by Irene Ingvor Zetterlund

1. Expression of an aphid-induced barley methyltransferase in Escherichia coli, purification and characterisation of the enzyme by Irene Ingvor Zetterlund

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. Hypothesis • The gene, characterized as encoding for an O-methyltransferase acting on caffeic acid, • might actually be encoding an N-methyltransferase, involved in gramine biosynthesis

9. 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

10. 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)

11. 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

12. 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

13. 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

14. Results: Transformation of E. coliDH5α-T1 and screening for recombinants • Toamplify the OMT sequence E. coliDH5α-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

15. 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

16. 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

17. Results: Transformation of E. coliER2566 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

18. 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

19. 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

20. 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.

21. 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

22. 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 1Concentration of the target protein

23. 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 the TLC-plates for liquid scintillation counting

24. Results: Assay of the methyltransferase activity • Methyl­transferase 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 Table 2 AMI and MAMI methylation products built per 1 mg protein, pmol/min, development in time Table 3 AMI and MAMI methylation products built per 1 mg protein, pmol/min, relative to the substrates concentration

25. 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

26. Discussion: Transformation of E. coliDH5α-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

27. Discussion: Purification of the target protein

28. 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

29. 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.

30. 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%

31. 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