WEL COME

Proteomics and its applications in
phytopathology
Abhijeet shankar
kashyap
PhD - 10333
plant pathology

Overview
INTRODUCTION
CONCEPT AND TOOL
PLANT PROTEOMICS IN INDIA
PROTEOMICS APPLICATIONS
CASE STUDIES
CHALLENGES
CONCLUSION

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

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

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

General classification for proteomics
Structural Proteomics
-Goal is to map the 3D
structure of protein
and protein
complex.eg 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

• 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

• 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

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

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.

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

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

Methods for
protein
identification

Mass Spectrometry

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

Protein identification – peptide mapping

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. (http://us.expasy.org/sprot)
• 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-
nbrf.georgetown.edu/)
• 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.
http://pir.georgetown.edu/uniprot/

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

Isolates individual peptide fragments for
2nd mass spec – can obtain peptide
sequence
Compare peptide sequence with
protein databases
(trypsin)
Tandem Mass Spectrometry

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

Plant proteomics in India

Plant proteomics research publications from India
•

Cond..

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

Proteomics: tool for crop improvement
Eldakak et al., 2013

Cond..
Eldakak et al., 2013

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)

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 )

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)

Important Host pathogen-related experiments using
proteomics
Nova et al 2012

CASE STUDY

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

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?

AIM: To elucidate the reason for one biocontrol agent superiority to the other in
controlling Fusarium oxysporum f sp radicis-lycopersici (FORL)

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

Results
Suppressive effect of EU07, FZB24 and QST713 on FORL:
In vitro antagonism bioassay
CK
QST713
FZB 24EU07

Genetic variability analysis of the Bacillus strains:
26 ISSR markers used
6 showed highest
polymorphism between the
3 strains
Dice’s genetic similarity matrix

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.

• • 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

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

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.

• 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

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

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.

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.

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.

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

Conclusion