# Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa

Raner José Santana Silva, Rafael Moyses Alves, Karina Peres Gramacho, Lucilia Helena Marcellino, Fabienne Micheli

► To cite this version:

Raner José Santana Silva, Rafael Moyses Alves, Karina Peres Gramacho, Lucilia Helena Marcellino, Fabienne Micheli. Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa. Plant Physiology and Biochemistry, 2020, 148, pp.142 - 151. 10.1016/j.plaphy.2020.01.009. hal-03489901

HAL Id: hal-03489901

https://hal.science/hal-03489901

Submitted on 21 Jul 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance

2

to the fungus *Moniliophthora perniciosa*

3

4

Raner José Santana Silva¹, Rafael Moyses Alves², Karina Peres Gramacho³, Lucilia Helena

5

Marcellino⁴, Fabienne Micheli¹,⁵,*

6

7

¹Universidade Estadual de Santa Cruz (UESC), Departamento de Ciências Biológicas (DCB),

8

Centro de Biotecnologia e Genética (CBG), Rodovia Ilhéus-Itabuna, km 16, 45662-900

9

Ilhéus-BA, Brazil.

10

²Empresa Brasileira de Pesquisa Agropecuária, Embrapa Amazônia Oriental, 66095-903

11

Belém-PA, Brazil.

12

³Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil.

13

⁴Empresa Brasileira de Pesquisa Agropecuária, Embrapa Recursos Genéticos e Biotecnologia,

14

Brasília-DF, 70770-917, Brazil.

15

⁵CIRAD, UMR AGAP, F-34398 Montpellier, France.

16

17

*Corresponding author: Dr Fabienne Micheli, UESC, DCB, Rodovia Ilhéus-Itabuna km16,

18

45662-900, Ilhéus-BA, Brazil. Phone: +55 73 3680 5196. Fax: +55 73 3680 5226. E-mail:

19

fabienne.micheli@cirad.fr

20

21

**Abstract:**

22

The cupuassu tree (*Theobroma grandiflorum*) is a crop of great economic importance to

23

Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu

24

fruit production is threatened by witches' broom disease caused by the fungus *Moniliophthora*

25

*perniciosa*. As elements of its defense mechanisms, the plant can produce and accumulate
pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three
PR proteins from cupuassu (TgPR3, TgPR5 and TgPR8) from cupuassu-*M. perniciosa* RNA-
seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are
**phylogenetically related to cacao** and to *Arabidopsis PR sequences involved in biotic and*
**abiotic stress.** The TgPR proteins' tridimensional structure was obtained through homology
modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can
interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene
expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes
infected by *M. perniciosa* at 8, 24, 48 and 72 hours after infection (hai). The TgPR genes
showed higher expression in resistant plants compared to the susceptible ones, mainly for
TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To
our knowledge, this is the first *in silico* and *in vitro* reports of cupuassu PR protein. The data
suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate
immune system through chitin receptors. Our results also suggest a putative role of
chitinase/chitosanase for the TgPR5/osmotin.

41

Keywords: Pathogenesis-related proteins, molecular docking, witches' broom disease, gene expression, chitin, chitosan

44

45

**1. Introduction**

The cupuassu (*Theobroma grandiflorum* [Willd. Ex Spreng.] Schum.) belongs to the
Malvaceae family; it is native to the Amazon region and is economically important to Brazil,
where it is cultivated [1, 2]. Cupuassu pulp and seeds are used in the cosmetics and food
industries, mainly for candy, ice cream, beverage and *cupulate* (a product similar to
chocolate) production [3]. *Cupulate* is an interesting alternative to chocolate production for an

46

47
Malvaceae
48
where it is cultivated [1, 2].
49
Cupuassu pulp and seeds are used in the cosmetics and food

48
where it is cultivated [1, 2]. Cupuassu pulp and seeds are used in the cosmetics and food

49
industries, mainly for candy, ice cream, beverage and cupulate (a product similar to

50
chocolate) production [3]. Cupulate is an interesting alternative to chocolate production for an
actual worldwide economic situation in which the production of cocoa (*Theobroma cacao*,
Malvaceae family) beans cannot supply the increased worldwide demand for chocolate [4, 5].
However, both cocoa and cupuassu cultures are affected by pests and pathogens that cause
damage, loss of production and even plant death. Among them, witches' broom disease,
caused by the fungus *Moniliophthora perniciosa*, is the main phytopathological problem for
cupuassu cultures [6].

Because they are attacked by a wide variety of pathogens, plants have developed
pattern-recognition receptors to recognize molecular patterns associated with pathogens
(PAMPs), which lead, among other outcome, to the production and accumulation of
pathogenesis-related proteins (PR proteins), which are considered crucial to plant defense
mechanisms [7-9]. The current classification divides PR proteins into 17 classes (PR1-PR17)
depending on their biological activity, physicochemical properties and/or sequence homology
[10, 11]. The PR-3, PR-4, PR-8 and PR-11 families correspond to different types of chitinases
(EC 3.2.1.14). Among them, the PR-3 proteins include glycoside hydrolase 19 (GH19)
chitinases, and the PR-8 family corresponds to glycoside hydrolase 18 (GH18) chitinases. The
PR-5 family classification corresponds to thaumatin-like protein (TLPs), whose biological
function in plants has yet to be established [12]. Among TLPs, the osmotins are
multifunctional proteins that may participate alongside chitinases in protein complexes
involved in plant defense against pathogens [8, 13]. GH18, GH19 and osmotin show
antifungal activity through cell wall hydrolysis or plasma membrane permeabilization [12,
14]; in the case of PR-5, it has been proposed that the existence of specific target receptors on
the membrane leads to fungus sensitivity or resistance [15]. The GH18 chitinases show a
characteristic barrel structure [16], while GH19 chitinases are composed of a high
concentration of α-helices and loops [14]. The TLP proteins have three well-characterized
domains: domain I is composed of β sheets and loops called a β-sandwich, domain II is
composed of α-helices and domain III is composed of two β-sheets [17]. Because chitin is present in the fungus cell wall and not in plant cells, it is considered an ideal PAMP and as one of the first barriers in the plant-pathogen battlefield [18, 19]. Chitin and chitosan, its deacetylated derivative, have been heavily studied, and their role in plant-pathogen interaction has received considerable attention, mainly because they are considered useful for biotechnological applications aimed at reducing fungus activity and/or development [19-21]. *Moniliophthora perniciosa*'s chitin metabolism, including associated enzymes such as chitin synthases and chitinases, has previously been studied [22-25].

Molecular studies of cupuassu related to plant-pathogen interaction are still poorly developed, and few works are related to resistance to witches' broom disease [26-28]. Moreover, unlike cocoa, the cupuassu genome has not been sequenced yet, and molecular analyses focused on genetics and genomics of resistance are only based on recent transcriptomic data [26, 28]. Here, we selected and analyzed three PR genes from cupuassu (TgPR3, TgPR5 and TgPR8) with putative chitinase or chitinase-associated functions as well as anti-fungal activity. We showed, in silico, the TgPR proteins' structural aspects and potential activity in relation to chitin and chitosan ligands, and, in vitro, the contrasting expression levels of the *TgPR* genes in resistant and susceptible cupuassu plants inoculated with *M. perniciosa*. Their expression profiles could be related to phylogenetic analysis using homologous PR sequences from *T. cacao* and *A. thaliana*, which are involved in response to biotic and abiotic stress. To our knowledge, this is the first PR protein report on cupuassu-*M. perniciosa* interaction. The overall data suggest that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin.
## 2.1 Identification and characterization of TgPR sequences

The PR sequences were selected from a previously obtained RNA-Seq database of cupuassu [28]. Open reading frame (ORF) detection was performed using the ORFinder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The proteins were analyzed with BLASTp against the Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) for conserved domain identification, protein sequence length checking and TgPR protein classification. Signal peptide was identified using the SignalP 4.1 software [29], while the molecular weight and isoelectric point were obtained using the Compute pI/Mw program [30]. The subcellular locations were predicted using the YLoc-HighRes Plants tool [31].

111

## 2.2 Phylogeny

Cacao and Arabidopsis nucleotide sequences that are homologous to TgPRs were obtained using local alignment (BLAST) from CocoaGenDB [32] and TAIR (https://www.arabidopsis.org), respectively. The global multiple alignment of all sequences was made using ClustalW available with MEGAX software [33]. The MEGAX software was used to construct a rooted phylogenetic tree using the maximum likelihood method with 1,000 bootstrap sampling steps, the Tamura-Nei model and the G+I substitution method. Phylogenetic trees were visualized and edited using the FigTree v1.4.4 tool (http://tree.bio.ed.ac.uk/software/figtree/).

121

## 2.3 Homology modeling of TgPRs and molecular docking with chitin and chitosan

The tri-dimensional (3D) structure of the TgPR proteins was obtained using the Swiss-Model server [34]. The crystal structures of homologous proteins available from the Protein Data Bank (PDB; https://www.rcsb.org/) were used as templates to build the structural models of
126 TgPR (Supplementary material 1). The stereochemical quality of the models was analyzed
127 with ANOLEA [35] and Procheck [36]. Water molecules were removed from and polar
128 hydrogens were added to the TgPR protein models, before calculation of their Gasteiger
129 charges; the results were exported in PDBQT format. The active sites were identified based
130 on TgPR protein alignment with PR proteins from other species (Supplementary materials 2
131 to 4), using the ClustalW tool [37]. The amino acid residues from the TgPR active sites of the
132 proteins as well as the grid box defining the docking region were marked using AutoDock
133 Tools v1.5.6 [38]. For docking, chitin (CID: 444514) and chitosan (CID: 71853) were used as
134 ligands; their structures were downloaded in SMILES format. Using the MarvinSketch
135 15.7.13.0 tool, the ligands were converted into 3D structures, checked for conformation and
136 saved in mol2 format. The Kollman charges were calculated using AutoDockTools v1.5.6,
137 and the results were exported in PDBQT format. The docking was done using the AutoDock
138 Vina software [39] with default parameters. The results were visualized in PyMOL v1.7.4
139 [40] and Discovery Studio 4.5.

140

## 2.4 Plant material

142 *TgPR* gene expression was analyzed in adult plants obtained from clonal cuttings of cupuassu
143 genotypes C174 (resistant to witches' broom disease) and C1074 (susceptible to witches'
144 broom disease), grown in the experimental station of CEPLAC (Belém, Pará, Brazil) [41].
145 Cupuassu apical meristems were inoculated with *M. perniciosa* using the droplet method (30
146 µL of suspension containing 1x10⁵ ml⁻¹ basidiospores) [42]. After inoculation, the stem apex
147 region was wrapped in a plastic bag (a water-saturated environment) to improve basidiospore
148 germination and plant infection by the fungus [43]. The control plants were submitted to the
149 same procedure, but the basidiospore suspension was replaced by distilled water. The apical
150 meristems of the inoculated C174 and C1074 genotypes were harvested 8, 24, 48 and 72
151

hours after inoculation (hai), while non-inoculated ones (control) were harvested at 8 hai
Three plants (biological replicates) were used for each genotype at each harvesting time. The
harvested samples were immediately frozen in liquid nitrogen and stored at -80 °C until use.

152

153

154

155

**2.5 Reverse transcription quantitative PCR (qPCR) analysis**

156

Each biological replicate (individual plant) was macerated in liquid nitrogen, and 3 mg of the
macerate was used to extract its total RNA using the RNAqueous® kit according to the
manufacturer's recommendations (Ambion). Total RNA (15 µl) was treated with the DNase I
kit according to the manufacturer's recommendations (Invitrogen). The cDNA was
synthesized using the RevertAid First Strand cDNA Synthesis Kit, a cDNA synthesis kit,
according to the manufacturer's recommendations (ThermoScientific). The cDNA was
quantified on a GeneQuant Pro Spectrophotometer UV/Vis Reader (Amersham). For qPCR,
the TgPR primers were designed with the Primer 3 plus tool [44] using previously described
parameters [45], and checked for dimer and hairpin formation using the Oligoanalyzer
(https://www.idtdna.com/calc/analyzer) (Supplementary material 5). Three reference genes
(acyl carrier protein/ACP, malate dehydrogenase/MDH and tubulin/TUB; Supplementary
material 5) previously identified in cupuassu [27] were analyzed for stability in three pooled
samples of each cupuassu genotype: i) a non-inoculated pool sample (3 plants); ii) a pool of 8
and 24 hai samples (6 plants); and iii) a pool of 48 and 72 hai samples (6 plants). Quantitative
PCR was performed in an Eppendorf thermocycler Realplex4. The reaction was made in a
final volume of 10 µl containing 5 µl of Taq READYMIX SYBR Green, 0.1 µl of ROX
(fluorescence signal normalizer), 0.5 µl of primers (forward and reverse, at a concentration of
10 mM each) and 4.4 µl of cDNA 1/20 (230 ng). The cycling conditions were 95°C for 2 min
followed by 45 cycles of 95°C for 15 s, 55°C for 15 s and 60°C for 45 s, followed by a
dissociation curve step. The fluorescence data and primer efficiency were analyzed using

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175
176 MINER 4.0 [46]. The gene-expression stability was calculated using NormFinder [47], which
177 determined the best combination of the reference genes ACP and MDH for relative expression
178 calculation (Supplementary material 6). The gene expression was analyzed in each biological
179 replicate (1 biological replicate = 1 plant) for each genotype (inoculated vs. non-inoculated) at
180 each harvesting time, using the qPCR cycling conditions described above and three technical
181 replicates. The relative expression was obtained using the REST-2009 software [48], which
182 considers the primer efficiency as well as the average ACP and MDH values for calculation.
183

183

184 3. Results

185 3.1 Identification and characterization of TgPR sequences

186 Analysis of the cupuassu RNA-seq allowed the selection of three sequences – codified as
187 C106, C68 and C356 – and showed homology with chitinases [28]. Sequence C106 showed
188 homology with chitinases from the GH19 family, which are involved in pathogenesis and are
189 related to the PR3 class (Table 1). For this reason, sequence C106 was re-named as *T. grandiflorum* pathogenesis-related protein 3 (TgPR3). The *TgPR3* ORF was 966 bp in length
190 and encoded for a 322 aa protein with a molecular weight and an isoelectric point of 35.5 kDa
191 and 6.64, respectively (Table 1). TgPR3 contained a signal peptide (26 aa) responsible for
192 extracellular addressing (86.48% of probability). The protein contained 17 and three predicted
193 phosphorylation and glycosylation sites, respectively (Table 1). The C68 sequence showed
194 homology with the thaumatin like protein (TLP-P) family, which belongs to the pathogenesis-
195 related protein 5 (PR5) class and includes chitinases and osmotins. The C68 sequence was
196 renamed *T. grandiflorum* pathogenesis-related protein 5 (TgPR5) (Table 1). The TgPR5's
197 ORF was 672 bp in length, and it encoded a 224 aa protein with a molecular weight of 24.2
198 kDa and an isoelectric point of 6.68 (Table 1). The protein contained a 23 aa signal peptide
199 responsible for extracellular addressing (99.7% probability). TgPR5 contains one predicted
200
glycosylation and eight predicted phosphorylation sites (Table 1). The protein encoded by the C356 sequence was classified as a chitinase from the GH18 family and as hevamine enzyme, which are chitinases belonging to the class of pathogenesis-related protein 8 (PR8). This sequence was renamed *T. grandiflorum* pathogenesis-related protein 8 (TgPR8). The TgPR8 ORF was 927 bp in length, and it encoded for a 309 aa protein with a molecular weight of 33.3 kDa and a pI of 4.27 (Table 1). TgPR8 contains a signal peptide (29 aa), which potentially addresses the protein to the extracellular (52.2% probability) or the vacuolar (34.5% probability) compartments. The TgPR8 protein showed one and 13 glycosylation and phosphorylation sites, respectively (Table 1).

210

211

## 3.2 Phylogeny

BLAST made from *TgPR* sequences allowed the identification of 11 and 14 homologous sequences of TgPR3, 30 and 28 of TgPR5 and 14 and one of TgPR8 in *T. cacao* and *A. thaliana*, respectively (Fig. 1). TgPR3 showed a high homology with *T. cacao* sequences Tc04_t029180 and Tc06_t000490 and with *A. thaliana* sequences AT3G16920.1 and AT1G05850.1 (Fig. 1A). TgPR5 was clustered with the sequences Tc00_t060970, Tc03_t026960, Tc03_t026980, Tc03_t026990, Tc03_t027000, Tc03_t027010 and Tc03_t027030 from *T. cacao* and with AT4G11650 (OSM34) from *A. thaliana* (Fig. 1B). TgPR8 was clustered with the sequences Tc01_t032120, Tc03_t017760, Tc03_t017780, Tc03_t017790, Tc10_t015260 and Tc10_t015330 from *T. cacao* as well as with AT5G24090 from *A. thaliana* (Fig. 1C).

222

223

## 3.3 Homology modeling and validation of TgPR's 3D structure

The TgPR3 protein's 3D structure (Fig. 2A) was obtained through homology modeling with the GH19 chitinase (PDB: 4TX7) from *Vigna unguiculata* (39% of identity and 91% of

225
coverage; Supplementary material 1). The TgPR3 protein 3D model showed that 87% of the amino acid residues was in the most favored regions, 11.8% was in the additional allowed regions, 0.6% was in the generously allowed regions and 0.6% was in the disallowed regions (Supplementary material 7A). The TgPR3 model contained 12 helices, 26 β-turns, eight γ-turns and three disulfide bonds (Fig. 2A, Supplementary material 8). The alignment of TgPR3 with other GH19 chitinases from other species (Supplementary material 2) allowed the identification of TgPR3's catalytic site containing the amino acid residues Lys70, Glu92 and Tyr125 (Fig. 2A). The TgPR5 protein's 3D structure (Fig. 2B) was obtained by homology modeling with an osmotin (PDB: 4L2J) from *Calotropis porcine* (75% of identity and 100% of coverage; Supplementary material 1). The TgPR5 protein 3D model showed 88.5% of the amino acid residues in the most favored regions, 11.1% in the generously allowed regions and 0.4% in the allowed regions, and it had no amino acid residues in disallowed regions (Supplementary material 7B). The TgPR5 model contained 14 β-sheets, five helices, 25 β-turns, two γ-turns, one bulge, five hairpins and seven disulfide bonds (Fig. 2B, Supplementary material 8). The alignment of TgPR5 protein with thaumatine-like proteins from other species (Supplementary material 3) allowed the identification of putative amino acid residues from the active cleft: Arg43, Glu83, Asp96 and Asp101 (Fig. 2B). The TgPR8 protein's 3D structure (Fig. 2C) was obtained by homology modeling with hevamine A (PDB: 1LLO) from *Hevea brasiliensis* (64% of identity and 99% of coverage; Supplementary material 1). The TgPR8 protein's 3D structure showed 78.8% of the amino acid residues in the most favored regions, 18.9% in the additional allowed regions, 0.9% in the generously allowed regions and 1.4% in disallowed regions (Supplementary material 7C). The TgPR8 model contained a barrel shape with nine β-sheets, 15 helices, three disulfide bonds, 16 β-turns, one γ-turn, four bulges, one hairpin, six α-β units and one PSI loop (Fig. 2C, Supplementary material 8). The alignment of TgPR8 with GH18 chitinases from other species (Supplementary material 4) allowed the
251 identification of motifs and conserved domains, including the predicted active site containing
252 the amino acid residues Tyr6, Phe32, Asp125, Glu127, Gln155, Gln178, Tyr180 and Trp253 (Fig. 2C).

253

254 **3.4 Molecular docking between TgPRs and chitin or chitosan**

255 The molecular docking of TgPR3 with chitin had an affinity energy of -6.4 kcal/mol (Supplementary material 9). It showed conventional hydrogen bond interactions with amino acid residues Ser92, Gly94, Glu112 and Tyr225, and carbon hydrogen bonds with amino acid residues Lys90 and Pro217 (Fig. 3). The molecular docking of TgPR3 with chitosan had an affinity energy of -7.4 kcal/mol (Supplementary material 9). It showed conventional hydrogen bond interactions with amino acid residues Lys90, Ser92, Asp118, Tyr119, Tyr145, Tyr148, Arg182, Leu224, Tyr225 and Asp236; carbon hydrogen bonds with amino acid residues Thr91, Tyr109 and Arg111; and alkyl interactions with the amino acid residue Pro143 (Fig. 3).

263 The molecular docking of TgPR5 with chitin had an affinity energy of -6.5 kcal/mol (Supplementary material 9) and showed conventional hydrogen bonds with the amino acid residues Thr40, Glu83, Gln88, Asp96 and Pro179; one carbon hydrogen bond with the amino acid residue Asp182; and one pi-sigma interaction with the amino acid residue Tyr176 (Fig. 3). The molecular docking of TgPR5 with chitosan had an affinity energy of -6.8 kcal/mol (Supplementary material 9) with conventional hydrogen bonds in the amino acid residues Thr40, Ala42, Tyr74, Gly75, Glu83, Tyr84, Asp96, Asp101, Tyr176 and Asp182 and one carbon hydrogen bond in the amino acid residue Cys154 (Fig. 3). The molecular docking of TgPR8 with chitin had an affinity energy of -7.0 kcal/mol (Supplementary material 9) and showed conventional hydrogen bonds with the amino acid residues Gln9, Ala81, Glu127, Gln155, Tyr180 and Tyr257 (Fig. 3). The docking of TgPR8 with chitosan had an affinity energy of -7.3 kcal/mol (Supplementary material 9) and showed conventional hydrogen bonds with the

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274
275 amino acid residues Gln9, Ser43, Met44, Asn45, Leu46, Gln155, Asn181, Asn182, Ala221, Trp253 and
276 Tyr257, and carbon hydrogen bonds with the amino acid residues Gln178 and Tyr180 (Fig. 3).

277

278 **3.5 Analysis of TgPR gene expression**

279 The primers PR3, PR5, PR8, ACP and MDH showed efficiency from 91% (MDH on the
280 C1074 genotype) to 102% (P3 on both the C174 and C1074 genotypes; Supplementary
281 material 10). For both genotypes and for all of the harvesting points, the PCR amplification
282 occurred at the same and unique melting temperature for each gene **and a unique band was
283 visible on electrophoresis agarose gel**, showing that only the corresponding gene was
284 amplified (Supplementary material 11). The relative expression of the *TgPR* genes was lower
285 in the susceptible genotype at all of the harvesting times, as compared to the resistant
286 genotype (Figs. 4A and B). The *TgPR3* gene showed two to three times higher expression in
287 the resistant genotype than in the susceptible one (Fig. 4B). Similarly, the *TgPR8* gene
288 showed two to four times higher expression in the resistant plants compared to the susceptible
289 ones (Fig. 4B). For both the *TgPR3* and *TgPR8* genes, the expression through the time course
290 disease slightly increased from 8 to 72 hai in the resistant plants (Fig. 4B). The *TgPR5* gene
291 showed a very high expression at 8 and 24 hai in the resistant genotype (about 25 of relative
292 expression; Fig. 4A), which decreased at 48 and 72 hai (about 2 of relative expression; Fig.
293 4B). Except for *TgPR5* 8 hai, the relative expression of the *TgPR* genes in the susceptible
294 genotype was almost constant (Fig. 4B).

295

296 **4. Discussion**

297 **4.1 TgPRs are structurally distinct and show a high energy affinity for chitosan**

298 The TgPR3 and TgPR5 proteins showed similar isoelectric points and share the same cellular
299 addressing (extracellular; Table 1), whereas the TgPR8 protein shows a more acidic
isoelectric point. Although the predictions for TgPR8 suggested extracellular and vacuolar addressing (Table 1), type GH18 chitinases are found in the apoplast [49]. Although GH19 (PR-3) chitinases are composed of α-helices and loops, they are highly thermostable [50]. GH19 are divided into loopful and loopless classes, where loopful chitinases have loops at both ends of the substrate-binding site, while loopless chitinases have a non-end loop structure, which is commonly found in bacteria [51]. The TgPR3 protein showed homology with the loopful GH19 class because of the presence of the loops near the substrate-binding site (Fig. 2A). Most GH18 proteins (PR-8) correspond to chitinases, although some have been reported to have xylanase-inhibiting activity or to have lost their chitinase activity [16]. As chitinases, the GH18 proteins have the same activity as GH19 but are extremely distinct, both structurally and evolutionarily [52]. One pattern that defines the chitinase activity in GH18 is the presence of a DXDXE motif [53]; the TgPR8 showed the DFDIE motif at positions 123-127 of the sequence, corroborating its classification and potential chitinase activity (Supplementary material 8). Osmotin antifungal activity is associated with the presence of the acidic cleft region [15, 54], which in TgPR5 protein is composed of the residues Glu<sub>83</sub>, Asp<sub>96</sub> and Asp<sub>101</sub> (Figure 1B). *In vitro* studies have shown that barley seeds' GH19 chitinase can efficiently catalyze tetrameric N-acetylglucosamine in dimers but that most GH19 chitinases prefer larger substrates [55]. These data corroborate our docking results, where the TgPR3 protein had an affinity of -6.4 kcal/mol with the chitin molecule (the smaller one) and -7.4 kcal/mol with the chitosan molecule (the larger one), showing that TgPR3 also has a higher affinity for larger substrates (Supplementary material 9, Fig. 3). TgPR8 protein has similar affinity energy for chitin and chitosan ligands (-7.0 kcal/mol and -7.3 kcal/mol, respectively; Supplementary material 9). The docking results reinforced the putative TgPR8 protein chitinase activity, with an interaction within the DFDIE motif, in which it is essential for the chitinase activity of this protein class [53]. Although structurally different and having
325

different catalytic mechanisms [52], the proteins TgPR3 and TgPR8 have a very close affinity
energy for the chitosan molecule. This may be related to their activity type, as both are
glycoside hydrolases [56]. The TgPR5 protein had an affinity energy of -6.5 kcal/mol with
the chitin molecule and -6.8 kcal/mol with the chitosan molecule (Supplementary material 9;
Fig. 3). These results show that TgPR5 can bind chitin molecules and may have potential
chitinase and antifungal activity.

326

327

328

329

330

331

332

## 4.2 TgPRs are homologous to other plant PR proteins involved in biotic and abiotic stress

333

334

TgPR3 was clustered with Tc06_t000490 and Tc04_t029180 (Fig. 1), which showed a high constitutive expression across treatments, Tc06_t000490 being up-regulated during cacao- *Phytophthora palmivora* interaction [57]. The AT3G16920 from *A. thaliana* that also clustered with TgPR3 (Fig. 1) is involved in response to mechanical stress [58] as well as to heat, salt and drought stress [59]. AT1G05850 is related to cellulose biosynthesis [60] and also to response to high temperatures, salt and drought stress [59]. Sequences Tc00_t060970, Tc03_t026990, Tc03_t027000, Tc03_t027010 and Tc03_t027030, which were clustered with TgPR5 (Fig. 1), showed increased expression levels during cacao interaction with the pathogenic fungi *P. palmivora* and *Colletotrichum theobromicola* [57]. The *A. thaliana* AT4G11650 sequence showed an increased expression with *Agrobacterium tumefaciens* at 24 hai [61]. This gene is also regulated by the ethylene and jasmonic acid-signalization pathway [62]. Transgenic Arabidopsis plants knocked-out for the transcription factor WRKY33 – which is mediated by ethylene and the jasmonic acid signalization pathway – showed a susceptibility pattern to *Botrytis cinerea* and a reduction of AT4G11650 expression level, while the wild-type plants showed increased AT4G11650 expression in response to the pathogen [62]. The *A. thaliana* sequence AT5G24090, homologous to TgPR8 (Fig. 1),

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349
350

showed an expression pattern specific to stress response; however, no expression was
observed during the plant development stage [63]. In *T. cacao*, the Tc03_g017760 gene
homologous with TgPR8 (Fig. 1) showed an increased expression level when the plant was
inoculated by *P. palmivora* or *Colletotrichum theobromicola* [57]. The PR gene's expression
in response to biotic and abiotic stress could be related to the phytohormone pathways that
regulate their expression. This could be related to the literature data about *M. perniciosa*,
which modifies the metabolism of *T. cacao* during compatible interactions through the
phytohormone pathway [64].

351

352

353

354

355

356

357

358

359

**4.3 TgPR genes were highly expressed in the cupuassu genotype resistant to M. perniciosa**

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

The relative expression of *TgPR3*, *TgPR5* and *TgPR8* was highly significant in the resistant genotype infected by *M. perniciosa*, at all of the harvesting points, but mainly at 8 and 24 hai for *TgPR5* (Fig. 3A). In the *T. cacao–M. perniciosa* interaction, which is closely related to and also deeper studied than the cupuassu-*M. perniciosa* pathosystem, the basidiospore germination and the beginning of the germination tube in the plant tissues were observed at 6 hai [65], a time period that is coherent with the high and early expression of the TgPRs (Figs. 3A and B). The very high expression observed for TgPR5 since 8 hai would corroborate other reports, in which the PR5 was overexpressed in resistant plant genotypes after pathogen infection [17, 66-68]. The osmotin's function and mode of action are still unclear, and different functions have been shown, such as in inhibiting beta-glucanase or xylanase [17, 69]. Here, we propose that TgPR5 may act as chitinase due to its strong *in silico* interaction with chitin and chitosan, as well as its extracellular localization (Fig. 2, Table 1). **Thus, we the resistant cupuassu genotype may have been able to recognize *M. perniciosa* through its innate immune system – probably through effectors [70] – and to generate a defense response**
against the pathogen from the beginning of the infection process (Fig. 5), which explains why the C174 resistant genotype does not present symptoms or disease features in the field [71]. In the susceptible cupuassu genotype, the TgPR genes showed reduced expression at all of the harvesting times (Fig. 3) showing that the pathogen can bypass the innate immune system of cupuassu and cause virulence. In *T. cacao*–*M. perniciosa* interaction, the fungus can interfere with the host’s metabolism and alter its gene expression [64]. The reduction of plant chitinase expression levels during the cupuassu–*M. perniciosa* interaction can be highly beneficial to the pathogen, which can grow and colonize the plant tissues (Fig. 5). Moreover, the reduction of cell wall degradation by chitinases (i.e. cell wall fragments) reduces the recognition by the plant’s innate immune system through chitin receptors [72, 73] (Fig. 5). In contrast, increased plant chitinase expression and secretion can cause serious damage to the pathogen, allowing the plant to recognize the pathogen, and triggering defense mechanisms and the plant’s resistance [73] (Fig. 5).

388

389

## 5. Conclusion

Here, we have shown the first *in silico* and *in vitro* analysis of pathogenesis-related proteins from *T. grandiflorum*. We show that TgPR3 and TgPR8 are putative chitinases with distinct structures, and that TgPR5 is a putative osmotin, all with affinity for chitin and chitosan molecules. These results suggest a new role for TgPR5/osmotin as chitinase/chitosanase in plant-pathogen interactions. TgPR expression highly increased in the resistant cupuassu genotype infected by *M. perniciosa* (mainly TgPR5 at the first infection times) and was low in the susceptible one. These results could be related to the phylogenetic analysis of the *TgPR* genes showing that *T. grandiflorum* sequences clustered with cacao and Arabidopsis *PR* sequences are involved in response to biotic and abiotic stress, some through phytohormone-
399

signaling pathways. These observations suggest that TgPRs could be involved in recognizing

400

mechanisms of a plant's innate immune system through chitin receptors.

401

402

**Availability of supporting data**

403

The data sets supporting the results of this article are included within the article and its

404

additional files.

405

406

**Abbreviations:** 3D: tridimensional; PAMP: molecular patterns associated with pathogens;

407

PR: pathogenesis related; TLP: thaumatin-like protein

408

409

**Competing interest**

410

No conflicts of interest to declare.

411

412

**Authors' contribution**

413

RJSS was responsible for the execution of all the experimental steps. RJSS, LHM and FM

414

analyzed and discussed the data. RJSS and FM wrote the manuscript. KPG and RMA were

415

responsible for plant material production and inoculation with *M. perniciosa*. RMA, LHM,

416

FM and KPG were responsible for the conception and design of the experiments. FM was

417

responsible for the financial support of the research. FM and LHM were responsible for the

418

advising of RJSS.

419

420

**Acknowledgements**

421

The work of RJSS was supported by the Fundação de Amparo à Pesquisa do Estado da Bahia

422

(FAPESB), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and

423

Embrapa Recursos Genéticos e Biotecnologia (Cenargen). This work was supported by
424 FAPESB project DTE0038/2013. KPG and FM received a Productivity grant from the
425 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank
426 Dr. Akyla Maria Martins Alves for advices during manuscript writing. This work was made in
427 the frame of the International Consortium in Advanced Biology (CIBA).

428

429 **References**

430 [1] R.M. Alves, M.D.V.d. Resende, B.d.S. Bandeira, T.M. Pinheiro, D.C.R. Farias, Avaliação e seleção de
431 progênies de cupuaçuzeiro (*Theobroma grandiflorum*), em Belém, Pará, Revista Brasileira de
432 Fruticultura, 32 (2010) 204-212.

433 [2] R.M. Alves, C.R.S. Silva, M.S.C. Silva, D.C.S. Silva, A.M. Sebbenn, Diversidade genética em coleções
434 amazônicas de germoplasma de cupuaçuzeiro [*Theobroma grandiflorum* (Willd. ex Spreng.) Schum.],
435 Revista Brasileira de Fruticultura, 35 (2013) 818-828.

436 [3] M.C. Costa, G.A. Maia, M.d.S.M. Souza Filho, R.W.d. Figueiredo, R.T. Nassu, J.C.S. Monteiro,
437 Conservação de polpa de cupuaçu [*Theobroma grandiflorum* (Willd. Ex Spreng.) Schum] por métodos
438 combinados, Revista Brasileira de Fruticultura, 25 (2003) 213-215.

439 [4] R. Sayid, Chocolate Could Run Out In 2020 Due To Worldwide Shortage of Cocoa, in: The Daily
440 Mirror online, 2013, pp. http://www.mirror.co.uk/news/world-news/chocolate-could-run-out-2020-
441 2913505.

442 [5] A. Wexler, World's sweet tooth heats up cocoa. Growing demand from emerging markets is
443 pushing up prices for key ingredient in chocolate, in: The Wall Street Journal, 2014, pp.
444 http://online.wsj.com/news/articles/SB10001424052702304703804579381234107085804.

445 [6] R.M. Alves, M.D.V. Resende, Avaliação genética de individuos e progênies de cupuaçuzeiro no
446 estado do Para e estimativas de parâmetros genéticos, Revista Brasileira de Fruticultura, 30 (2008)
447 696-701.

448 [7] J. Sels, J. Mathys, B.M.A. De Coninck, B.P.A. Cammue, M.F.C. De Bolle, Plant pathogenesis-related
449 (PR) proteins: a focus on PR peptides, Plant Physiology and Biochemistry, 46 (2008) 941-950.

450 [8] L.C. Van Loon, Induced resistance in plants and the role of pathogenesis-related proteins,
451 European Journal of Plant Pathology, 103 (1997) 753-765.

452 [9] S. Ali, B.A. Ganai, A.N. Kamili, A.A. Bhat, Z.A. Mir, J.A. Bhat, A. Tyagi, S.T. Islam, M. Mushtaq, P.
453 Yadav, S. Rawat, A. Grover, Pathogenesis-related proteins and peptides as promising tools for
454 engineering plants with multiple stress tolerance, Microbiological Research, 212-213 (2018) 29-37.

455 [10] A. Edreva, Pathogenesis-related proteins: Research progress in the last 15 years, Gen Appl Plant
456 Physiology, 31 (2005) 105-124.

457 [11] M. Sinha, R.P. Singh, G.S. Kushwaha, N. Iqbal, A. Singh, S. Kaushik, P. Kaur, S. Sharma, T.P. Singh,
458 Current overview of allergens of plant pathogenesis related protein families, ScientificWorldJournal,
459 2014 (2014) 543195-543195.

460 [12] Hakim, A. Ullah, A. Hussain, M. Shaban, A.H. Khan, M. Alariqi, S. Gul, Z. Jun, S. Lin, J. Li, S. Jin,
461 M.F.H. Munis, Osmotin: A plant defense tool against biotic and abiotic stresses, Plant Physiology and
462 Biochemistry, 123 (2018) 149-159.

463 [13] J. Grenier, A. Asselin, Sorne Pathogenesis-Related Proteins Are Chitosanases with Lytic Activity
464 Against Fungal Spores, MOLECULAR PLANT-MICROBE INTERACTIONS, 3 (1990) 401-407.

465 [14] T. Kawase, S. Yokokawa, A. Saito, T. Fujii, N. Nikaidou, K. Miyashita, T. Watanabe, Comparison of
466 Enzymatic and Antifungal Properties between Family 18 and 19 Chitinases from S. coelicolor A3(2),
467 Bioscience, Biotechnology, and Biochemistry, 70 (2006) 988-998.
468

469

[15] K. Min, S.C. Ha, P.M. Hasegawa, R.A. Bressan, D.-J. Yun, K.K. Kim, Crystal structure of osmotin, a plant antifungal protein, Proteins: Structure, Function, and Bioinformatics, 54 (2004) 170-173.

470

471

472

[16] D.N. Patil, M. Datta, A. Dev, S. Dhindwal, N. Singh, P. Dasauni, S. Kundu, A.K. Sharma, S. Tomar, P. Kumar, Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases, PLoS ONE, 8 (2013) e63779.

473

474

[17] J.-J. Liu, R. Sturrock, A.M. Ekramoddoullah, The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function, Plant Cell Reports, 29 (2010) 419-436.

475

476

[18] T. Pusztahelyi, Chitin and chitin-related compounds in plant-fungal interactions, Mycology, 9 (2018) 189-201.

477

478

[19] A. Sánchez-Vallet, J.R. Mesters, B.P.H.J. Thomma, The battle for chitin recognition in plant-microbe interactions, FEMS Microbiology Reviews, 39 (2015) 171-183.

479

480

[20] M. Malerba, R. Cerana, Recent Applications of Chitin- and Chitosan-Based Polymers in Plants, Polymers, 11 (2019).

481

482

483

[21] M.S. Riaz Rajoka, L. Zhao, H.M. Mehwish, Y. Wu, S. Mahmood, Chitosan and its derivatives: synthesis, biotechnological applications, and future challenges, Applied Microbiology and Biotechnology, 103 (2019) 1557-1571.

484

485

486

487

[22] D.S. Gomes, M.A. Lopes, S.P. Menezes, L.F. Ribeiro, C.V. Dias, B.S. Andrade, R.M. de Jesus, A.B.L. Pires, A. Goes-Neto, F. Micheli, Mycelial development preceding basidioma formation in Moniliophthora perniciosa is associated to chitin, sugar and nutrient metabolism alterations involving autophagy, Fungal Genetics and Biology, 86 (2016) 33-46.

488

489

490

[23] M.A. Lopes, D.S. Gomes, M.G. Koblitz, C.P. Pirovani, J.C. Cascardo, A. Goes-Neto, F. Micheli, Use of response surface methodology to examine chitinase regulation in the basidiomycete *Moniliophthora perniciosa*, Mycol Res, 112 (2008) 399-406.

491

492

493

494

[24] C.S. Souza, B.M. Oliveira, G.G. Costa, A. Schriefer, A. Selbach-Schnadelbach, A.P. Uetanabaro, C.P. Pirovani, G.A. Pereira, A.G. Taranto, J.C. Cascardo, A. Goes-Neto, Identification and characterization of a class III chitin synthase gene of *Moniliophthora perniciosa*, the fungus that causes witches' broom disease of cacao, J Microbiol, 47 (2009) 431-440.

495

496

497

[25] D. Penman, G. Britton, K. Hardwick, H.A. Collin, S. Isaac, Chitin as a measure of biomass of Crinipellis perniciosa, causal agent of witches' broom disease of Theobroma cacao, Mycological Research, 104 (2000) 671-675.

498

499

500

501

[26] L. Ferraz Dos Santos, R. Moreira Fregapani, L.L. Falcão, R.C. Togawa, M.M.d.C. Costa, U.V. Lopes, K. Peres Gramacho, R.M. Alves, F. Micheli, L.H. Marcellino, First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao, PLoS ONE, 11 (2016) e0151074-e0151074.

502

503

504

505

[27] L. Ferraz dos Santos, R.J. Santana Silva, D. Oliveira Jordão do Amaral, M.F. Barbosa de Paula, L. Ludke Falcão, T. Legavre, R.M. Alves, L.H. Marcellino, F. Micheli, Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum, PLoS ONE, 11 (2016) e0160646.

506

507

508

509

510

[28] L.L. Falcao, J.O.S. Werneck, R.C. Togawa, M.M.d.C. Costa, P. Grynberg, O.B.d. Silva Junior, R.M. Alves, P.S.B. Albuquerque, L.H. Marcellino, Analyses of cupuassu (*Theobroma grandiflorum*) transcriptome during interaction with *Moniliophthora perniciosa*, the causal agent of Witches' Broom disease., in: S.-E. CPATU (Ed.) 7th BRAZILIAN BIOTECHNOLOGY CONGRESS and 2nd BIOTECHNOLOGY IBERO-AMERICAN CONGRESS, Brasília, DF, Brazil, 2018.

511

512

[29] T.N. Petersen, S. Brunak, G. von Heijne, H. Nielsen, SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat Meth, 8 (2011) 785-786.

513

514

515

[30] E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel, B. A., Protein Identification and Analysis Tools on the ExPASy Server, in: J.M.W. (ed) (Ed.) The Proteomics Protocols Handbook, Humana Press 2005, pp. 571-607.

516

517

[31] S. Briesemeister, J. Rahnenführer, O. Kohlbacher, YLoc--an interpretable web server for predicting subcellular localization, Nucleic Acids Research, 38 (2010) W497-W502.

518

519

[32] X. Argout, J. Salse, J.-M. Aury, M.J. Guiltinan, G. Droc, J. Gouzy, M. Allegre, C. Chaparro, T. Legavre, S.N. Maximova, M. Abrouk, F. Murat, O. Fouet, J. Poulain, M. Ruiz, Y. Roguet, M. Rodier-
520 Goud, J.F. Barbosa-Neto, F. Sabot, D. Kudrna, J.S.S. Ammiraju, S.C. Schuster, J.E. Carlson, E. Sallet, T. Schiex, A. Dievart, M. Kramer, L. Gelley, Z. Shi, A. Bérard, C. Viot, M. Boccara, A.M. Risterucci, V. Guignon, X. Sabau, M.J. Axtell, Z. Ma, Y. Zhang, S. Brown, M. Bourge, W. Golser, X. Song, D. Clement, R. Rivallan, M. Tahi, J.M. Akaza, B. Pitollat, K. Gramacho, A. D'Hont, D. Brunel, D. Infante, I. Kebe, P. Costet, R. Wing, W.R. McCombie, E. Guiderdoni, F. Quetier, O. Panaud, P. Wincker, S. Bocs, C. Lanaud, The genome of Theobroma cacao, Nature Genetics, 43 (2011) 101-108.

526 [33] S. Kumar, G. Stecher, M. Li, C. Knyaz, K. Tamura, MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms, Molecular Biology and Evolution, 35 (2018) 1547-1549.

528 [34] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics, 22 (2006) 195-201.

530 [35] F. MELO, d. Melo, e. melo, g. melo, ANOLEA: a www server to assess protein structures, *Intell. Sys. Mol. Biol.*, 97 (1997) 110-113.

532 [36] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, Journal of Applied Crystallography, 26 (1993) 283-291.

534 [37] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, D.G. Higgins, Clustal W and Clustal X version 2.0, Bioinformatics, 23 (2007) 2947-2948.

537 [38] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, Journal of Computational Chemistry, 30 (2009) 2785-2791.

540 [39] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry, 31 (2010) 455-461.

543 [40] W.L. DeLano, The PyMOL Molecular Graphics System, in, 2002.

544 [41] E.D. Cruz, R.M. Alvez, R.L. Benchimol, Avaliação de clones de cupuacuzeiro (Theobroma grandiflorum (Willd. ex Spreng) K. Schumm.) quanto a tolerância a vassoura-de-bruxa (Crinipellis perniciosa (Stahel) Singer). in: Embrapa Amazônia Oriental. Comunicado técnico, 28, Embrapa CPATU, Belém, PA, Brazil, 2000, pp. 4 p.

548 [42] S. Surujdeo-Maharaj, P. Umaharan, D.R. Butler, T.N. Sreenivasan, An optimized screening method for identifying levels of resistance to *Crinipellis perniciosa* in cocoa (*Theobroma cacao*), Plant Pathol, 52 (2003) 464-475.

551 [43] G. Frias, L. Purdy, R. Schmidt, An inoculation method for evaluating resistance of cacao to *Crinipellis perniciosa*, Plant Disease, 79 (1995) 787-791.

553 [44] A. Untergasser, H. Nijveen, X. Rao, T. Bisseling, R. Geurts, J.A.M. Leunissen, Primer3Plus, an enhanced web interface to Primer3, Nucleic Acids Research, 35 (2007) W71-W74.

555 [45] B. Thornton, C. Basu, Real-time PCR (qPCR) primer design using free online software, Biochemistry and Molecular Biology Education, 39 (2011) 145-154.

557 [46] S. Zhao, R.D. Fernald, Comprehensive algorithm for quantitative real-time polymerase chain reaction, J Comput Biol, 12 (2005) 1047-1064.

559 [47] C.L. Andersen, J.L. Jensen, T.F. Ørntoft, Normalization of Real-Time Quantitative Reverse Transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets, Cancer Research, 64 (2004) 5245-5250.

562 [48] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Research, 30 (2002) e36-e36.

565 [49] N. Takahashi-Ando, M. Inaba, S. Ohsato, T. Igawa, R. Usami, M. Kimura, Identification of multiple highly similar XIP-type xylanase inhibitor genes in hexaploid wheat, Biochemical and Biophysical Research Communications, 360 (2007) 880-884.

568 [50] S. Martínez-Caballero, P. Cano-Sánchez, I. Mares-Mejía, A.G. Díaz-Sánchez, M.L. Macías-Rubalcava, J.A. Hermoso, A. Rodríguez-Romero, Comparative study of two GH19 chitinase-like proteins from Hevea brasiliensis, one exhibiting a novel carbohydrate-binding domain, The FEBS Journal, 281 (2014) 4535-4554.
572

573

574

[51] T. Ohnuma, N. Umemoto, T. Nagata, S. Shinya, T. Numata, T. Taira, T. Fukamizo, Crystal structure of a "loopless" GH19 chitinase in complex with chitin tetrasaccharide spanning the catalytic center, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1844 (2014) 793-802.

575

576

[52] F. Fukamizo, Chitinolytic Enzymes: Catalysis, Substrate Binding, and their Application, Current Protein & Peptide Science, 1 (2000) 105-124.

577

578

579

[53] T. Masuda, G. Zhao, B. Mikami, Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity, Bioscience, Biotechnology, and Biochemistry, 79 (2015) 45-50.

580

581

582

[54] H. Koiwa, H. Kato, T. Nakatsu, J.i. Oda, Y. Yamada, F. Sato, Crystal structure of tobacco PR-5d protein at 1.8 Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins11

Edited by R. Huber, Journal of Molecular Biology, 286 (1999) 1137-1145.

583

584

585

[55] M.M. Chaudet, T.A. Naumann, N.P.J. Price, D.R. Rose, Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays, Protein Science, 23 (2014) 586-593.

586

587

588

[56] N.A. Udaya Prakash, M. Jayanthi, R. Sabarinathan, P. Kangueane, L. Mathew, K. Sekar, Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases, Journal of Molecular Evolution, 70 (2010) 466-478.

589

590

591

[57] A.S. Fister, L.C. Mejia, Y. Zhang, E.A. Herre, S.N. Maximova, M.J. Guiltinan, Theobroma cacao L. pathogenesis-related gene tandem array members show diverse expression dynamics in response to pathogen colonization, BMC Genomics, 17 (2016) 363-363.

592

593

594

[58] K. Koizumi, R. Yokoyama, K. Nishitani, Mechanical load induces upregulation of transcripts for a set of genes implicated in secondary wall formation in the supporting tissue of Arabidopsis thaliana, Journal of Plant Research, 122 (2009) 651.

595

596

597

[59] Y. Kwon, S.-H. Kim, M.-S. Jung, M.-S. Kim, J.-E. Oh, H.-W. Ju, K.-i. Kim, E. Vierling, H. Lee, S.-W. Hong, Arabidopsis hot2 encodes an endochitinase-like protein that is essential for tolerance to heat, salt and drought stresses, The Plant Journal, 49 (2007) 184-193.

598

599

600

601

[60] C. Sánchez-Rodríguez, S. Bauer, K. Hématy, F. Saxe, A.B. Ibáñez, V. Vodermaier, C. Konlechner, A. Sampathkumar, M. Rüggeberg, E. Aichinger, L. Neumetzler, I. Burgert, C. Somerville, M.-T. Hauser, S. Persson, Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis, The Plant Cell, 24 (2012) 589-607.

602

603

604

[61] R.F. Ditt, K.F. Kerr, P. de Figueiredo, J. Delrow, L. Comai, E.W. Nester, The Arabidopsis thaliana Transcriptome in Response to Agrobacterium tumefaciens, Molecular Plant-Microbe Interactions®, 19 (2006) 665-681.

605

606

[62] Z. Zheng, S. Qamar, Z. Chen, T. Mengiste, Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens, Plant Journal, 48 (2006).

607

608

609

[63] Y. Takenaka, S. Nakano, M. Tamoi, S. Sakuda, T. Fukamizo, Chitinase Gene Expression in Response to Environmental Stresses in Arabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance, Bioscience, Biotechnology, and Biochemistry, 73 (2009) 1066-1071.

610

611

612

613

[64] P.J.P.L. Teixeira, D.P.d.T. Thomazella, O. Reis, P.F.V. do Prado, M.C.S. do Rio, G.L. Fiorin, J. José, G.G.L. Costa, V.A. Negri, J.M.C. Mondego, P. Mieczkowski, G.A.G. Pereira, High-resolution transcript profiling of the atypical biotrophic interaction between *Theobroma cacao* and the fungal pathogen *Moniliophthora perniciosa*, The Plant Cell, 26 (2014) 4245-4269.

614

615

[65] K. Sena, L. Alemanno, K.P. Gramacho, The infection process of *Moniliophthora perniciosa* in cacao, Plant Pathology, (2014) 1272-1281.

616

617

618

[66] D. Prasath, I. El-Sharkawy, S. Sherif, K.S. Tiwary, S. Jayasankar, Cloning and characterization of PR5 gene from Curcuma amada and Zingiber officinale in response to Ralstonia solanacearum infection, Plant Cell Reports, 30 (2011) 1799.

619

620

621

[67] D. PRASATH, A. BALAGOPAL, V. MAHANTESH, O.B. ROSANA, S. JAYANSANKAR, M. ANANDARAJ, Comparative study of pathogenesis-related protein 5 (PR5) of different Zingiberaceae species., Indian Journal of Biotechnology, 13 (2014) 178-185.
622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

[68] A. El-kereamy, I. El-sharkawy, R. Ramamoorthy, A. Taheri, D. Errampalli, P. Kumar, S. Jayasankar, Prunus domestica pathogenesis-related protein-5 activates the defense response pathway and enhances the resistance to fungal infection, PLoS ONE, 6 (2011) e17973-e17973.

[69] J. Grenier, C. Potvin, J. Trudel, A. Asselin, Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucans, The Plant Journal, 19 (1999) 473-480.

[70] C.S. Barbosa, R.R.d. Fonseca, T.M. Batista, M.A. Barreto, C.S. Argolo, M.R.d. Carvalho, D.O.J.d. Amaral, E.M.d.A. Silva, E. Arévalo-Gardini, K.S. Hidalgo, G.R. Franco, C.P. Pirovani, F. Micheli, K.P. Gramacho, Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations, BMC Genomics, 19 (2018) 509-509.

[71] R.M. Alves, M.D.V. Resende, B.S. Bandeira, T.M. Pinheiro, D.C.R. Farias, Evolução da vassoura-de-bruxa e avaliação da resistência em progênies de cupuaçuzeiro, Revista Brasileira de Fruticultura, 31 (2009) 1022-1032.

[72] H. Böhm, I. Albert, L. Fan, A. Reinhard, T. Nürnberger, Immune receptor complexes at the plant cell surface, Current Opinion in Plant Biology, 20 (2014) 47-54.

[73] H. Kaku, Y. Nishizawa, N. Ishii-Minami, C. Akimoto-Tomiyama, N. Dohmae, K. Takio, E. Minami, N. Shibuya, Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor, Proceedings of the National Academy of Sciences of the United States of America, 103 (2006) 11086-11091.
641 Tables

642

<table><caption>643 Table 1. TgPR gene and protein sequence characteristics.</caption><thead><tr><th rowspan="2">Name</th><th colspan="4">Gene data</th><th colspan="7">Protein data</th></tr><tr><th>Identification*</th><th>mRNA*<br>(bp)</th><th>ORF<br>size (bp)</th><th>Protein<br>size<br>(aa)</th><th>Signal<br>peptide<br>(aa)</th><th>Molecular<br>weight<br>(kDa)</th><th>pI</th><th>Subcellular localization<br>(probability)</th><th>Phosphorylation<br>sites</th><th>Glycosylation<br>sites</th></tr></thead><tbody><tr><td>TgPR3</td><td>C106</td><td>1239</td><td>966</td><td>322</td><td>26</td><td>35.5</td><td>6.64</td><td>Extracellular (86.48%)</td><td>S37 S41 S114 S123 S193<br>T91 T99 T185 T205 T219 T240<br>Y62 Y119 Y124 Y126 Y145 Y167</td><td>N20 N171 N207</td></tr><tr><td>TgPR5</td><td>C68</td><td>950</td><td>672</td><td>224</td><td>23</td><td>24.2</td><td>6.68</td><td>Extracellular (99.7%)</td><td>S118 S139 S161 S176 T143 T256 Y137 Y239</td><td>N163</td></tr><tr><td>TgPR8</td><td>C356</td><td>1111</td><td>927</td><td>309</td><td>29</td><td>33.3</td><td>4.27</td><td>Extracellular (52.2%)<br>Vacuolar (34.5%)</td><td>S77 S95 S147 S266<br>T15 T41 T88<br>Y66 Y99 Y134 Y149 Y162 Y227</td><td>N145</td></tr></tbody></table>

<footnote n="644">644 * Data from RNA-seq as described by [28].</footnote>
645 **Figure legends**

646 **Figure 1.** Phylogenetic tree obtained with the *TgPR* nucleotide sequences and their
647 homologues from *T. cacao* and *A. thaliana*. **A.** Phylogenic tree from *TgPR3*. **B.**
648 Phylogenic tree from *TgPR5*. **C.** Phylogenic tree from *TgPR8*. Sequence names
649 beginning with *Tc* and *AT* correspond to *T. cacao* and *A. thaliana*, respectively. The
650 yellow square highlights the *TgPR's* closest phylogenetic branches.

651 **Figure 2.** The 3D structure of TgPR proteins. **A.** TgPR3's 3D structure, with the amino
652 acid residues Lys70 Glu92, and Tyr125 of the active site highlighted. **B.** TgPR5's 3D
653 structure with the amino acid residues Arg43, Glu83, Asp96 and Asp101 of the active
654 site highlighted. **C.** TgPR8's 3D structure with the amino acid residues Tyr6, Phe32,
655 Asp125, Glu127, Gln155, Gln178, Tyr180 and Trp253 of the active site highlighted.

656 **Figure 3.** Representations (2D and 3D) of docking between TgPRs and chitin/chitosan.

657 **Figure 4.** Relative expression of TgPR genes in resistant (R) and susceptible (S)
658 cupuassu genotypes 8, 24, 48 and 72 hours after inoculation (hai) with *Moniliophthora*
659 *perniciosa*. **A.** Full-scale representation. **B.** Reduced scale for the best visualization of
660 lower relative expression.

661 **Figure 5.** Putative scheme of the early steps (6–72 hai) of the interactions between *T. grandiflorum* plants and the hemibiotrophic fungus *M. perniciosa*. **A.** Resistant interaction. **B.** Susceptible interaction. PTI: PAMP-triggered immunity; ETI: effector-triggered immunity; PRR: pattern-recognition receptors; PR: pathogenesis-related protein; PCD: programmed cell death.
A

<figure><img src="image_2.png" /></figure>

3.0

C

<figure><img src="image_3.png" /></figure>

0.7

B

<figure><img src="image_4.png" /></figure>

2.0
A

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

B

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

C

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

TgPR3

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

TgPR5

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

TgPR8

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

Chitosan

<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>

<figure><img src="image_16.png" /></figure>

H-Bonds

Donor

Acceptor

Interactions

<figure><img src="image_17.png" /></figure>

Conventional Hydrogen Bond

<figure><img src="image_18.png" /></figure>

Carbon Hydrogen Bond

<figure><img src="image_19.png" /></figure>

Pi-Alkyl

<figure><img src="image_20.png" /></figure>

Unfavorable Donor-Donor

<figure><img src="image_21.png" /></figure>

Pi-Sigma
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_25.png" /></figure>