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 O-

methyltransferase 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 The gene, characterized as encoding for an O-methyltransferase acting on caffeic acid,O-methyltransferase acting on caffeic acid,

– might actually be encoding an N-might actually be encoding an N-methyltransferase, involved in gramine methyltransferase, involved in gramine biosynthesisbiosynthesis

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 self-cleavage in presence of DTT

The target protein releases from the chitin-bound intein tag

Methods

RT-PCR and PCR Cloning of the target gene into the vectorTransformation of the competent cellsAgarose 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 OMT-gene

Results: Synthesis of the coding region of the OMT gene

The total RNA was isolated from barley green tissue

RNA was reverse transcribed into single-stranded 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 - 6706 and 1801 bp – Nco I - 7380, 680 and 447 bp – Nde I - 8507 bp – Sap I - 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/NSDS-PAGE analysis showed bands 100

kDa100 kDa = OMT-intein fusion proteinPositive control - ER2566 transformed with

pMYB5 vector Negative control –

– uninduced E1E2 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 hRT, O/N15oC, 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

Cleavage reaction conditions

Concentration of the target protein, μg/ml

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 TLC-plates standing in TLC solvent

The regions with the reactions products were scraped from theTLC-plates for liquid scintillation counting

Results: Assay of the methyltransferase activity

Methyl transferase activity was measured by estimation of the amount of 3H-labelled product produced with methyl-3H-SAM

3H count per minute was calculated into built product per 1 mg protein

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 2 AMI and MAMI methylation products built per 1 mg protein, pmol/min, development in time

Concentration, mM

0,75 mM 1,5 mM 3 mM

AMI 37,03 18,71 21,34

MAMI 2,38 30 1,53

Table 3 AMI and MAMI methylation products built per 1 mg protein, pmol/min, relative to the substrates concentration

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

pH30 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