# The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance

Akyla Maria Martins Alves, Sara Pereira Menezes, Eline Matos Lima, Karina Peres Gramacho, Bruno Silva Andrade, Monaliza Macêdo Ferreira, Carlos Priminho Pirovani, Fabienne Micheli

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## To cite this version:

Akyla Maria Martins Alves, Sara Pereira Menezes, Eline Matos Lima, Karina Peres Gramacho, Bruno Silva Andrade, et al.. The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance. Plant Physiology and Biochemistry, 2019, 142, pp.472 - 481. 10.1016/j.plaphy.2019.08.005. hal-03488311

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Submitted on 20 Jul 2022

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1 **The selenium-binding protein of *Theobroma cacao*: an important protein to control**

2 **biotic and abiotic stresses in plants a thermostable protein involved in the witches'**

3 **broom disease resistance**

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5 Akyla Maria Martins Alves¹, Sara Pereira Menezes¹, Eline Matos Lima¹, Karina Peres

6 Gramacho², Bruno Silva Andrade3, **Monaliza Macêdo Ferreira¹, Carlos Priminho**

7 **Pirovani¹**, Fabienne Micheli¹,⁴,*

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9 ¹Universidade Estadual de Santa Cruz (UESC), Departamento de Ciências Biológicas (DCB), Centro de Biotecnologia e Genética (CBG), Rodovia Ilhéus-Itabuna, km 16, 45662-900 Ilhéus-BA, Brazil.

12 ²Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brasil.

13 ³Universidade Estadual do Sudoeste da Bahia (UESB), Av. José Moreira Sobrinho, Jequié, Bahia, 45206-190, Brazil

15 ⁴CIRAD, UMR AGAP, F-34398 Montpellier, France

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17 **\*Corresponding author:** Dr Fabienne Micheli, UESC, DCB, Rodovia Ilhéus-Itabuna km16, 45662-900, Ilhéus-BA, Brazil. Phone: +55 73 3680 5196. Fax: +55 73 3680 5226. E-mail: fabienne.micheli@cirad.fr

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26 **Abstract**

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from *Theobroma cacao* (*TcSBP*) was first identified from a cocoa-*Moniliophthora perniciosa* cDNA library. The present study provides details on the *TcSBP* gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Specific *cis*-elements related to hormone signaling and response to light were identified in the *TcSBP* promotor region. Homology modeling and molecular docking were performed and showed that the *TcSBP* has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant *TcSBP*, which also presented thermostable behavior. RT-qPCR analysis showed that *TcSBP* was differentially expressed in resistant vs susceptible cacao varieties inoculated by *M. perniciosa* and its expression was probably due to hormone induction via *cis*-regulating elements present in its promotor. The presence of the CSSC conserved domain in *TcSBP* structure suggested that *TcSBP* acted by altering oxidation/reduction of proteins during H₂O₂ production and programmed cell death (PCD) in the final stages of the witches' broom disease. Moreover, *TcSBP* was able to bind *in silico* the selenite and potentially may be able to bind heavy metals. For these reason, the *TcSBP* appeared to be a good candidate for biotechnological applications aiming control of biotic and abiotic stresses in plants. To our knowledge, this is the first *in silico* and *in vitro* analysis of the SBP from cacao.

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49 **Keywords: *Moniliophthora perniciosa***, selenium, molecular docking, gene expression

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## 1. Introduction

Cacao is an endemic plant of tropical forests in South America and is grown mainly for the production of cocoa liquor, butter and powder for the chocolate industry [1]. However, cocoa production has been severely damaged by diseases caused by fungi and oomycetes [2]. In Brazil, the witches' broom disease caused by the basidiomycete *M. Moniliophthora perniciosa* caused drastic economic and social changes in the affected areas [2]. *Moniliophthora perniciosa* has two distinct phases of development: a biotrophic phase characterized by a monokaryotic and intercellular mycelium, and a necrotrophic phase characterized by a dikaryotic and intracellular mycelium [3, 4]. The transition from biotrophic to necrotrophic phase is characterized by significant accumulation of H₂O₂ and by programmed cell death (PCD). In cacao plants susceptible to *M. perniciosa*, the PCD occurs initially as a defense mechanism and then is deflected by the fungus for its own profit, allowing sporulation and further propagation [3]. Thus, the study of compounds, genes and proteins that induce PCD and/or facilitate facilitating the transition from biotrophic to necrotrophic phase [3, 4] is important to better understand the cacao-*M. perniciosa* pathosystem and to develop strategies for witches' broom disease control.

The selenium-binding protein (SBP) is known as a tumor suppressor involved in the regulation of cell proliferation, senescence, migration and apoptosis [5] and has been studied as a target for the treatment of various types of cancer in animals and human, generally in related studies [6]. In human, it has been reported that the expression of the SBP gene was reduced in tumor tissues when compared to healthy ones, and for this reason, this gene has been indicated as a good predictor of clinical outcome of cancer [5]. SBP was also was related to resistance to abiotic and biotic stresses in plants [7, 8].

**In several plant species, studies showed the correlation between SBP level and tolerance**
76 to metal toxicity – e.g. by Se/selenite ($SeO_3^{2-}$), cadmium and sulfur – suggesting that
77 SBP participated to metal detoxification mechanism. In several plant species, studies
78 showed the correlation between SBP level and tolerance to metal toxicity (e.g. by
79 Se/selenite $SeO_3^{2-}$, cadmium and sulfur) suggesting that SBP participated to metal
detoxification mechanism [8]. In Arabidopsis, SBP1 was considered a general stress-
80 responsive gene [9]. In transgenic rice plants (*Oryza sativa*), the overexpression of
81 *OsSBP* increased the plant resistance to the rice blast disease caused by the fungus
82 *Pyricularia grisea*, and to the bacterial blight due to *Xanthomonas oryzae* pv. oryzae [7,
83 10]. The SBP function was related to its ability to bind **covalently** Se **covalently** [5] and
84 to the regulation of Se and $SeO_3^{2-}$ metabolism [5].

86 The selenium-binding protein gene from *Theobroma cacao* (*TcSBP*) was first
87 identified from a cocoa-*Moniliophthora perniciosa* cDNA library [11]. Here, we
88 identified and analyzed *in silico* and *in vitro* the unique SBP sequence found in the *T.
cacao* genome. The present study provides details on the *TcSBP* gene, including
89 promotor analysis, as well as on the corresponding protein. The protein modeling and
90 docking analyses revealed that TcSBP interacts with selenite at its CSSC conserved
91 domain. Moreover, we showed that TcSBP presented differential expression pattern
92 between resistant and susceptible cacao plants infected by *M. perniciosa*. The
93 expression results associated to the structural analysis of the gene and protein, allowed a
94 better understanding of the SBP role in cacao plants submitted to biotic and abiotic
95 stresses. To the best of our knowledge, this is the first study of SBP in plants infected by
96 hemibiotrophic fungi. The aim of this study was to understand the role of TcSBP during
97 the cacao-*M. perniciosa* interaction as well as to identify possible action mechanisms of
98 this protein associated to its ligation to selenite.
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101 **2. Methods**

102 **2.1 In silico analysis of TcSBP**

103 The *TcSBP* cDNA was identified from a library of *T. cacao* meristem (genotype
104 TSH1188) infected by *M. perniciosa* [11]. The complete sequence of the entire gene
105 and of the corresponding proteins was obtained from the CocoaGenDB database
106 (http://cocoa-genome-hub.southgreen.fr/). Open reading frame (ORF) detection was
107 performed using the ORFinder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).
108 Sequence homology search was made with BLAST [12] on the National Center for
109 Biotechnology Information (NCBI). Multiple sequence alignment was performed with
110 the ClustalW2 software [13]. The prediction of theoretical isoelectric point (pI) and
111 molecular weight (MW) was obtained using the Expasy Molecular Biology Server
112 (www.expasy.org)ProtParam tool (https://web.expasy.org/protparam/ [14]). Post-
113 translational events were predicted using the NetPhos 2.0 Server to identify putative
114 sites of phosphorylation (Ser/Thr/Tyr) [15] and NetNGlyc 1.0 Server
115 (http://www.cbs.dtu.dk/services/NetNGlyc/) to identify putative sites of N-
116 glycosylation. Predictions of subcellular localization were conducted with the programs
117 TargetP 1.1 server [16] and PSORT [17]. Signal peptide presence was analyzed using
118 the SignalP 4.0 Server [18]. The TcSBP was used, together with the sequences of
119 the homolog proteins from other plants and mammals, to construct an unrooted
120 phylogenetic tree by the neighbor joining method with the ClustalW2 [13] and MEGA 6
121 [19] programs. For the dendrogram analysis, only complete sequences with high identity
122 (>70%) from plants and mammals were considered. The tree was constructed using the
123 neighbor-joining method of ClustalW, with 1000 bootstraps. The search for regulatory
124 sequences of *TcSBP* was performed in the CocoaGenDB database. A region of 1500 bp
125 upstream the UTR was identified and selected for cis-element analysis. The presence of
126 the cis-regulatory elements in the promoter region of *TcSBP* was analyzed using the
127 plantCARE (sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html) software [20].

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129 **2.2 Molecular modeling of TcSBP and docking analysis**

130 The prediction of the three-dimensional (3-D) model of the TcSBP protein was
131 obtained using the Swiss-Model server and the Automated Protein Homology-
132 modeling, which relies on the high similarity of target-template [21]. The crystal
133 structure of the *Sulfolobus tokodaii* SBP was used as template (Protein Data Bank code:
134 2ECE) to build the structural model of TcSBP. The stereochemical quality of the
135 models was evaluated using the Procheck [22] and Anolea programs [23]. The 3-D
136 model visualization was obtained using the PyMol (The PyMOL Molecular Graphics
137 System, Version 1.5.0.4 Schrödinger, LLC.) and Discovery Studio 4.0. For docking
138 analysis of TcSBP 3-D model (receptor) with inorganic selenium compounds (ligands),
139 selenite and sodium selenite were search by keyword in the PubChem databases
140 (http://pubchem.ncbi.nlm.nih.gov). The 2D structure of these compounds were copied
141 in Similes and saved in PDB format using the Marvin program, and then used for
142 molecular docking. The ligand and receptor molecules were prepared using the
143 AutoDockTools 1.5.6 [24]. The grid definition, adjusted to active site, was set up
144 manually according to the recommendations of the program manual [24, 25]. The ligand
145 and receptor structures were then saved in pqbqt format to be used for docking
146 calculations. AutoDock Vina was used to perform Docking Scoring for each ligand-
147 receptor complex [25]. Before running each Docking calculation, a configuration file
148 was generated with information about grid size and coordinates, and indicating the
149 ligand and receptor files. The reports for each calculation were analyzed to obtain
150 affinity energy (kCal/Mol) values for each ligand conformation in its respective
complex. PyMol program was used to verify the ideal complex by considering all stereochemical aspects previously evaluated and the free-energy results. Then the best ligand i.e., that fit best in the active site, was selected.

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## 2.3 Plant material

Plant material was obtained as previously described [26]. Seeds of *T. cacao* genotypes Catongo (susceptible to *M. perniciosa*) and TSH1188 (resistant to *M. perniciosa*) were germinated and grown at CEPLAC/CEPEC (Ilhéus, Bahia, Brazil) greenhouses. Twenty to thirty days after germination, the apical meristems of the plantlets were inoculated by the droplet method with a basidiospore suspension of *M. perniciosa* (inoculum from isolate 4145 maintained in the CEPLAC/CEPEC phytopathological *M. perniciosa* collection under number 921 of the WFCC; http://www.wfcc.info/index.php/collections/display). After inoculation, the plantlets were kept for 24 h at 25 ± 2°C and 100% humidity. Rate of disease fixation based on presence/absence of symptoms in each genotype, was evaluated 60 days after inoculation (dai); disease rate was 45% and 80% for TSH1188 and Catongo, respectively. Moreover, the presence of *M. perniciosa* in the plant material was checked by semi-quantitative RT-PCR using specific *M. perniciosa* actin primers [26]; both genotypes presented fungus incidence (data not shown) coherent with previous data obtained in the same conditions of plant culture and inoculation [4, 26]. Apical meristems were harvested at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 dai. Non-inoculated plants (controls) were kept and harvested under the same conditions at 72 hai, 60 dai and 90 dai. For each genotype and at each harvesting time (for inoculated and non-inoculated plants), 20 samples were collected (1 sample = 1 apical meristem of 1 cacao plantlet). The 20 samples collected from one genotype at
one harvesting time were pooled; thus 9 inoculated and 5 non inoculated (control)
samples were immediately frozen in liquid nitrogen and stored at -80°C until use.

Pooling samples before RNA extraction has the advantage of reducing the variation
caused by biological replication and sample handling [26].

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2.4 Reverse transcription quantitative PCR analysis

Total RNA was extracted from macerated samples using the RNAqueous Kit®
(Ambion) according to the manufacturer's instructions, with modifications as
previously described [26]. The synthesis of cDNA was carried out using Revertaid First
Fisrt Strand cDNA Synthesis Kit according to the manufacturer's instructions (Thermo
Scientific). The cDNA quantification was carried out on the GeneQuant pro UV/Vis
spectrophotometer (Amersham). For the qPCR analysis, two cacao endogenous
reference genes were used: the malate dehydrogenase (MDH) and β-actin (ACT),
previously identified as *T. cacao* housekeeping genes and tested in our experimental
conditions (same plant material, same equipment [26]; Supplementary material 1).

Specific *TcSBP* primers were also defined (Supplementary material 1). The expression
analysis of *TcSBP* was performed using standard settings of the ABI PRISM 7500 and
Sequence Detection System (SDS) software, version 1.6.3 (Applied Biosystems). The
qPCR reaction consisted of 10 ng/µl of cDNA, 1 µM of each primer from reference or
targets genes (Supplementary material 1) and 11 µl of Power SYBR Green Master Mix
(Applied Biosystems) in a total volume of 22 µl. Cycling conditions were: 50°C for 2
min then 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30s and
60°C for 1 min. To verify that each primer pair produced only a single PCR product, a
dissociation analysis was carried out from 60°C to 95°C and analyzed with the
Dissociation Curve 1.0 program (Applied Biosystems). The expression analysis of
201 *TcSBP* in meristems of cacao plantlets inoculated or not with *M. perniciosa*, was
202 obtained by RT-qPCR using 6 *experimental technical* replicates. The relative expression
203 was analyzed with the comparative Ct method (2<sup>-ΔΔCt</sup>) using MDH and ACT as
204 endogenous reference genes and non-inoculated plants (control) as calibrator. Statistical
205 analysis was made using the SASM-Agri software which tested the experiments as a
206 completely randomized design. *t*-test and *F*-test (ANOVA) were applied with a critical
207 value of 0.01. The Scott-Knott (P≤0.01) test was employed for mean separation when *F*-
208 values were significant.

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210 **2.5 Obtaining of the recombinant TcSBP**

211 Total RNA was extracted as described above (§ 2.4) from a pool of all samples of
212 TSH1188 *T. cacao* seedlings inoculated with the fungus *M. perniciosa* (see §2.3 and
213 *TcSBP* expression data). The synthesis of cDNA was carried out as described above (§
214 2.4). To develop a *TcSBP* cloning strategy in pET-28a(+) vector (used for heterologous
215 expression), specific primers (F 5'-AAGACAGGGCATATGGCCGGTAACG-3' and R
216 5'-CGGTAAAGAGTGAAGCTTCACAGCAAG-3' containing NdeI and HindIII
217 restriction sites) were designed using the Custom Primers-OligoPerfect™ Designer
218 (Invitrogen), OligoAnalyser 3.1 (IDT SciTools/Integrated DNA Technologies, Inc.,
219 California), and Webcutter (http://heimanlab.com/cut2.html) programs. The *TcSBP*
220 amplification reaction was performed in a total volume of 25 µl containing 1 µl of the
221 cDNA sample, 2.5 µl of 10X PCR buffer, 0.75 µl of MgCl₂ (1.5 mM), 1 µM of each
222 primer (0.2 mol/µl), 1U Recombinant Taq DNA Polymerase (Invitrogen) and ultrapure
223 water (qsp 25 µl). The reaction was conducted in a Veriti 96-well Thermal Cycler
224 (Applied Biosystems) and consisted of the following steps: 4 min at 94°C; 30 seconds at
225 94°C; 1 min at 58°C; 4 min at 72°C; 7 min at 72°C; final wait of 15°C. The PCR
product was analyzed on a 1% agarose gel stained with GelRed (Biotium). Purification of the PCR product was performed using the GFX™ PCR DNA and Gel Band Purification Kit according to the manufacturer's recommendations (GE Healthcare). The purified PCR product, as well as the pET-28a(+) vector, were digested at 37°C for 4 h by NdeI and HindIII in a final volume reaction of 120 µL containing 96 µl of DNA, 12 µl of the Tango buffer, 4.5 µL of each enzyme and 3 µl of ultrapure water. After purification using the™ PCR DNA and Gel Band Purification Kit (GE Healthcare), the reactions were treated with the FastAP enzyme according to the manufacturer's recommendations (Thermo Fisher Scientific). Cloning step was performed using 90 µl of insert, 10 µl of pET-28a(+) vector, 10 µl of T4-DNA ligase, 15 µl of 10X buffer and 25 µl of ultrapure water. Cloning reaction was used to transform *Escherichia coli* C43 strain by heat shock method [27] and the transformed colonies were selected on LB medium containing kanamycin (50 µg/ml). Transformed colonies were confirmed by PCR using the same primers and conditions as described above. For protein induction, PCR-confirmed colony was grown on LB medium at 37°C under agitation at 180 rpm until OD<sub>600nm</sub> was reached 0.6 to 1.0, then incubated with 0.4 µmol/l of IPTG at 18°C for 16 h under agitation, and finally centrifuged at 180 rpm. The pellet was resuspended in 50 µg/ml of lysozyme, kept on ice for 20 min, and then sonicated in an ice bath with 10 pulses of 10s (on), 25 pulses of 10 seconds (off) and 70% amplitude, until breaks the pellet viscosity. The lysate material was centrifuged at 5000 rpm, 4°C for 45 min and the supernatant was used for recombinant protein purification on TALON Superflow cobalt ion (Co<sup>2+</sup>) (GE Healthcare). The recombinant protein was eluted with 150 mM of imidazole and submitted to dialysis in phosphate buffer (5mM). rTcSBP purification was checked on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [28].
Electrophoresis ran for 2 h at 150 V with the cube immersed in ice, and the proteins were detected on electrophoresis gel by Coomassie Blue G250 staining [29]. The concentration of the rTcSBP was determined by the Bradford method [30] on the microplate reader ELX 800 (Biotek) using the bovine serum albumin (BSA) for the standard curve calculation.

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## 257 Circular dichroism

The circular dichroism (CD) was carried out on a spectropolarimeter (model J-815, Jasco, MD) equipped with a Peltier temperature controller and a cell support with a thermostat connected to a thermostatic bath. The CD of rTcSBP was performed at a concentration of 0.355 µg µL⁻¹ of the protein in sodium phosphate buffer (10 mmol L⁻¹, pH 7.0). Three CD spectra (experimental replicates) of rTcSBP were measured in wavelength range of 190-240 nm at 25°C and 95°C. The thermal stability analysis of rTcSBP was also evaluated using unfolding (refraction denaturation from 25 to 95°C) and refolding (variation from 95 to 25°C) at 205 nm in sodium phosphate buffer (10 mmol L⁻¹, pH 7.0). The rTcSBP structure was also analyzed in presence of sodium selenite (1:1 molar ratio) at 190-240 nm and 25°C. The secondary α-helix and β-sheet structures were estimated using the K2D2 server (http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d2/).

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## 271 3. Results

### 272 3.1 *In silico* analysis of *TcSBP*

*In silico* analysis on CocoaGenDB revealed the presence of a unique sequence encoding TcSBP located on the chromosome 4 (Fig.1). The *TcSBP* gene is 4774 bp in length, contains 7 exons and 6 introns and has an ORF of 1431 bp (Fig.1). *TcSBP*

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encodes a protein of 476 amino acids, with a MW of 52732.7 Da and a putative pI of
5.74. Twenty-one putative phosphorylation and no N-glycosylation sites were observed
in the TcSBP protein sequence (Fig. 2A). TcSBP does not contain any signal peptide,
and its subcellular location is unknown (data not shown). The alignment of TcSBP with
SBPs from other organisms revealed high identity (>70%) with plant species and human
and allowed the identification of the CSSC, DELHH and HGD conserved region
(Fig.2A). The full-length amino acid sequences of plants and mammalian SBPs
available in the databases were used to build an unrooted dendrogram (Fig. 3;
Supplementary material 2). Plant and mammalian SBPs were separated and formed two
distinct branches in the dendrogram (Fig. 23). In silico analysis of the TcSBP promoter
region revealed the presence of several conserved cis-elements characteristic of
different plant species (Table 1). Among these cis-elements, TATA-box and GA-motif
related with transcription start, and with cis-acting in promoter and enhancer regions,
respectively, were found (Table 1). Elements related to light response and circadian
control were encountered (CAAT-box and Circadian, respectively) as well as elements
related to response to abiotic stress or external stimuli (anaerobic induction/ARE, low-
temperature response/LTR) were found. Finally, several cis-elements related to
hormone signaling were found such as ABRE (abscisic acid responsiveness), TATC-
box (gibberellin responsiveness), TCA (salicylic acid responsiveness) and TGA-box
(auxin-responsive element) (Table 1).

## 3.2 Homology modeling of TcSBP and molecular docking of TcSBP with selenite

The modeling of the TcSBP was based in the X-ray structure of the SBP from
*Sulfolobus tokodaii* (StSBP). The alignment of the amino acid sequence of TcSBP with
the StSBP (Fig. 32A) presented 38% of identity, which is superior to the reliable cut-off
of 25% [31]. And the structure identity between StSBP and TcSBP was 43.91% (X-ray, 301 2.0Å; data not shown). The structural modeling of TcSBP revealed the presence of 7 α- 302 helices and numerous β-strands and P-loops (Fig. 32B). The Ramachandran plots 303 showed that 99.9% of the amino acids of the TcSBP model were located in the most 304 favored regions (Supplementary material 3). The TcSBP showed important amino acid 305 residues organized on domains (DELHH, HGD, CSSC/CSSA) based on its high identity 306 (>70%) with SBPs from other species (Fig. 32A). The tridimensional structure of 307 TcSBP obtained by homology modeling showed that the protein contains 8 α-helices (in 308 red, Fig. 32B) and 30 β-strands (in blue, Fig. 32B). The CSSC conserved motif of 309 TcSBP was showed in the surface format structure (in pink, Fig. 32C) and as stick 310 representation (in pink, Fig. 32D). Putative phosphorylation sites were present at the 311 superficies of the protein structure; interestingly, phosphorylation sites were present in 312 the CSSC domain of the protein (Fig. 2C). The interaction between the conserved motif 313 CSSC of TcSBP and the selenite occurred with a setting of -1.9 kCal/mol (Fig. 2E). No 314 interaction between TcSBP and selenate was observed (data not shown). 315

316

317 **3.3 Production of rTcSBP and circular dichroism**

The rTcSBP was expressed in *E. coli* C43, purified on affinity column, and 318 visualized on SDS-PAGE gel (see Material and Methods). The purified rTcSBP showed 319 molecular weight (56 kDa) coherent with the predicted one (Supplementary material 4). 320 Circular dichroism spectra of rTcSBP in phosphate buffer pH 7.0 showed that the 321 protein contained both α-helix and β-strand elements (Fig. 4A). The rTcSBP structure 322 spectra showed a lower peak at ~205 nm, mainly at 95°C (Fig. 4A). The rTcSBP 323 secondary structure variation according to the temperature (unfolding and refolding) at 324 205 nm showed that the rTcSBP was duly folded at 25°C and denatured from 25°C to 325

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326 95°C (unfolding, Fig. 4B), while the refolding occurred from 95°C to 35°C with a fully
327 recovery of the secondary structure at 35°C (0% of denaturation; Fig. 4B). Circular
328 dichroism of rTcSBP in presence and absence of sodium selenite showed that the
329 sTcSBP interacts with selenite but that this interaction did not influence the protein
330 structure (Fig. 4C).

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332 **3.34 Differential expression of TcSBP in resistant and susceptible cacao genotypes**
333 **infected by M. perniciosa**

334 The expression of the *TcSBP* gene was analyzed for each cacao genotype, i.e.
335 TSH1188 (resistant to witches' broom disease) and Catongo (susceptible), infected or
336 not (control) with *M. perniciosa* (Fig. 5). For both genotypes and for all the harvesting
337 point, the PCR amplification occurred at the same and unique melting temperature for
338 each gene showing that only the corresponding gene was amplified (Supplementary
339 material 5). At 24 hai, *TcSBP* was significantly more expressed in inoculated
340 susceptible variety than in the control, while its expression did not differ between
341 inoculated resistant variety and the control plants. At 48 and 72 hai no significant
342 difference of *TcSBP* expression between the resistant and susceptible varieties was
343 observed, however, at 72 hai, *TcSBP* had a lower expression in the inoculated plants
344 (whatever the genotype) than in the control plants. At 8 dai, the resistant variety showed
345 the highest expression of *TcSBP* in relation to the other times after inoculation. At this
346 time the susceptible variety exhibits a relative *TcSBP* expression lower than in the
347 control plants. At 15 dai, the relative *TcSBP* expression in the resistant variety did not
348 differ from the control, while in the susceptible variety the expression was lower than in
349 the control plants. At 30 and 45 dai, the *TcSBP* expression was also significantly
350 different between the varieties; *TcSBP* presented significantly higher expression in the
susceptible variety. At 30 and 45 dai, in the resistant variety, *TcSBP* expression did not differ between inoculated and the control plants. At 60 and 90 dai, no significant difference in *TcSBP* expression was observed in the studied varieties.

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## 4. Discussion

Here, we characterized *in silico* a SBP gene from *T. cacao* (Fig. 1 and 2). This gene is present in the cacao genome in a unique copy located in the chromosome 4 (Fig. 1). The corresponding protein (476 amino acids; Fig. 2A) presented a secondary structure containing α-helices and β-strands (Fig. 2B and Fig4A) and is thermostable *in vitro* (Fig. 2A and B). Interestingly, *TcSBP* showed a high level of identity with the SBP from *Sulfolobus tokodaii* (Fig. 2A), which is a thermophilic archaea able to survive in high temperature environments (e.g. 70°C) [32]. The promotor region of *TcSBP* contained several *cis*-elements related to biotic stress and to hormone signaling such as abscisic acid, gibberellin, salicylic acid and auxin (Table 1). Some of these hormones are known to be highly involved in witches' broom disease development [11, 33-35]. Several genes related to plant hormone signaling were altered in cacao plants in response to *M. perniciosa* infection, such as gene involved in gibberellin response as well as auxin-responsive genes, both up-regulated in green broom [35]. It has been suggested that the up-regulation of auxin-responsive genes in infected cacao plants is a consequence of the biosynthesis of this hormone by *M. perniciosa* [33-35]. In various plant pathogens, the production of auxin has been correlated to the reduction of the plant defenses mediated by salicylic acid, and consequently to the pathogenicity and to the biotrophic stage development [33, 35]. Here, we observed that the *TcSBP* gene was significantly more expressed in the susceptible variety during both the asymptomatic and the green broom stages (Fig. 4). Thus, the *TcSBP* expression may be related to its
376 induction by hormones present at these stages as the consequence of the infection by the
377 fungus (Fig. 4), as previously observed for SBP genes from other species [10]. In rice,
378 the SBP expression increased rapidly in response to jasmonic acid and salicylic acid and
379 also responded to abscisic acid and paraquat, both responsible for ROS reactive oxygen
380 species generation [10].

381 The witches' broom disease development has been previously associated to
382 increased H₂O₂ levels in both resistant and susceptible cacao varieties, however with
383 distinct temporal and functional patterns [3, 36]. Once produced at the beginning of the
384 infection by the resistant variety, H₂O₂ contributes to the infection control and to plant
385 resistance. In advanced stages of the disease in the susceptible variety, it promotes the
386 pathogen development and the finalization of its life cycle [36]. Interestingly, the *TcSBP*
387 expression also presented distinct temporal patterns; it was higher at 8 and 15 dai in the
388 resistant variety, and from 30 dai until the end of the disease in the susceptible variety
389 (Fig. 4). Considering that *TcSBP* contains a CSSC conserved domain (*in silico* analysis,
390 Fig. 2E), which is known to act as an active redox center controlling
391 oxidation/reduction of protein *in vivo* [10], it could be suggested that *TcSBP* gene may
392 play a role in protecting cells from oxidative stress in swelling ramification phase in the
393 resistant genotype, and regulating the PCD in the susceptible one during the green
394 broom stage (Fig. 4; [3, 36]). This results could be related to the known SBP function,
395 i.e. its capacity to inhibit the cell proliferation, and consequently its involvement in
396 apoptosis mechanism [5]. In rice, *OsSBP* was involved in the accumulation of reactive
397 oxygen intermediates produced by NADPH oxidase, and the observed H₂O₂
398 accumulation was due to the reduction of scavenging enzyme activity [7].

399 Finally, the *TcSBP* protein contains multiple predicted phosphorylation sites
400 (Fig. 2A); protein phosphorylation is a ubiquitous mechanism for the temporal and
spatial regulation of proteins involved in almost every cellular process [37]. *TcSBP* contains three domains, DELHH, HGD and CSSC, highly conserved among the SBPs from other organisms (Fig. 2A); the CSSC domain contained predicted phosphorylation sites (Fig. 2C). It has been suggested that the characteristic CSSC motif has a strong affinity for selenium and heavy metals [38]. In *T. cacao*, we observed that the conserved domain CSSC was able to bind, *in silico*, the selenite (Fig. 2E). The binding of *rTcSBP* with the selenite was confirmed *in vitro* by circular dichroism, showing that this interaction did not modify the protein structure (Fig. 4C). Some phosphorylation sites (the stable ones) are known to be important for the regulatory function of the protein [39] and, here, it may be suggested that the phosphorylation at the CSSC domain may be related to the binding with selenite, as well as to the protein function as regulator of oxidation/reduction during the plant-pathogen interaction. Moreover, the conserved domain CSSC potentially may be a binding site for other metals as observed in other organisms [38]. This could be interesting considering the concerns about high concentrations of heavy metals (e.g. Pb, Cd, Cu) found in beans of cacao as well as in its products (including chocolate). Contamination of cacao is attributed to the increasing use of phosphate fertilizers but also to several kinds of industrial processes close to the cacao production areas [40].

## 5. Conclusion

Here we showed the first *in silico* and *in vitro* analysis of the SBP from cacao. The *rTcSBP* is a thermostable protein able to bind *in silico* and *in vitro* the selenite and potentially may be able to bind heavy metals. *TcSBP* is differentially expressed in resistant vs susceptible cacao varieties inoculated by *M. perniciosa* and its expression is probably due to hormone induction (e.g. IAAindole acetic acid, ASsalicylic acid,
426 gibberellins) via cis-regulating elements present in its promotor (e.g. TATC-box, TCA,
427 TGA-box). The presence of the CSSC conserved domain in TcSBP structure suggested
428 that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production
429 and PCD in the final stages of the witches' broom disease. Moreover, TcSBP was able
430 to bind in silico the selenite and potentially may be able to bind heavy metals. For these
431 reasons, the TcSBP appeared to be a good candidate for biotechnological applications
432 aiming control of biotic (e.g. pathogens) and abiotic (e.g. heavy metals) stresses.
433

433

434 **Availability of supporting data**

435 The data sets supporting the results of this article are included within the article and its
436 additional files.

437

438 **Abbreviations**

439 ACT: actin; dai: days after inoculation; hai: hour after inoculation; pI: isoeletric point;
440 MDH: malate dehydrogenase; MW: molecular weight; ORF: open reading frame; PCD:
441 programmed cell death; SBP: selenium-binding protein; Se: selenium; UTR:
442 untranslated regions.

443

444 **Competing interest**

445 No conflicts of interest to declare.

446

447 **Authors' contribution**

448 AMMA was responsible for the execution **of all the** experimental steps; AMMA, SPM,
449 BSA and FM analyzed and discussed the data; AMMA and FM wrote the manuscript;
450 SPM and EML gave support in qPCR experiment; BSA gave support in modeling and
451 docking analyses; KPG was responsible for plant material production and inoculation
452 with *M. perniciosa*; **MMF and CPP supported the circular dichroism analysis**; FM and
453 BSA were responsible for the conception and design of the experiments; FM was
454 responsible for the financial support of the research and for the advising of AMMA,
455 EML and SPM.

456

457 Acknowledgements

458 The work of AMMA was supported by the Fundação de Amparo à Pesquisa do Estado
459 da Bahia (FAPESB). The work of SPM, EML **and MMF** was supported by the
460 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). FM, KPG
461 **and CPP** were supported by a Productivity grant from Conselho Nacional de
462 Desenvolvimento Científico e Tecnológico (CNPq). This research was supported by the
463 FAPESB project (DTE0038/2013) coordinated by FM. The authors thank Francisca
464 Feitosa Jucá (UESC/Ceplac) and Louise Araújo Sousa (Ceplac) for technical help in
465 plant inoculation experiments, Dr. Gesilvado Santos (UESB) for advices and discussion
466 about SBP role. This research was made in the frame of the International Consortium in
467 Advanced Biology (CIBA).

468

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<table><caption>579 Table 1. Cis-elements present in the promoter region of TcSBP gene.</caption><thead><tr><th>cis-elements name</th><th>Organism</th><th>Sequence</th><th>Function</th></tr></thead><tbody><tr><td rowspan="3">ABRE</td><td>Arabidopsis thaliana</td><td>ACGTG</td><td rowspan="3">Abscisic acid responsiveness</td></tr><tr><td>Hordeum vulgare</td><td>GCAACGTGTC</td></tr><tr><td>Arabidopsis thaliana</td><td>CACGTG</td></tr><tr><td>ARE</td><td>Zea mays</td><td>AAACCA</td><td>Anaerobic induction</td></tr><tr><td>TATC-box</td><td>Oryza sativa</td><td>TATCCCA</td><td>Gibberellin-responsiveness</td></tr><tr><td>TCA-element</td><td>Nicotiana tabacum</td><td>CCATCTTTTT</td><td>Salicylic acid responsiveness</td></tr><tr><td>TGA-box</td><td>Glycine max</td><td>TGACGTAA</td><td>Auxin-responsive element</td></tr><tr><td>Circadian</td><td>Lycopersicon esculentum</td><td>CAAAGATATC</td><td>circadian control</td></tr><tr><td rowspan="3">CAAT-box</td><td>Brassica rapa</td><td>CAAAT</td><td rowspan="3">Light responsive</td></tr><tr><td>Hordeum vulgare</td><td>CAAT</td></tr><tr><td>Glycine max</td><td>CAATT</td></tr><tr><td>GA-motif</td><td>Glycine max</td><td>AAGGAAGA</td><td>Cis-acting in promoter and enhancer regions</td></tr><tr><td>LTR</td><td>Hordeum vulgare</td><td>CCGAAA</td><td>Low-temperature responsiveness</td></tr><tr><td rowspan="6">TATA-box</td><td>Arabidopsis thaliana</td><td>TATAAA</td><td rowspan="6">Transcription start</td></tr><tr><td>Arabidopsis thaliana</td><td>TATAAA</td></tr><tr><td>Oryza sativa</td><td>TATAAGAA</td></tr><tr><td>Lycopersicon esculentum</td><td>TTTTA</td></tr><tr><td>Lycopersicon esculentum</td><td>TTTTA</td></tr><tr><td>Arabidopsis thaliana</td><td>TATAA</td></tr></tbody></table>

580

581 Figure legends

582 **Figure 1.** Structural features of TcSBP sequence. Lengths of the regions are indicated at
583 the scale.

584 **Figure 2.** Homology modeling of TcSBP and molecular docking. **A.** Alignment of the
585 amino acid sequence of TcSBP (XP_007034202.1) with SBPs from *Homo sapiens* (HsSBP1, AAB02395.1, UniProtKB:Q13228) and *Sulfolobus tokodaii* (StSBP,
586 WP_010977998.1, UniProtKB:Q976Y0). Gaps introduced to get the best alignment are
587 indicated by (-), (*) represents identical amino acids between all sequences, (.) and (:)
588 represent conserved substitutions and semi-conserved substitutions, respectively. The
589
conserved motifs DELHH, CSSC and HGD are highlighted in blue, yellow and black, respectively. Phosphorylation sites are indicated by green squares. B. Tridimensional structure of TcSBP obtained by homology modeling with StSBP from *Sulfolobus tokodaii* (PDB code 2ECE.pdb). C. Surface representation of TcSBP with the indication of the conserved motifs CSSC in pink and of putative phosphorylation sites in green. D. Stereo view using sticks representation with focus on the conserved CSSS motif (in pink). E. Docking between TcSBP and selenite at the TcSBP CSSC site.

**Figure 3.** Dendrogram of TcSBP and SBP from other species. The green and red boxes indicate plant and mammalian SBPs, respectively. The amino acids sequences were derived from the GenBank under the following accession numbers: VvSBP, *Vitis vinifera* (XP_002267004.1; GI:225433514); CsSBP, *Citrus sinensis* (XP_006492530.1; GI:568879129); StSBP, *Solanum tuberosum* (XP_006360921.1; GI:565390388); PtSBP, *Populus trichocarpa* (XP_006373096.1; GI:566212227); RcSBP, *Ricinus communis* (XP_002520613.1; GI:255559184); TcSBP, *Theobroma cacao* (XP_007034202.1; GI:1063488813); CsSBP, *Cucumis sativus* (XP_004138463.1; GI:449441386); MsSBP, *Medicago sativa* (AJ401228.1; CAC67501.1); GmSBP, *Glycine max* (KHN24031.1; GI:734383687); PvSBP, *Phaseolus vulgaris* (XP_007163807.1; GI:593801540); ShSBP, *Sarcophilus harrisii* (XP_003773388.1; XP_003769976.1; GI: 395535935); MmSBP, *Mus musculus* (AAA40104.1; GI:200952); RnSBP, *Rattus norvegicus* (NP_543168.1; NP_001316822.1; GI: 1049480106); HgSBP, *Heterocephalus glaber* (XP_004875930.1; XP_004854049.1; GI: 512984154); LaSBP, *Loxodonta africana* (XP_010587740.1; GI:731468243); BmSBP, *Bos mutus* (XP_005895013.1; GI:555965912); TtSBP, *Tursiops truncatus* (XP_004328520.1; XP_019775099.1; GI:1131274601); PtSBP, *Pan troglodytes* (XP_001172033.2; GI:332810264); HsSBP,
615 *Homo sapiens* (AAB02395.1; GI:1374792); GggSBP, *Gorilla gorilla*
616 *gorilla* (XP_004026682.1; GI: 426331426).

617 **Figure 4.** Circular dichroism analysis of rTcSBP. A. Spectra of the rTcSBP secondary structure at 190-240 nm. Black and dashed lines corresponded to the rTcSBP structure at 25°C and 95°C, respectively. B. Unfolding (black line) and refolding (dashed line) of rTcSBP in phosphate buffer (pH 7.0) at 205 nm. C. Secondary structure of rTcSBP in presence of sodium selenite at 25°C. The grey line corresponds to sodium phosphate buffer scan, yellow line to sodium selenite scan, orange line to rTcSBP scan, and blue line to rTcSBP with sodium selenite scan (1:1 ratio).

624 **Figure 45.** Relative expression of *TcSBP* in cacao meristems inoculated or not (control) with *M. perniciosa*. A. Representation of the plant symptoms and fungus phase during the infection time course in Catongo genotype. B. Relative expression observed in resistant (TSH1188) and susceptible (Catongo) varieties. Different letters indicate significant statistical difference between samples by the Scott-Knott test (p≤0.01). *t*-test were applied with a critical value of 0.01: upper case letters correspond to statistics between each of the genes on different harvesting times for each genotype while lower case letters correspond to statistics between the three genes for each harvesting time. hai: hours after inoculation; dai: days after inoculation. (*) indicates the times that were harvested also in the non-inoculated (control) plants.
<figure><img src="image_3.png" /></figure>

DNA

Chromosome 4

56

1063

196

859

228

153

232

150

146

273

514

282

402

4774 bp

Untranslated region

Exons

Introns

Open Reading Frame
<figure><img src="image_4.png" /><figcaption>A</figcaption></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>

E

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

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