Mol Biol Rep DOI 10.1007/s11033-013-2640-1
Recombinant b-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth Dahyana Santos Britto • Carlos Priminho Pirovani • Bruno Silva Andrade • Tassiara Pereira dos Santos • Cristina Pungartnik • Ju´lio Cezar M. Cascardo Fabienne Micheli • Abelmon S. Gesteira
•
Received: 6 August 2012 / Accepted: 2 May 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract In this work, we identified a gene from Theobroma 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 b1,3-1,4-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 propidium 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.
This article is dedicated in memory of Ju´lio Cezar M. Cascardo.
Introduction
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 Gene´tica, Universidade Estadual de Santa Cruz (UESC), Rodovia Ilhe´us-Itabuna, km 16, Ilhe´us, 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
Keywords Glycosyl hydrolases Witches’ broom Pathogenesis related protein Antifungal activity Abbreviations ORF Open reading frame PB Phosphate buffer PI Propidium iodide
Carbohydrates constitute the major class of organic compounds in plant tissues and are involved in numerous physiological processes such as growth, signaling, metabolism, symbiosis and plant defense [1]. These carbohydrates, 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-glucan 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
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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 b1,4-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-1,3-1,4D-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)-bglucanase 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–pathogen 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 CaMV35S 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 gloeosporioides; fungal growth and leaf necrosis were reduced in the transgenic plants when compared to controls. Cacao (Theobroma cacao L.) is an important commodity 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 glucanase expression in heterologous system and the first analysis showing the antifungal activity of a b-1,3-1,4glucanase.
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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 conserved 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. Phylogenetic 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 species 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 phylogenetic Bayesian analyses were performed in MRBAYES 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 submitted to an alignment with the TcGlu2 sequence using TCOFFEE [29], to find conserved regions and motifs. Separately, another TCOFFEE alignment with 1ZM1—a b-1,31,4-D-glucanase of Fibrobacter succinogenes previously
Mol Biol Rep
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/ ˚ in vacuum. Molecular dynamics simulation of 2.71 A 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, PROCHECK 3.6 [33] and ANOLEA [34, 35] were used to evaluate 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 (NovagenÒ), 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-thiogalactoside 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 (ClontechÒ 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 (Promega). The resulting tryptic digests were vacuum concentrated (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 acetonitrile 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 analyzed 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). Afterwards, 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 control 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 (LeicaÒ) 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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Mol Biol Rep b 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-1,3-1,4glucanases, 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 b1,3-1,4-D-glucanase (XP_002516793), G. max endo-1,3-1,4-b-D-glucanase (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 substitutions 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
as well as under fluorescent filter using the IM50 software (LeicaÒ).
Results Sequence analysis The sequence TcGlu2 was identified as a probable b-1,31,4-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 incomplete ORF of 582 nucleotides encoding a protein of 194 amino acids residues (Fig. 1a, filled triangle). The corresponding 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).
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, ˚ , and Gly102, Phe103, Glu104, Asp105), a space of 2831 A 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
Phylogeny of T. cacao glucanases The Bayesian consensus phylogram positioned the glucanases 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
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
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Mol Biol Rep Fig. 3 TcGlu molecular modeling. a Alignment between TcGlu2 and 1ZM1 sequences. The colors blue and green correspond 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)
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
Fig. 4 SDS-PAGE analysis of TcGlu2 expressed in bacteria. a Expression of the recombinant TcGlu2 (with His-Tag) in E. coli Rosetta (DE3). Line 1 pET28a without insert and without induction, line 2 pET28a without insert 5 h after induction, line 3 pET28aTcGlu2 without induction, line 4 pET28a-TcGlu2 5 h after induction. The arrow indicates a TcGlu2 protein band. b Purified TcGlu2 under native conditions
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
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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 control (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 fluorescence emitted by the hyphae indicated that membrane
Mol Biol Rep 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. PB phosphate buffer; CP control proteins corresponding to purified protein extract from E. coli expressing pET28a without insert
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 incubation (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-glucanase 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. communis (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, respectively) 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. succinogenes (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-1,31,4-glucanase from barley with sequences of other plant b1,3-1,4-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 corresponding 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 posttranslational modification involved in signaling, regulation of the protein function, protein stability and sub-cellular localization [37, 38]. Some PR protein, such as the CaPR10 from hot pepper (Capsicum annuum) was phosphorylated in response to the biotic stress due to the tobacco mosaic virus (TMV-P0), and this post-translational
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Mol Biol Rep Fig. 6 Membrane permeability of Moniliophthora perniciosa hyphae. 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
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).
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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-
Mol Biol Rep
glucanase isoenzyme II from Hordeum vulgare (accession number AAA32962; [41]), 8 % of identity with the b-1,4glucanase (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 mechanical wounding [13]. Recombinant incomplete TcGlu2 inhibited the M. perniciosa mycelium growth (Fig. 5) suggesting an antifungal/ antimicrobial activity of this enzyme. However, in the literature, antimicrobial activity was mainly observed in b-1,3glucanases. For example, Ji and Kuc [43] showed that a b1,3-glucanase from cucumber inhibited spore germination and mycelium growth of the necrotrophic fungus Colletotrichum 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 comparison 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-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. The use of PI,
known to be a marker of cell viability, revealed that the antifungal activity of TcGlu2 may be related to cell membrane 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 Cientı´fico e Tecnolo´gico (CNPq, Brazil) and the International Foundation for Science (IFS). The work of DSB and TPS was supported by the Fundac¸a˜o de Amparo a` Pesquisa 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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