Cloning, expression and antimicrobial activity of a

thaumatin – like protein from Pinus sylvestris

Ilze Šņepste, Vilnis Šķipars, Dainis Ruņģis Latvian State Forest Research Institute “Silava”,

Rīgas st 111, Salaspils, Latvia

P. sylvestris in Latvia

• Scots pine (Pinus sylvestris) covers 47 % of „Latvijas Valsts meži” total forest area.

• Needle cast (caused by Lophodermium spp.) and root rot (caused by Heterobasidion annosum) are significant diseases of Pinus sylvestris, which may become a more frequent, and severe problem in the future due to climatic changes and forest regeneration practices.

• Needle cast can affect pine trees of all ages, but is a significant disease in forest nurseries and young plantations because it can be fatal to young Pinus sylvestris seedlings. Developement of needle cast is favored by warm, rainy autumn and mild winter.

• In forest nurseries were applied fungicides (″Amistar″) but not all seedlings will be protected from L. seditiosum. Use of fungicides in forests are forbbiden and its necessitates the search for new, harmless means of disease control.

Induced resistance (IR)

• Because of the fact that plants cannot move away from recurring abiotic and biotic stresses they need to be able to respond and adapt to them.

• Induced resistance is an increased expression of natural defense mechanisms in plants against repeated exposure to adverse factors.

• Induced resistance is non-specific (broad-spectrum) and are systemic or local.

• One of the most important inducible defense mechanisms is the biosynthesis of pathogenesis – related (PR) proteins (17 groups) (Velazhahan et al., 1999).

• Many PR – 5 proteins are induced in plants as a response to infection by pathogens, osmotic stress, treatment with abscisic acid, ethylene, salicylic acid, methyl jasmonate and wounding.

• Members of the PR – 5 group are called thaumatin – like proteins (TLPs) and have been shown to have antifungal activity against a broad spectrum of fungal pathogens.

Thaumatin - like protein

• TLP have been discovered in a wide range of organisms (nematodes, insects, fungi, plants).

• High stability under extreme thermal and pH conditions, resistance to protease degradation.

• All plant PR – 5 proteins with known antifungal activity have acidic cleft from 5 highly conserved amino acids (R, E, D, D, D).

• This conserved amino acids are believed to be involved in binding to β-1,3-glucan on the fungal cell membrane, which leads to permeabilization of the fungal cell membrane and disruption of the osmotic balance inside hyphal cells, resulting in cell rupture.

Liu and Sturrock, 2010

• The inhibitory effects of TLPs can differ depending on the fungal species tested (fungi produce cell wall ß-1,3-glucans with different degrees of ß-1,6-glucosyl substitution).

Previous results from our laboratory have shown that TLP gene expression increases in P. sylvestris after inoculation with Heterobasidion annosum, therefore the present study was undertaken to investigate TLP expression and antifungal activity, suggesting that it may play an important role in P. sylvestris defense response.

Aims and methods

Identification and analysis of the Pinus sylvestris TLP sequence

• The Pinus taeda TLP mRNS, cds sequence (GenBank Acc. No. EF532603) was used to design primers for amplification of the full - length TLP gene from P. sylvestris.

• Genomic P. sylvestris DNA was isolated using a CTAB method (Murray and Thompson 1980).

• Analysing ORF, P. sylvestris had shorter TLP protein sequence than P.taeda.

• PCR and cDNA sequencing were used to confirm the size of intron in the PsTLP gene and to verify the predicted intron position.

ORF of the P. taeda TLP

ORF of the P. sylvestris TLP

http://www.ncbi.nlm.nih.gov/projects/gorf/

Open reading frame (ORF) is the part of a reading frame that has the potential to code for

a protein or peptide.

1. P.sylvestris gDNA → 2. P.taeda mRNA →

• Under normal conditions, TLP are expressed at low levels in certain organs at specific development stages.

• MJa treatment has been reported to induce expression of the TLP in Pinus moticola (Piggott N. et al. 2004) and application of MJa suppresses pathogen growth therefore verify the gene sequence and position of the intron, RNA and DNA were extracted from methyl jasmonate (MJa) treated plants.

• P. sylvestris seedlings (1 year old) were treated with: – 5, 10, 30, 50, 100 mM MJa + 0,1% Tween 80,

– Control – H2O; 0,1% Tween 80,

– Each seedling received 2,5 - 5 ml of MJa solution,

– Needles were taken prior to and 2 – 4 weeks after treatment.

• In this study, all MJ treatments significantly affected seedling growth.

• During the study period, 100% of the 30mM MJ, 50mM MJ and 100mM MJ treated seedlings died.

• These results indicate that these doses of MJ applied exogenously on seedlings were too high, which reduced seedlings growth and showed a phytotoxic effect, such as needle browning.

10 – 100 mM MJa (5 ml)

30 mM MJa (2.5 ml)

2 weeks post treatment 3 weeks post treatment

Intron verification

The PsTLP gene has a 145bp intron.

Verification of introne in P. sylvestris seedlings (after treatment).

Verification of intron in the P. sylvestris TLP gene. PCR amplicons length from genomic DNA are 818bp and from cDNA – 673bp. 1; 2 – gDNS, 3;4 – before treatment (cDNA); 5-8 – after treatment with MJa, 9-negative control (cDNA).

Nucleotide sequence analysis of the TLP genomic DNA and cDNA identified an open

reading frame (ORF) of 850 bp, which includes one 145bp intron.

1 GTGTTGAGTTATACTGTTTAACCTGCTGCCTCGCCATTTGCAATGGGGCGAAGAACATGC 60

----------------------------------------- M G R R T C

61 GCAGGGTCCCTTTGGATCACAGCAATAGTAACTTTATTAGCTCTTAATGTTTATCTGCAA 120

A G S L W I T A I V T L L A L N V Y L Q

121 Ggtaacgtgccgctctctcaattactctacaatatcactatactgattgctttgatcaat 180

----------------------------------------------------------

181 aaatcatgaattatatagcaggcttatgtattcatttgtcaaagtagaaatattaataga 240

-----------------------------------------------------------

241 ttttgtttggtgaactataagtgcagGTATAGAAGGTGCGACAATGACCGTCAAAAACCA 300

------------------------- G I E G A T M T V K N Q

301 GTGCTCATACACAGTTTGGGCGGCTGGCAGTCCGGGCGGCGGTAAGCAACTGGGGCAGGG 360

C S Y T V W A A G S P G G G K Q L G Q G

361 TGAGACATGGAGTTTTGATGTTCCAGCGGGAACAACAGGAGGCAGAATCTGGGGGCGTAC 420

E T W S F D V P A G T T G G R I W G R T

421 CGGTTGCTCCTTCGACGCTGGCAGTGGTACAGGTAGCTGTACCACTGGTGACTGTGGTGG 480

G C S F D A G S G T G S C T T G D C G G

481 GTTGCTCAATTGTCAAGGCTATGGAAGCGTCCCTGCAACGCTTTTTGAATATGGTCTGAA 540

L L N C Q G Y G S V P A T L F E Y G L N

541 TAAGTACCAGAATCAAGACTTCTATGATATCTCTCTTGTCGATGGCTTCAATATCCCTCT 600

K Y Q N Q D F Y D I S L V D G F N I P L

601 CTCTGTAACTCCCTCAGACTCTTCGTGCAAAGTAATAGGTTGCACCACCGACATAAATGC 660

S V T P S D S S C K V I G C T T D I N A

661 TGTGTGCCCTGCAGAACTGAAAGTGACCGATGGATGCAAGAGTGCTTGTGCGGAGTTCAA 720

V C P A E L K V T D G C K S A C A E F N

721 TACCCCTCAATACTGCTGTACCGGAGACTATGAAAACAACTGCTCTCCCACCAACTATTC 780

T P Q Y C C T G D Y E N N C S P T N Y S

781 GCAATTCTTCAAGGGGCAGTGCCCACAGGCATACAGCTATGCCAAGGACGATGCCACCAG 840

Q F F K G Q C P Q A Y S Y A K D D A T S

841 CACCTTCACCTGCCCTTCTGGTGCTAATTACAACGTCGTCTTCTGTGGTTAGGAAGATAT 900

T F T C P S G A N Y N V V F C G * --------

901 TTACGTAATATCTTCCTACTCATATAGTAACAATAG 936

---------------------------------------------------------------

Fig.x. Nucelotide and deduced amino acid sequence of the P. sylvestris TLP gene. The TLP gene contains one introne. The TLP consists of 234 amino acids.

PCR gDNA 818 bp → ←PCR cDNA 673 bp

Before treatment

After treatment with MJa cDNA

gDNA

10/5/30/50 NC

Controls/ MJa treatment/ - /NC

←PCR cDNA 673 bp

• The TLP gene has an open reading frame, encoding a protein of 234 amino acids with molecular weight approximately 24.6 kDa,

• Sequence has 16 cysteine residues (C) which are characteristic of L-type TLPs.

• The sequence has 5 highly conserved amino acids residues (R, E, D, D, D)* responsible for the acidic cleft with known antifungal activity.

• The deduced protein has a thaumatin family signature (PS00316): G-x-[GF]-x-C-x-T-[GA]-D-C-x(1,2)-[GQ]-x(2,3)C sequence.

• N – terminal signal peptide sequence is underlined.

• Sequence has 7 aa mismatches comparing with P. taeda (cDNA sequence was highly similar to the TLP mRNA, cds from Pinus taeda (99%)).

• TLP gene expression increases in one year old P. sylvestris seedlings after MJ treatments. TLP gene expression was higher 2 weeks after MJ application than before treatment.

• High concentrations of MJ were deadly for young seedlings. Expression level of the TLP gene in needles was lower in plants treated with higher MJ concentrations.

– 10 sample pairs before and after treatment in each treatment group

– Non - treated plants were used as a control

– Samples were collected 2 weeks post treatment, showed varying results

Real time PCR was used to compare TLP gene expression in seedlings before and after MJ treatment.

Results of expression analyses of the TLP gene. In the sample names, the first symbol indicates sample type (10mM MJa; 30 mM MJa;, second – sample number and symbol after comma indicates treatment stage (0 – before treatment, 2 – two weeks after treatment).

Expression of recombinant TLP

Champion pET Directional TOPO Expression Kit (Invitrogen)

• The TLP gene was amplified and cloned into the expression vector pET100 using BL21 Star bacteria.

• Weak TLP expression, protein presence in the insoluble fraction and reduced growth of E.coli that expressed protein, indicated that TLP expression in BL21 cells was toxic to bacteria cells.

Time (h)

• Optimization of the recombinant TLP protein was achieved by cold induction through decreasing expression temperature (25oC) and adding 1% glucose resulted in a lowered expression levels of the recombinant protein in E. coli cells, which reduced the toxicity to the host cells.

• Adding 1 % glucose to the bacterial culture medium may help to repress basal expression of T7 RNA polymerase and stabilize vector construct.

• Decreasing temperature, higher amounts of PsTLP was synthesized, indicating that the PsTLP is not as toxic to the E. coli cells at lower levels of expression.

M – protein MW marker A – no plasmid/no IPTG B – no plasmid/with IPTG C – with plasmid/no IPTG D – with plasmid/with IPTG (TLP)

TLP (24,6 kDa) + N-terminal peptid (3 kDa) = 27,6 kDa.

Different expresion temperature

The presence of a possible TLP even in the non - induced culture indicated some background protein expression.

Initially, cells were induced with 1mM IPTG at 37oC at OD600 = 0.5.

After optimization, induction was done at OD600 = 1,5 (more E. coli cells that could synthesise the PsTLP).

M – protein MW marker A – no plasmid/no IPTG B – no plasmid/with IPTG C – with plasmid/no IPTG D – with plasmid/with IPTG (TLP)

5x dilution

M/A/B/C/D A/B/C/D

5x dilution

M/A/B/C/D/ A/B/C/D

Different induction time

Different IPTG concentration

• Cells were induced at 25oC overnight using different concentrations of IPTG (0.05, 0.1, 0.5 mM) without glucose.

• Different concentration of IPTG had no a significant effect on expressed protein amount.

Fig.x. Analysis of TLP obtained by running aliquots from the same samples in parallel using 2100 Bioanalyzer and SDS-PAGE.

Purification with Ni-NTA

Grown at 25oC ~ 60h with different IPTG conc. M – protein MW marker A – 0,1mM IPTG B – 0,5mM IPTG C – 0,05mM IPTG D – with plasmid/no IPTG E – with plasmid/no IPTG/no 1% glucose

M /A /B /C / A/ B/ C/ D/ E/ M

5x dilution

Recombinant TLP purification with Ni-NTA and enterokinase

• TLP expresed with 6x His tag for protein purification, • Protein purification in native conditions with imidazole, • Imidazole inhibits enterokinase activity, • Imidazole removal with Sephadex G-50, • In antimicrobial activity assay we used TLP with 6x His tag.

His-tag cleavage

E1/E2/E3/+enterokinase Eluates + enterokinase after incubation

L/FT/W1/W2/W3

L - lysate FT- flow-through W- wash E- eluate

A B C

D E F

G H I

J K L

M N

Fig.X. Inhibition of fungi growth by recombinant TLP. (A) Trichoderma sp., (B) A. aerolatum, (C) B. adusta, (D) H. parviporum 17, (E) H. parviporum 162, (F) C. borealis, (G) G.sepiarium, (H) H.annosum 50, (I) H. annosum V Ma15, (J) L.seditiosum, (K) L. conigenum, (L) R.bicolor, (M) S.brinkmannii, (N) S.sanguinolentum. Clockwise from top: 1 - 20µg; 2 – 50 µg; 3 – 300 µg of protein; 4 – control (Ni – NTA eluation buffer).

• S. brinkmanni, C. borealis and R. bicolor were the most sensitive to TLP, inhibited at 20 µg of TLP in the wells.

• H. annosum 50, Trichoderma sp. and L. seditiosum were inhibited only at 100 µg of TLP.

• The longest inhibition time was detected in R. bicolor (14 days), G. sepiarium (14 days), H. parviporum (10 days), H. annosum (10days) and L. seditiosum (14 days) cases.

• There was less activity on faster growing fungi B. adusta and Trichoderma sp..

S. brinkmanni, C. borealis, R. bicolor

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Parts of these fungi are significant pathogen in spruce in Latvia. Arhipova N. et al. 2011 study shows that about one-fifth Picea abietis trees in Latvian forest are infected by butt rot and stem decay. H. parviporum was the most common among decay-causing fungi, isolated from stumps (11,1%) and stems (55,3%). S. sanguinolentum was the second most common basidiomycete. Both of these fungi TLP inhibit relatively strong and for a long time.

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H. annosum, Trichoderma sp., L. seditiosum

Thank you!

This research was funded by European Regional Development

Fund project No 2DP/2.1.1.2.0/10/APIA/VIAA/021.