry
  16. How does a mass spectrometer work? • Ionization method – MALDI – Electrospray (Proteins must be charged and dry) • Mass analyzer – MALDI-TOF – Quadrapole – Ion trap Create ions Separate ions Detect ions • Mass spectrum • Database analysis
  17. Protein identification – peptide mapping
  18. Databases Available for Id of MS Spectra • SWISS-PROT–nr database of annotated protein sequences. Contains additional information on protein function, protein domains, known post-translational modifications, etc. ( • TrEMBL-computer-annotated supplement of Swiss-Prot that contains all the translations of EMBL nucleotide sequence entries not yet integrated in Swiss-Prot. • • PIR-International–nr annotated database of protein sequences. (http://www- • NCBInr –translated GenBank DNA sequences, Swiss-Prot, PIR. • ESTdb –expressed sequence tag database (NIH/NSF) • UniProt –proposed new database. Will joint Swiss-Prot, TrEMBL, PIR.
  19. Programs Used to Identify Mass Spectra 3 main types programs available 1.Use proteolytic peptide fingerprint for protein Id (ie MALDI- TOF data). • PeptIdent,MultiIdent, ProFound 2.Programs that operate with MALDI-TOF or MS-MS spectra or combination of both • PepSea, MASCOT, MS-Fit, MOWSE 3.Programs that operate with MS-MS spectra only • –SEQUEST,PepFrag, MS-Tag,Sherpa
  20. Isolates individual peptide fragments for 2nd mass spec – can obtain peptide sequence Compare peptide sequence with protein databases (trypsin) Tandem Mass Spectrometry
  21. Comparison of MALDI-TOF and MS/MS MALDI-TOF • Sample on a slide • Spectra generate masses of peptide ions • Protein Id by peptide mass fingerprinting • Expensive • Good for sequenced genomes TANDEM MS • Sample in solution • MS-MS spectra reveal fragmentation patterns – amino acid sequence data possible • Protein Id by cross- correlation algorithms • Very Expensive • Good for unsequenced genomes
  22. Plant proteomics in India
  23. Plant proteomics research publications from India •
  24. Cond..
  25. Proteome Mining Identifying as many as possible of the proteins in your sample Protein Profiling(large scale identification) Identification of proteins in a particular sample as a function of a particular state of the organism or cell Proteomics in plant pathology Post-translational modifications Identifying how and where the proteins are modified Protein-protein interactions Protein- network mapping Determining how the proteins interact with each other in living systems Proteomics in crop improvement Protein quantitation or differential analysis eg. isotope-coded affinity tags (ICAT) Proteomics Applications
  26. Proteomics: tool for crop improvement Eldakak et al., 2013
  27. Cond.. Eldakak et al., 2013
  28. Proteomics studies relevant to plant pathology • To Establish proteomic map. eg in case of B. cinerea by Using 2-DE and MALDI-TOF/TOF MS/MS, 306 proteins were identified which have a crucial role in the pathogenicity process (Acero et al.,2009) • To characterize fungal strains and find new biomarkers in host pathogen interactions. • To compare the proteome diversity of host and non host plants. (Copper et al 2007)
  29. cond.. • To study mode of action of toxins eg. Proteomics-based anlysis reveals Verticillium dahliae toxin induces cell death by modifying host proteins synthesis ( Xie et al.,2013) • Proteomic technique have been used to study ETI.(Hurley et al., 2014) • Development of proteomic based fungicides, new strategies for environmentally friendly control of plant diseases. (Acero et al., 2011 )
  30. Cond.. • Proteomic help in identification of Resistance mechanism. Eg Maize kernels resistance mechanisms against Aspergillus flavus identified by comparative proteomics (Nogueir et al ., 2013) • To study biocontrol mechanism • eg.Seven cell-wall degrading enzymes, chitinase, cellulase, xylanase, β-1,3-glucanase, β-1,6-glucanase, mannanase, and protease, were revealed by proteomics and these target proteins are directly related to biocontrol mechanism of T. harzianum (Tseng et al., 2008)
  31. Important Host pathogen-related experiments using proteomics Nova et al 2012
  33. Fusarium oxysporum f.sp. radicis-lycopersici : A major and common soil borne pathogen causing damage to tomato crop. Chemical methods alone are not sufficient and also hazardous for the living beings feeding on crop produce Alternative strategies required to control soilborne plant pathogens Exploitation of beneficial microorganisms (Biopesticides) such as Bacillus spp. exhibiting antagonistic relation with the pathogens would be most safe and rational way of managing the damages. Introduction
  34. Previous study revealed EU07 strain had highest inhibitory effect on FORL than the existing commercial strain QST713. The plants inoculated with EU07 showed good plant height and development when compared to the QST713. Eventhough two strains belongs to same species had differential antagonistic effect on FORL. What might be the reason?
  35. AIM: To elucidate the reason for one biocontrol agent superiority to the other in controlling Fusarium oxysporum f sp radicis-lycopersici (FORL)
  36. Experimental set up Protein Identification by MALDI-TOF/TOF and Bioinformatics Two-dimensional Gel Electrophoresis Analysis of B. subtilis EU07 and FZB24 GC-MS Analysis of the Volatile Organic Compounds from B. subtilis Strains Determined Antifungal Activities of Volatiles from B. subtilis against FORL Measured effect of temperature and enzyme on inhibition Inhibitory activity of the cell free supernatant of B.subtilis against FORL Molecular analysis of QST713, EU07 and FZB24, and their genetic comparison In vitro Bioassay of pathogen inhibition effect of Bacillus subtilis
  37. Results Suppressive effect of EU07, FZB24 and QST713 on FORL: In vitro antagonism bioassay CK QST713 FZB 24EU07
  38. Genetic variability analysis of the Bacillus strains: 26 ISSR markers used 6 showed highest polymorphism between the 3 strains Dice’s genetic similarity matrix
  39. Inhibitory activity of cell-free supernatant of B.subtilis against FORL • The cell free supernatants of EU07 had an effective antifungal function and contained some biologically active constituent against FORL. Measuring effect of temperature and enzyme on inhibition • Antifungal activity of the cell free supernatant remained stable after heating at 60, 90 and 100 C for 1 hr, but not at 121 C. • Disk diffusion test result suggest that the inhibitory activity of the cell-free supernatant is partly dependent on proteinase activity.
  40. • • All the three strains produced antifungal volatile compounds and inhibited the mycelial growth of FORL. • The VOCs from the strain is determined by SPME-GC/MS. • 2,3-Butanediol metabolite was detected and confirmed by the Voges-Proskauer assay. Determining Antifungal Activity of volatiles from B.subtilis against FORL Sealed disc bioassay
  41. Effect of Bacillus strains on mycelial morphology EU07 had a greater effect on mycelial morphology , VOC exhibited morphological aberrations such as irregular, distorted, disrupted, shrivelled and swollen mycelia
  42. SDS-PAGE Separation and MS/MS Analysis of Secretory Proteins from EU07 and FZB24 Secretory proteins in the cell free supernatant of EU07 and FZB24 were separated by SDS-PAGE. The bands of interest in each lane were cut into three section for in gel digestion and MS/MS data was used in mascot search By searching the B. subtilis tryptic peptide database (obtained from NCBI), 37 proteins from EU07 and 43 proteins from FZB24 were Identified.
  43. • Of 37 proteins from EU07, 20 had a notably high identification score , most of them being assigned to a protease function. Inhibitory effect depends on the proteinase activity
  44. Identification of differentially expressed protein of Bacillus subtilis EU07 and FZB24 Proteins in EU07 and FZB24 cells were separated by 2D-gel electrophoresis (IEF and SDS-PAGE) Protein profiles obtained by scanning, digitizing and image analysis More than 3500 spots were detected on each of the 2-D gels
  45. 1. Cut protein spot 2. Protein digestion 3. Peptide purification4. Spot onto MALDI chip 5. MALDI-TOF analysis 6. Peptide fragment fingerprint Protease Peptide mass fingerprinting was carried out using MALDI-TOF-MS Comparative analysis of two strains results in 48 spots with four fold differential expression The 42 most significant proteins were identified as either up- or down-regulated in EU07 relative to FZB24 strain. The differentially expressed proteins identified here are found to be associated with metabolism, protein degradation, translation, signal transduction, DNA repair, energy production and protein folding.
  46. EU07 also expresses new unknown proteins displayed similarity with putative phage related protein, pre-neck-related protein, phosphatase, and S-adenosylmethionine (SAM)- dependent methyltransferase, which may play a significant role in the signal transduction and recognition mechanism in the control of pathogenic fungi. SO, they recommended EU07 strain as a better biopesticide for the control of FORL as compared to that of FZB24 strain.
  47. CONCLUSION B. subtilis displays genetic variation Inhibitory products from B. subtilis was influenced by enzymes and temperature VOCs produced by B. subtilis have an inhibitory effect on FORL The majority of the secreted proteins from B. subtilis may function as proteases Proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation,translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum.
  48. Challenges • Complexity – some proteins have >1000 variants • Need for a general technology for targeted manipulation of gene expression • Limited throughput of todays proteomic platforms • Lack of general technique for absolute quantitation of proteins
  49. Conclusion