Transcript Expression of an aphid-induced barley methyltransferase in

Expression of an aphid-induced barley
methyltransferase in Escherichia coli,
purification and characterisation of the
enzyme
by Irene Ingvor Zetterlund
Aim
 To test the hypothesis that OMT is involved
in gramine biosynthesis
 To clone the OMT gene into an expression
vector with purpose to synthesize the
enzyme in E. coli
 To characterize the enzyme kinetically
Background: Barley
(Hordeum vulgare)
 Is an important cereal in Sweden
 Is cultivated on the large area of arable land,
about 400 000 ha
 Is used in the malting industry and for
livestock feed
 All kind of farm animals can be fed on it
Background: Bird cherry-oat
aphid (Rhopalosiphum padi)
 One of the most serious barley pests
 Transmit virus infections, i. a. barley yellow dwarf
virus
 Phloem-feeding insects, cause little tissue damage
 Are perceived by plants as pathogens
 Breed by sexual reproduction and parthenogenesis
 Overwinter as eggs on its primary host, bird cherry
 In summer make use of diverse grasses as secondary
hosts, among them barley
Background: Plant defence
reactions
 Alkaloids – a big group of N-containing secondary
metabolites, have strong physiological effects in
defence against herbivores
 Aphids induce pathogen-defence response
 Jasmonic acid signalling pathway induces
expression of a wide range of defense genes
 One of them was identified as an Omethyltransferase gene
Background: Plant defence
reactions - Gramine
 Gramine - indole proto-alkaloid, secondary metabolite in
barley and some other species in the grass family Poaceae
 Induced in barley upon aphid infestation
 Found in epidermis and in mesophyll parenchyma
 Missing in the vascular bundles
 The higher gramine amount
the lower vulnerability of
barley to aphids
 Synthesized from tryptophan
via 3-aminomethylindole
 NMT catalyzes SAM-dependent
conversion of AMI to MAMI
and from MAMI to gramine
Background: OMT
 One gene, induced by the aphid, is encoding an O-
methyltransferase, OMT
 It is also induced by the jasmonic acid signalling
pathway
 OMTs generally
– methylate caffeic acid
– lead to lignin precursors
– or various classes of flavonoids
 But not all of barley cultivars had OMT gene in their
genome
 In the barley varieties missing the gene, gramine was
not found either
 In all gramine-containing lines OMT was present
Hypothesis
 The gene, characterized as encoding for
an O-methyltransferase acting on caffeic
acid,
– might actually be encoding an Nmethyltransferase, involved in gramine
biosynthesis
Methods: IMPACT-CN Protein
Purification System
 IMPACT = intein mediated purification with




an affinity chitin-binding tag
A target protein is fused to a self-cleavable
intein tag
A chitin-binding domain in intein tag allows
purification of the target protein on the chitin
column
The intein tag undergoes specific selfcleavage in presence of DTT
The target protein releases from the chitinbound intein tag
Methods
 RT-PCR and PCR
 Cloning of the target gene into the vector
 Transformation of the competent cells
 Agarose and SDS-PAGE gel electrophoresis
 Western blotting
 Bradford microassay for protein
quantification
 Silica gel thin-layer chromatography (TLC)
Materials: Growth and treatment
of plants
 H. vulgare, variety Lina, susceptible to the aphids
 Sown in November 2003
 Grown in a growth chamber at 26oC, long day, (18
h light/6 h darkness)
 5-day-old barley plants were harvested
 Their green tissue was treated with 45 μM
jasmonic acid for 24 hours to induce the OMTgene
Results: Synthesis of the coding
region of the OMT gene
 The total RNA was isolated from barley
green tissue
 RNA was reverse transcribed into singlestranded cDNA using the First-Strand
Synthesis System for RT-PCR
 To amplify the coding region of the OMT
gene by PCR primers OMTcloneF and
OMTcloneR2 were used
 A product of about 1100 bp was visualized
by 2% agarose gel electrophoresis
Results: Cloning of the OMT
gene into the pTYB 12 vector
 The plasmid pTYB12, chosen as a vector - allows the
fusion of the cleavable intein tag to the N-terminus of a
target protein
 The plasmid - digested with
the restriction nucleases SmaI
and NdeI
 The DNA fragment - digested
with restriction nuclease NdeI
 The digested DNA - ligated
into the pTYB12 using the
BioLabs Quick Ligation Kit
Results: Transformation of E.
coli DH5α-T1 and screening for
recombinants
 To amplify the OMT sequence E. coli DH5α-T1 were
transformed with the new plasmid pTYB12-OMT
 The recombinant cells were selected on Petri dishes
with LB medium containing 100 μg/ml ampicillin
 96 randomly chosen colonies were
inoculated in a microtitre plate in LB/amp
medium
 PCR test for inserts using intern primers
OMT F1 and OMT R1 obtained 3 clones
Results: Control of the inserts
 To confirm the obtained recombinant clones, digesting
reactions with restriction nucleases Kpn I, Nco I, Nde I and
Sap I were carried out over night at 37oC
 The digested DNA was analyzed on 1 % agarose gel
 Plasmid 1 gave the expected fragment pattern and thus was
chosen as the pTYB12-OMT plasmid
 The digesting reaction with restriction
nucleases resulted in bands as follow:
–
–
–
–
Kpn I
Nco I
Nde I
Sap I
- 6706 and 1801 bp
- 7380, 680 and 447 bp
- 8507 bp
- 7810 and 697 bp
Results: Control of the insert
 The plasmid was controlled for the right insert by
PCR with 3 pairs of primers: OMT clone F and
OMT clone R2 (1); OMT F1 and OMT R1 (2),
and Intein Forward and T7 Terminator Reverse (3)
 Bands of the correct sizes were visible on 2 %
agarose gel, lane 1- 1100 bp, 2 - 348 bp and 3 1300 bp
 To make sure that there was no error in the
sequence of the cloned fragment, the plasmid
pTYB12-OMT was sequenced at Cybergene
 The sequence proved to be identical to the
one published earlier
Results: Transformation of E.
coli ER2566 and screening for
recombinants
 The E. coli strain ER2566 was provided by Impact-CN as a host





strain for the expression of a target gene cloned in the pTYB12
vector
ER2566 have a chromosomal copy of the T7 RNA polymerase gene
inserted into the lacZ gene, and therefore under the control of the
lacZ promoter
Expression of T7 RNA polymerase is suppressed in the absence of
IPTG, by the binding of lac I repressor to the lac promoter
The transcription of the fusion protein takes place
when IPTG is accessible
Transformed cells ER2566 were selected on Petri
dishes with LB/amp medium
To control the protein induction ER2566 was
transformed with the pMYB5 vector
Results: Induction of protein
expression
 Induced with 0,5 mM IPTG at RT O/N
 SDS-PAGE analysis showed bands 100 kDa
 100 kDa = OMT-intein fusion protein
 Positive control - ER2566 transformed with
pMYB5 vector
 Negative control –
– uninduced E1
 E2 and E6 chosen to
continue the experiment
Results: Optimizing of the
protein induction conditions
 Different conditions were verified:
– Induction with 0,5 mM and 1 mM IPTG
– Temperature and time:
37oC, 4 and 6 h
RT, O/N
15oC, O/N
 SDS-PAGE analysis showed
the strongest band about
100 kDa for the induction with
1 M IPTG at RT O/N
Results: Western Blot
 Protein bands were transferred onto PVDF membrane by
semi-dry transfer apparatus
 Immunoblotting:
– primary antibodies - against the chitin binding domain
– Secondary antibodies - Goat Anti-Rabbit HRP
 The protein was detected using the ECL Plus Western
Blotting kit and chemiluminescence in the CCD-camera
 The strongest bands of about 100 kDa
in lanes 4 – 7
 E6 induced at RT with 1 mM IPTG
showed the strongest band,
conditions were the best for the protein
expression.
Results: Purification of the
target protein




Purified using the IMPACT-CN Protein Purification System
1 l cell culture was induced with 1 mM IPTG at RT O/N
Cells were broken by sonication
Clarified cell extract, obtained by centrifugation, was loaded onto
chitin column
 Cleavage reaction - started by
adding Cleavage Buffer with DTT
 The protein was eluted using
the Column Buffer
 SDS-PAGE analysis showed band ~
43 kDa corresponding to the purified
methyltransferase
Results: Purification of the
target protein - conditions
 Different conditions for the on column cleavage reaction
were tested: at 4oC and RT for 24 and 40 hours
 Elution with the Column Buffer containing 0,5 M and 1 M
NaCl
– The highest protein concentration – elution with 0,5 M NaCl
 The protein concentration was measured
spectrophotometrically, using Bradford microassay
method for protein quantification
Table 1 Concentration of the target protein
Concentration of the target protein, μg/ml
Cleavage reaction
conditions
Eluant 1
Eluant 2
Eluant 3
RT, 24 h
240,00
18,80
0,00
RT, 40 h
162,12
31,14
4,81
4oC, 24 h
66,10
0,00
0,00
Results: Silica gel thin-layer
chromatography
 For determination of the kinetic parameters of the methyltransferase
were used as substrates:
– AMI, MAMI and caffeic acid
 The methylation reactions were
started and stopped by adding of:
– Start – SAM+3H-SAM (95+5)
– Stop - stop buffer
 The methylation products were
separated by means of TLCplates standing in TLC solvent
 The regions with the reactions
products were scraped from the
TLC-plates for liquid
scintillation counting
Results: Assay of the
methyltransferase activity
 Methyltransferase activity was measured by estimation of
the amount of 3H-labelled product produced with methyl3H-SAM
 3H count per minute was calculated into built product per 1
mg protein
Table 2 AMI and MAMI methylation
products built per 1 mg protein,
pmol/min, development in time
Incubation
time,
min
0 min
30 min
60 min
AMI
0
226,3
148
MAMI
0
52,6
71,2
Lina, AMI
0
-
16,6
Table 3 AMI and MAMI methylation
products built per 1 mg protein, pmol/min,
relative to the substrates concentration
Concentration,
mM
0,75 mM
1,5 mM
3 mM
AMI
37,03
18,71
21,34
MAMI
2,38
30
1,53
Results: Assay of the
methyltransferase activity
 The reactions with the enzyme extract from
barley green tissue did not show any activity
 The reactions with the methyltransferase
purified by IMPACT-CN obtained some built
product, but the data are questionable.
Table 5 Caffeic acid methylation products built per 1 mg protein, pmol/min,
development in time
pH
30 min
60 min
Enzyme
7,5
0
38,5
Enzyme
9,0
18,0
0
Lina
7,5
0
0
Lina
9,0
0
0
Discussion: Transformation of
E. coli DH5α-T1
 E. coli had difficulties to survive after its
uptake of the plasmid with the insert OMT
 Few recombinant colonies were obtained
and the survivors turned out to have
mutations in the OMT sequence
 The third transformation resulted in a
frameshift mutation
 The fourth transformation was succesful
Discussion: Purification of the
target protein
Discussion: Assay of the
methyltransferase activity
 The methylation of AMI:
– highest after 30 min incubation, decreasing later
– that contradicts the kinetic development in time as a logarithmic
function
 The methylation of MAMI:
– increases in time
– highest after 60 min incubation
 An explanation - the scraped samples were contaminated
and thus are not trustworthy
 The methyltransferase activity was analyzed relatively to
AMI and MAMI concentration:
– production of MAMI from AMI is inversely proportional to the
substrate concentration
Discussion: Assay of the
methyltransferase activity
 Enzyme activity with caffeic acid as substrate - very
little activity.
 The purified enzyme was going through several
freeze-thaw cycles between the first measurement
with AMI and MAMI as substrate and those with
caffeic acid.
 This could have resulted in the loss of enzymatic
activity.
 These experiments have to be repeated with freshly
purified enzyme.
Summary
 The enzyme exhibit little activity with caffeic acid
but did methylate AMI and MAMI
 Thus it might be involved in gramine synthesis by
methylating AMI and MAMI rather than acting as
caffeic acid OMT
 Described as an O-methyltransferase, but a
sequence similarity with other OMTs is only 40%
Conclusion
 The enzyme carries out the transfer of a methyl
group from S-adenosylmethionine to AMI,
methylating it to MAMI and a methyl group from
SAM to MAMI, with the formation of gamine, in
fact acting as an N-methyltransferase in gramine
biosynthesis
 This work supports the idea that the
methyltransferase gene accession number U54767
should be classified as an NMT-gene involved in
gramine biosynthesis