Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth

Recombinant b-1,3-1,4-glucanase from Theobroma cacao impairsMoniliophthora perniciosa mycelial growth

Dahyana Santos Britto • Carlos Priminho Pirovani • Bruno Silva Andrade •

Tassiara Pereira dos Santos • Cristina Pungartnik • Julio Cezar M. Cascardo •

Fabienne Micheli • Abelmon S. Gesteira

Received: 6 August 2012 / Accepted: 2 May 2013

� Springer Science+Business Media Dordrecht 2013

Abstract In this work, we identified a gene from Theo-

broma cacao L. genome and cDNA libraries, named

TcGlu2, that encodes a b-1,3-1,4-glucanase. The TcGlu2

ORF was 720 bp in length and encoded a polypeptide of

239 amino acids with a molecular mass of 25.58 kDa.

TcGlu2 contains a conserved domain characteristic of b-

1,3-1,4-glucanases and presented high protein identity with

b-1,3-1,4-glucanases from other plant species. Molecular

modeling of TcGlu2 showed an active site of 13 amino

acids typical of glucanase with b-1,3 and 1,4 action mode.

The recombinant cDNA TcGlu2 obtained by heterologous

expression in Escherichia coli and whose sequence was

confirmed by mass spectrometry, has a molecular mass of

about 22 kDa (with His-Tag) and showed antifungal

activity against the fungus Moniliophthora perniciosa,

causal agent of the witches’ broom disease in cacao. The

integrity of the hyphae membranes of M. perniciosa,

incubated with protein TcGlu2, was analyzed with propi-

dium iodide. After 1 h of incubation, a strong fluorescence

emitted by the hyphae indicating the hydrolysis of the

membrane by TcGlu2, was observed. To our knowledge,

this is the first study of a cacao b-1,3-1,4-glucanase

expression in heterologous system and the first analysis

showing the antifungal activity of a b-1,3-1,4-glucanase, in

particular against M. perniciosa.

Keywords Glycosyl hydrolases � Witches’ broom �Pathogenesis related protein � Antifungal activity

Abbreviations

ORF Open reading frame

PB Phosphate buffer

PI Propidium iodide

Introduction

Carbohydrates constitute the major class of organic com-

pounds in plant tissues and are involved in numerous

physiological processes such as growth, signaling, metab-

olism, symbiosis and plant defense [1]. These carbohy-

drates, which present large structural and functional

diversity, are synthesized, modified and degraded by a

great variety of enzymes. The ‘‘carbohydrate-active

enzymes’’ are classified based on amino acid sequences

and represent 130 different families which can be accessed

in the CAZy website (Carbohydrate Active Enzymes

database; http://www.cazy.org/; [2]). In particular, b-glu-

can degradation in nature is catalyzed by b-glucanases and

endoglucanases that depolymerize b-1,3-1,4-D-glucans.

These enzymes are organized in four categories: (i) specific

This article is dedicated in memory of Julio Cezar M. Cascardo.

D. S. Britto � C. P. Pirovani � T. P. dos Santos � C. Pungartnik �J. C. M. Cascardo � F. Micheli � A. S. Gesteira

Centro de Biotecnologia e Genetica, Universidade Estadual de

Santa Cruz (UESC), Rodovia Ilheus-Itabuna, km 16, Ilheus,

BA 45662-900, Brazil

B. S. Andrade

Universidade Estadual do Sudoeste da Bahia (UESB), Av. Jose

Moreira Sobrinho, Jequie, BA 45206-190, Brazil

F. Micheli (&)

CIRAD-BIOS, UMR AGAP, Avenue Agropolis TA96/03,

34398 Montpellier Cedex 5, France

e-mail: [email protected]

A. S. Gesteira

Embrapa Mandioca e Fruticultura, Caixa Postal 007, Cruz das

Almas, BA 44380-000, Brazil

123

Mol Biol Rep

DOI 10.1007/s11033-013-2640-1

b-1,3-1,4-D-glucanases or true lichenases (EC 3.2.1.73)

that strictly cleave b-1,4-glycosidic linkages adjacent to a

3-O-substituted glucose residue but are inactive against b-

1,4-glucans; (ii) endo-b-1,4-D-glucanases (EC 3.2.1.4),

which hydrolyze b-1,4-glycosidic bonds other than those

targeted by lichenases; (iii) b-1,3(4)-D-glucanases (EC

3.2.1.6) active on b-1,3-1,4-D-glucans and b-1,3-D-glucans;

and (iv) b-1,3-D-glucanases or laminarinase (EC 3.2.1.39)

[2, 3]. The b-1,3-1,4-D-glucanases (lichenases) have been

identified in various microorganisms [4–7] and fungi [8–

10], mainly for industrial applications. However, b-1,3-1,4-

D-glucanases from plants have been little studied, and these

studies focused mainly on plant development [11–13]. In

2009, Akiyama et al. showed that an endo-(1,3;1-4)-b-

glucanase gene from rice (OsEGL2) had its expression

significantly increased in response to methyl jasmonate,

abscissic acid and mechanical wounding [13]. The

mechanical wounding also increased the leaf elongation

rate in rice seedlings in comparison to the control [13]. To

our knowledge, there is only few data about involvement of

b-1,3-1,4-D-glucanases in plant defense or in plant–patho-

gen interactions. Other plant glycosyl hydrolases are well

known to be involved in such responses to pathogens such

as chitinases and b-1,3-glucanases and may be classified as

pathogenesis-related (PR) proteins [1]. Among various

examples, some of them are related to cacao disease

resistance [14, 15]. A PR10 protein of cacao (TcPR-10),

although it does not belong to the glysosyl hydrolase

family, showed in vitro and in vivo antifungal activity

against mono- and dicaryotic mycelium and basidiospores

of M. perniciosa, and against Saccharomyces cerevisiae

[14, 16]. On the other hand, a chitinase gene of cacao

(TcChi1) was used under the control of a modified CaMV-

35S promoter for Agrobacterium-mediated transformation

of cacao somatic embryo cotyledons [15]. The TcChi1

transgenic cacao plants showed an in vivo antifungal

activity against the foliar pathogen Colletotrichum gloeo-

sporioides; fungal growth and leaf necrosis were reduced in

the transgenic plants when compared to controls.

Cacao (Theobroma cacao L.) is an important com-

modity cultivated primarily to provide cacao liquor, butter,

and powder for the chocolate industry and unfortunately

has been frequently the target of several fungal diseases

[17]. For this reason, we focused our attention on a gene of

b-1,3-1,4-glucanase from T. cacao (TcGlu2) as a candidate

for disease control. Here, we report the molecular cloning

of TcGlu2, the heterologous expression of the recombinant

protein and its antifungal activity against Moniliophthora

perniciosa, the causal agent of the witches’ broom disease.

To our knowledge, this is the first study of a cacao glu-

canase expression in heterologous system and the first

analysis showing the antifungal activity of a b-1,3-1,4-

glucanase.

Materials and methods

Identification and analysis of genomic and cDNA

sequences of TcGlu2

The TcGlu2 cDNA was identified from a cDNA library of

susceptible cacao (Catongo variety) inoculated by M.

perniciosa [18] and later the complete sequence was

encountered in the T. cacao genome [19]. The Open

reading frame (ORF) of the nucleotide sequence was

determined using the ORFinder program (Lasergene,

Madison, WI, USA). For function homology analysis, the

sequence was compared with the public sequence database

using BLAST [20]. ClustalW was used for multiple

nucleotide or amino acid sequence alignment [21]. NetPhos

2.0 Server [22] and InterProScan [23] were used for

identification of putative phosphorylation sites and con-

served domains, respectively.

Phylogeny

All b-1,3-1,4-glucanases from T. cacao were obtained at

CocoaGenDB website (http://cocoagendb.cirad.fr/gbrowse/

cgi-bin/gbrowse/theobroma/); TcGlu2 and two more

sequences homologous to this one were found. Phyloge-

netic analyses were performed with the BLAST program

version 2.2.27 [20] using: (i) TcGlu2 and the two other

amino acid sequences of b-1,3-1,4-glucanases from T.

cacao; and (ii) b-1,3-1,4-glucanases from other plant spe-

cies similar to T. cacao glucanases. Amino acid sequences

were aligned using BLOSUM matrix [24] in ClustalW2

(http://www.ebi.ac.uk/Tools/msa/clustalw2/) [25]. The phy-

logenetic Bayesian analyses were performed in MRBA-

YES 3.1.2 [26] using the mixed evolutionary model. Three

independent runs were conducted (each with four chains)

for 2 9 106 generations, sampling every 100 generations.

For this analysis we used one of Brachypodium distachyon

glucanase sequences (XP 0035684871) as outgroup, because

of its more distant evolution relationship with T. cacao.

Molecular modeling of TcGlu2

The structure of the complete ORF of TcGlu2 was built by

Comparative Modeling approach. Initially, TcGlu2 amino

acid sequence was subjected to the Swiss Model Workspace

server, using an automated modeling approach [27, 28] to

find templates that could be used to construct the TcGlu2

model. Additionally, other templates deposited in Protein

Data Bank, were searched. All templates found were sub-

mitted to an alignment with the TcGlu2 sequence using

TCOFFEE [29], to find conserved regions and motifs. Sep-

arately, another TCOFFEE alignment with 1ZM1—a b-1,3-

1,4-D-glucanase of Fibrobacter succinogenes previously

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crystallized and considered as a good template for molecular

modeling [30]—was performed in order to find conserved

active site regions between the two sequences. The 3D model

was generated using Swiss Model Workspace [27, 28] in an

alignment mode. Afterwards, the initial model was prepared

by AMBER 11 package [31, 32], using LEAP and SANDER

for structure refinement. The structure was fully minimized

with 500 steps of steepest descent followed by 500 more

steps of conjugate gradient to an RMS gradient of 0.01 kcal/

2.71 A in vacuum. Molecular dynamics simulation of

refined structure was performed in vacuum using f99 force

field at 300 K for 1 ns, restricted to residues 90–145—

according to TcGlu2/1ZM1 alignment. Finally, PRO-

CHECK 3.6 [33] and ANOLEA [34, 35] were used to eval-

uate stereo chemical and energy quality of the final model.

The final structure of TcGlu2 and the crystallographic

structure of 1ZM1 were structurally aligned by Pymol

1.5.0.4 (The PyMOL Molecular Graphics System, 2012)

using the script fitting.py, and restricted to the amino acids

fragments 90–145 in the TcGlu2 sequence, and 58–148 in the

1ZM1 sequence. The active site of TcGlu2 was estimated

using superimposition and alignment data, and observing

which 1ZM1 catalytic amino acids best fit with the target

model amino acids.

Expression of recombinant TcGlu2

The TcGlu2 cDNA was amplified by PCR using the TcGlu-F

(50-GGCGGGATCCATATGTCAGGTCCGCAGTGC-30)and TcGlu-R (50-GGCGGTCGACTCAGGGTTTAGCTTT

TAAG-30) primers. The PCR product was cloned into the

NdeI and SalI sites of the plasmid pET28a (Novagen�),

and the resulting in frame fusion plasmid was transformed

into Escherichia coli strain Rosetta (DE3). Overexpression

of the TcGlu2 tagged with six histine residues at the

N-terminus was induced by 1 mM of isopropyl-b-D-thiog-

alactoside at 37 �C. To establish the kinetics of TcGlu2

induction, bacteria were collected and protein content

analyzed 1, 2 and 3 h after induction. Larger quantities of

TcGlu2 that are needed for protein purification were

obtained 4 h after induction. For recombinant protein

purification, bacteria were centrifuged and washed once in

equilibration buffer [50 mM phosphate buffer (PB),

300 mM NaCl, pH 7.4], suspended in lysis buffer

(50 mM PB, 300 mM NaCl, 0.1 mg/plate lysozyme, pH

7.4) and kept at room temperature for 1 h. The sample was

kept in ice and sonicated (Gex Ultrasonic processor 130,

130W; 8 pulses of 30 s each, 75 % output, 30 s intervals),

and the resulting lysate was centrifuged for 20 min at

11,000g. The supernatant containing soluble proteins was

loaded onto Talon resin metal affinity column (Clontech�

Laboratories), eluted with 150 mM imidazole and dialyzed

against 500 ml of 50 mM PB, pH 7.0. The purified TcGlu2

protein was digested with trypsin (25 ng/ll) at 37 �C for

12 h according to the manufacturer’s instructions (Pro-

mega). The resulting tryptic digests were vacuum con-

centrated (Concentrator 5301, Eppendorf), desalted using a

pre-Symmetry column (Waters, Mildford, MA, USA) C18

(5, 180 lm in inner diameter 9 20 mm long), and then

fractioned by C18 reverse phase chromatography column

(100 mm 9 100 lm, 1.7 lm particles) on the nanoAcquity

UPLC (WATERS) for 50 min under 0.6 ll min-1 aceto-

nitrile flux. The following gradient was used: 1 % for

1 min, 1–50 % in 40 min, 50–85 % in 5 min, 85 % for

2 min, 85–1 % in 1 min, 1 % for 2 min. Afterwards, the

peptides were deionized at 3,000 V and split on positive

mode with minimum relative intensity of 10 counts on

Micromass ESI-Q-TOF (WATERS). Spectra were ana-

lyzed using the ProteinLynx Global Server 4.2 (WATERS)

and compared with the NCBI database (http://www.ncbi.

nlm.nih.gov/BLAST/).

M. perniciosa growth conditions

M. perniciosa strain ALF553 cultures (CCBM000257,

UEFS, Feira de Santana, Brazil) were grown and toxicity

tests were performed on M. perniciosa pseudo-colonies as

previously described [36]. Dikaryotic cultures were grown

in CPD (2 % glucose, 2 % peptone) in liquid media,

without agitation at 25 �C, for 5–7 days. Agar 2 % was

added when solid media were used.

Antifungal activity of TcGlu2

TcGlu2 (6, 12 and 32 lg/plate) or PB (control) or purified

protein extract from E. coli expressing pET28a without insert

was spread onto 20 ml of CPD agar plates (control). After-

wards, 1 ml of dikaryotic M. perniciosa broken hyphae [36]

was spread one the same plates which were further incubated

for 7 days at 25 �C. M. perniciosa survival was defined as the

percentage of grown pseudo-colonies (treated/PB con-

trol 9 100). The experiment was made in triplicate.

Membrane viability analysis

To check for viability and membrane integrity, fungal

hyphae were incubated with both TcGlu2 (8 lg/ll) and

2 lM of propidium iodide (PI) at 25 �C. After incubation

intervals ranging from 30 min to 1 h, PI-labeled, hyphae

were washed twice in 0.05 mM PB and then observed

under fluorescence microscope DMRA2 (Leica�) attached

with PI filter. The following negative controls were used:

(i) assay with TcGlu2 boiled for 1 h at 100 �C; (ii) assay in

presence of NaN3 (inhibitor of metabolism); and (iii) assay

at 4 �C (inhibition of protein transport). Images were

captured using 409 and 1009 objectives under bright field

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A

B

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as well as under fluorescent filter using the IM50 software

(Leica�).

Results

Sequence analysis

The sequence TcGlu2 was identified as a probable b-1,3-

1,4-glucanase with an ORF of 720 nucleotides encoding a

protein of 239 amino acids residues. The protein contains a

domain conserved between b-1,3-1,4-glucanases (from

amino acid 5–238) and 11 putative phosphorylation sites

on serines (S17, S86, S115, S119, S160, S176 and S221) and

tyrosine (Y69, Y222 and Y237) (Fig. 1a). The complete

protein has a calculated molecular mass of 25.58 kDa and

an isoelectric point of 5.85. The cDNA obtained from

cDNA libraries already published [18] presents an incom-

plete ORF of 582 nucleotides encoding a protein of 194

amino acids residues (Fig. 1a, filled triangle). The corre-

sponding predicted protein, without the His-Tag, has a

calculated molecular mass of 20.61 kDa (22.2 kDa with

the His-Tag) and an isoelectric point of 6.82. The amino

acid alignment of TcGlu2 with different members of the

glycosylated hydrolase family (b-1,3-1,4 glucanases)

indicated high similarity between the sequences (Fig. 1b).

Phylogeny of T. cacao glucanases

The Bayesian consensus phylogram positioned the glu-

canases found in T. cacao genome in relation to those

found in other plant families (Fig. 2). T. cacao glucanase

(g009650) and TcGlu2 (g022720) formed a polyphyletic

clade with glucanases of Ricinus communis and Glycine

max. T. cacao (g022730) presented itself externally over

the tree, forming a polyphyletic clade with R. communis

glucanase and with a group of R. communis (XP 0025167911)

and G. max (XP 0035428951) glucanases.

TcGlu2 structure

TcGlu2 is a single b-1,3-1,4-D-glucanase chain presenting

7 a-helices, 10 b-sheets and 4 salt bridges (Fig. 3b). After

TCOFFEE alignment with 1ZM1 sequence, a high level of

identity between both sequences was found, in particular

with the active site region of 1ZM1 (Fig. 3a). The active

site of TcGlu2 is composed of 13 amino acids (Trp93,

Leu94, Lys95, Asp96, His97, Gly98, Pro99, Asp100, Lys101,

Gly102, Phe103, Glu104, Asp105), a space of 2831 A, and

presents a-helices and loop structures (Figs. 3c, 1a).

Expression analysis of recombinant protein TcGlu2

and mass spectrometry sequence validation

The TcGlu2 cDNA was successfully cloned in pET28a

plasmid and expressed in E. coli Rosetta (DE3) after 1 h of

induction. The highest yield of the recombinant protein was

obtained after 5 h of induction (Fig. 4a, line 4), whereas no

visible band was observed in the control (pET28a without

insert) (Fig. 4a, line 2). The soluble enzyme purified from the

cytosolic fraction of lysed cells showed a molecular mass of

25 kDa—with His-Tag (Fig. 4b). Trypsin digestion and

Fig. 2 Bayesian phylogenetic analysis, using amino acid data.

Bayesian consensus phylogram of T. cacao b-1,3-1,4-glucanases

and glucanases from other plant species

Fig. 1 Nucleotide and amino acid sequences of TcGlu2. a Nucleotide

and deduced amino acid sequences of TcGlu2. The asterisk represents

the ORF termination codon. Putative phosphorylation sites are squared

on the amino acid sequence. The conserved domain between b-1,3-1,4-

glucanases, is underlined. Putative active site is shaded in grey. The

filled triangle represents the beginning of the lost region in TcGlu2

cDNA. b Comparison of predicted amino acid sequence of TcGlu2 with

those of Vitis vinifera b-1,3-1,4-D-glucanase (XP_002275697), R.communis b-1,3-1,4-D-glucanase (XP_002524812), R. communis b-

1,3-1,4-D-glucanase (XP_002516793), G. max endo-1,3-1,4-b-D-glu-

canase (XP_003550071), Arabidopsis thaliana b-1,3-1,4-D-glucanase

(AAM61180), E. guineensis endo-1,3-1,4-b-D-glucanase (ACF06491),

Brachypodium distachyon endo-1,3-1,4-b-D-glucanase (XP_00356

8483), Brachypodium distachyon endo-1,3-1,4-b-D-glucanase

(XP_003568487). Gaps introduced to get the best alignment are

indicated by dashes, (asterisks) represents identical amino acids

between all sequences, (dot) and (colon) represent conserved substitu-

tions and semi-conserved substitutions, respectively. Identity percent

between TcGlu2 and the other sequences is indicated at the right of each

sequence end. Conserved aspartate residues potentially involved in

recognition and/or cleavage of a specific substrate are shaded in grey

b

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mass spectrometry identified four peptides from purified

TcGlu2: peptide 1: LAVLLVSDVFGYDAPNLR; peptide 2:

LVIDALK; peptide 3: EALIPAAVLLHPSFVTVDDIK;

and peptide 4: VPIAILGAEIDQLSPPALVK. Blastp of

these segments against public databanks showed significant

homology with plant b-1,3-1,4-glucanase (for the three

largest segments, i.e., segments 1, 3 and 4; Table 1). The 4

peptides covered 27 and 34.3 % of the incomplete and

complete protein, respectively.

In vitro antifungal activity of purified TcGlu2

The survival rate of dikaryotic hyphae of M. perniciosa

incubated with the TcGlu2 protein (6, 12 and 32 lg) was

assessed on solid medium (Fig. 5). The survival rate of M.

perniciosa is about 13 and 7 % when incubated with 6 and

12 lg of TcGlu2, respectively; the survival rate is zero

when incubated with 32 lg of protein, i.e., there is no

fungal growth at this dose. Under control conditions (PB

and protein extract purified from E. coli expressing pET28a

without insert) the fungus growth was 100 % (Fig. 5).

Fungistatic activity test of TcGlu2 on M. perniciosa

hyphae, using 2, 4 and 6 lg of protein by plate showed that

only the hyphae incubated with 6 lg of protein showed

significant inhibition of growth in comparison to the con-

trol (data not shown).

Membrane integrity

The hydrolytic activity of the TcGlu2 protein was analyzed

in the presence of PI (Fig. 6). The presence of red fluo-

rescence emitted by the hyphae indicated that membrane

Fig. 3 TcGlu molecular

modeling. a Alignment between

TcGlu2 and 1ZM1 sequences.

The colors blue and greencorrespond to bad aligned

regions, yellow and orange to

average aligned regions and the

color red indicates good aligned

regions. Cons. consensus.

b Theoretical structure of

TcGlu2. c Theoretical structure

of TcGlu2 detailing active site

amino acids in pink. (Color

figure online)

Fig. 4 SDS-PAGE analysis of TcGlu2 expressed in bacteria.

a Expression of the recombinant TcGlu2 (with His-Tag) in E. coliRosetta (DE3). Line 1 pET28a without insert and without induction,

line 2 pET28a without insert 5 h after induction, line 3 pET28a-

TcGlu2 without induction, line 4 pET28a-TcGlu2 5 h after induction.

The arrow indicates a TcGlu2 protein band. b Purified TcGlu2 under

native conditions

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damages were caused by the hydrolase activity of the

protein. The cells incubated with the protein were assayed

within 30 min and 1 and 6 h in different conditions (see

‘‘Materials and methods’’ section; Fig. 5). At 25 �C, a

slight red coloration was observed after 30 min of incu-

bation (Fig. 6a) and highly increased at 1 h after incubation

(Fig. 6b). At 25 �C in presence of NaN3 0.05 % or at 4 �C,

no fluorescence was observed (Fig. 6c, d, respectively). No

fluorescence emission was observed when the experiment

was made with boiled TcGlu2 (Fig. 6e).

Discussion

Here, we reported the characterization of b-1,3-1,4-glu-

canase cDNA of cacao and its corresponding protein,

named TcGlu2. The TcGlu2 ORF was 720 bp in length and

encoded a polypeptide of 239 amino acids with a molecular

mass of 25.58 kDa. This molecular mass is in accordance

with the one of the b-1,3-1,4-glucanase of R. communis

(25.83 kDa; accession number XP002524812), R. com-

munis (25.8 kDa; accession number XP_002516793) and

Elaeis guineensis (25.88 kDa; accession number

ACF06491) encountered as similar to TcGlu2 after mass

spectrometry analysis (Table 1). TcGlu2 also contains a

conversed domain characteristic of b-1,3-1,4-glucanases

(Fig. 1a) and presented high protein identity (from 53.9 to

70.5 % for XP_003568487 and XP_002516793, respec-

tively) with b-1,3-1,4-glucanases from other plant species

(Fig. 1b). The phylogeny analysis of TcGlu2 with T. cacao

and other plant species glucanases showed that T. cacao

sequences formed a clade with R. communis and G. max

glucanases (Fig. 2), corroborating the results of Argout

et al. (2011), which described a high sharing of genes

between T. cacao, Vitis vinifera, G. max, Arabidopsis

thaliana and Populus trichocarpa. The alignment of

TcGlu2 with the b-1,3-1,4-D-glucanase from F. succinog-

enes (1ZM1) showed that the active site regions of these

structures share common features and folding (Fig. 3). The

structural alignment of TcGlu2 and 1ZM1 showed that the

active site of TcGlu2 presents several amino acids folded in

the same way that the 1ZM1 active site (Fig. 3c).

According to Tsai et al. [30], 1ZM1 presents bonds

between glucose residues ?1, makes eight hydrogen bonds

with four amino acids (Asp58, Glu60, Gln70 and Asn72), and

has one van der Waals stacking interaction with Trp148.

TcGlu2 presents three amino acids (Asp100, Glu104 and

Lys101) performing hydrogen bonds with the substrate and

Trp93—instead van der Waals stacking interaction in the

case of Trp148 of 1ZM1 (Fig. 3c). This result confirms that

TcGlu2 is a glucanase with b-1,3 and 1,4 action mode. Tsai

et al. [30] also compared the primary sequence of b-1,3-

1,4-glucanase from barley with sequences of other plant b-

1,3-1,4-glucanases and b-1,3-glucanases, and showed that

the tyrosine 177 (Y177) was an important residue conserved

in all the b-1,3-1,4-glucanases. The structural analysis

results suggest that the tyrosine residue is involved in the

recognition of mixed b-1,3 and b-1,4 linked polysaccharide

[30]. In several known b-1,3-glucanases the structural

position equivalent to the tyrosine residue was occupied by

a glycine or an aspartate [30]. In TcGlu2—and in the other

b-1,3-1,4-glucanases highly homologous to TcGlu2—an

aspartate residue (D156) is present at the position corre-

sponding to Y177 (Fig. 1b).

Putative phosphorylation sites were observed in the

specific b-1,3-1,4-glucanase conserved domain of TcGlu2

(Fig. 1a). As already known, the phosphorylation is a post-

translational modification involved in signaling, regulation

of the protein function, protein stability and sub-cellular

localization [37, 38]. Some PR protein, such as the CaPR-

10 from hot pepper (Capsicum annuum) was phosphory-

lated in response to the biotic stress due to the tobacco

mosaic virus (TMV-P0), and this post-translational

Table 1 Peptides of TcGlu2 after analysis by mass spectrometry

Peptide number Amino acid sequence Identity (%) E-value Organism Accession number Function

1 LAVLLVSDVFGYDAPNLR 82 1.10-04 Elaeis guineensis ACF06491 b-1,3-1,4-glucanase

2 LVIDALK – – – – –

3 EALIPAAVLLHPSFVTVDDIK 94 1.10-06 R. communis XP002524812 b-1,3-1,4-glucanase

4 VPIAILGAEIDQLSPPALVK 85 1.10-06 R. communis XP002516793 b-1,3-1,4-glucanase

Fig. 5 Effect of TcGlu2 on Moniliophthora perniciosa growth. PBphosphate buffer; CP control proteins corresponding to purified

protein extract from E. coli expressing pET28a without insert

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modification altered the CaPR-10 function [39]. The

expression of active recombinant TcGlu2 protein in a

bacterial system is supported by the absence of putative

N-glycosylation sites (N-X-S/T type) [40] in the amino

acid sequence (Fig. 1a).

To our knowledge a small number of physiological data

related to b-1,3-1,4-glucanases were encountered, and

unfortunately, TcGlu2 presented few identity with those for

which such physiological reports exist. For example,

TcGlu2 presents 8 % of identity with Glb2, a b-1,3-1,4-

Fig. 6 Membrane permeability

of Moniliophthora perniciosahyphae. TcPR-10 was incubated

with M. perniciosa hyphae at 25

or 4 �C with or without NaN3,

and membrane integrity was

checked by using PI uptake

assay. Hyphae were observed

under fluorescence microscope

at 30 min and 1 h and images

were taken under phase contrast

and PI filter

Mol Biol Rep

123

glucanase isoenzyme II from Hordeum vulgare (accession

number AAA32962; [41]), 8 % of identity with the b-1,4-

glucanase (TcGlu1) from T. cacao (accession number

AY487173 [42]), and 15.5 and 13 % of identity with

OsEGL1 and OsEGL2 b-1,3-1,4-glucanases from rice,

respectively (accession numbers AAV37460 and

BAB85436, respectively; [13]) (data not shown). Glb2, as

other barley glucanases [3, 11] is possibly involved in cell

wall organization during seed germination [41]. In cacao,

the expression of TcGlu1 was induced in leaves after

treatment by the Necrosis and Ethylene inducing Protein 1

and after infection by the fungus Phytophthora megakarya,

and may be associated with senescence process [42]. In

rice, the OsEGL1 gene was expressed in response to

methyl jasmonate, abscissic acid, ethephron and mechani-

cal wounding [13].

Recombinant incomplete TcGlu2 inhibited the M. per-

niciosa mycelium growth (Fig. 5) suggesting an antifungal/

antimicrobial activity of this enzyme. However, in the lit-

erature, antimicrobial activity was mainly observed in b-1,3-

glucanases. For example, Ji and Kuc [43] showed that a b-

1,3-glucanase from cucumber inhibited spore germination

and mycelium growth of the necrotrophic fungus Colleto-

trichum lagenarium, and Sela-Buurlage et al. [44] showed

that the b-1,3-glucanase from tobacco was active against the

fungus Fusarium solani, resulting in lysis of the hyphal tips

and growth inhibition. In both works, there was a synergistic

activity of the b-1,3-glucanase studied and a chitinase from

the same organism (cucumber vs tobacco) [43, 44].

According to Jach et al., [45] co-overexpression of genes

coding for b-1,3-glucanase and chitinase from barley in

tobacco plants led to a significant increase of plant defense

when infected by the fungus Rhizoctonia solani in compar-

ison to the non-transformed plants. Similar results were

observed by introducing a maize b-1,3-glucanase (M-GLU)

into tomato and by submitting the transformed plant to

inoculation with Alternaria solani spores (causal agent of the

early blight disease); compared to control (non-transformed)

plants, the transgenic lines carrying M-GLU showed

enhanced resistance to early blight disease [46]. According

to the sequence structure of TcGlu2 (D instead of Y residue

as the position 156 of the substrate recognition site) and to the

its functional characteristics as antifungal molecule, this

glucanase presents more similarities with b-1,3-glucanase

than with the b-1,3-1,4-glucanase family to which it belongs,

but on the other hand, the structure of the active site of

TcGlu2 suggests a b-1,3 and 1,4 glucanase activity. It is

interesting to note that the 198–239 region of the protein is

not necessary for the antifungal activity. Moreover, to our

knowledge, this is the first study of a cacao b-1,3-1,4-glu-

canase expression in heterologous system and the first

analysis showing the antifungal activity of a b-1,3-1,4-glu-

canase, in particular against M. perniciosa. The use of PI,

known to be a marker of cell viability, revealed that the

antifungal activity of TcGlu2 may be related to cell mem-

brane damages (Fig. 6). Because the PI fluorescence was

observed only 1 h after TcGlu2 application (Fig. 6b), it may

be suggested that the destruction of the membrane was a

consequence of substrate-specific recognition of TcGlu2 to

polymers of the fungus hyphae, and that TcGlu2 may have

acted by permeabilization or by direct destruction of the

fungus membrane, as observed for other antifungal proteins

in plants [43]. Furthermore, according to the data presented

here, TcGlu2 may be a good candidate to increase T. cacao

resistance to the fungus M. perniciosa by a similar approach

as the one developed for a class I chitinase gene [15].

Acknowledgments This research was supported by the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq,

Brazil) and the International Foundation for Science (IFS). The work

of DSB and TPS was supported by the Fundacao de Amparo a Pes-

quisa da Bahia (FAPESB, Brazil). We thank Dr. Alan Pomella (Grupo

Farroupilha, Brazil) for kindly providing the M. perniciosa strain and

Dr. Claudia Fortes Ferreira (Embrapa, Cruz das Almas-BA, Brazil)

for critical reading of the manuscript.

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