BACKGROUND
the production and accumulation of pathogenesis-related proteins  in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense
<cit> .
the pr proteins are defined as plant proteins induced in pathological situations not necessary implied with a direct interaction with the pathogen
<cit> .
however, several characterized pr proteins showed enzymatic activities related to antimicrobial properties.
among the  <dig> pr protein families already described, at least  <dig> present enzymatic activity such glucanases , osmotins and thaumatins , protease inhibitors , lysozymes , peroxidase , ribonucleases  and chitinases .
focusing on pr- <dig> proteins, several studies showed the involvement of this family in plant defense responses regulated by signal molecules, such as salicylic acid , abscissic acid , jasmonate  and ethylene .
in rice, maize and wheat, the pr- <dig> gene expression was induced in the first hours  after the contact with the pathogenic fungus and/or the fungal elicitors
<cit> .
moreover, it has been shown that the activation of wheat pr- <dig> genes depends on both sa and ja dependent metabolic pathways
<cit> .
in dicotyledones, the induction of the pr- <dig> gene expression was also observed in the initial stages of disease in the case of red pepper infected by the pepper mild mottle virus <cit>  or tobacco infected by the tabacco mosaic virus <cit> , as well as in response to ethephon – an et-releasing compound in chinese cabbage
<cit> .
in apple, the mdpr- <dig> gene expression was associated to the plant defense response against botryosphaeria dothidea, through signalization pathways dependent of sa and ja, as well as to several physiological functions such as flower formation
<cit> .
the presence of a c-terminal conserved barwin domain – first identified in proteins obtained from barley seeds, and containing  <dig> conserved cystein residues forming disulfide bridges
<cit>  – corresponds to the characteristic structure of the pr- <dig> proteins.
moreover, most of the pr- <dig> proteins have a signal peptide and some of them show transmembrane structure in the n-terminal region
<cit> .
some pr- <dig> proteins also present a c-terminal extension domain involved in protein targeting to the vacuole
<cit> .
it has been shown that the barley barwin protein is able to slightly interact with the oligosaccharide β- tetramer of n-acetylglucosamine, an analog of chitin
<cit> .
the pr- <dig> protein classification is based on the presence  or absence  of the chitin ligation domain
<cit>  also known as hevein-like domain – from the name of hevein, a small antifungal molecule present in hevea brasilienses latex
<cit> .
the pr- <dig> proteins could show chitinase, rnase and/or dnase activities as well as antifungal properties; these functions could be related  one to another and have been detected in both class i and ii proteins.
the class i tobacco pr- <dig> protein ntcbp <dig> showed antifungal activity against trichoderma viride and fusarium solani by causing germinative tube lyses and fungal growth inhibition
<cit> .
in fig, the class i fapr- <dig> protein as well as its truncated form fapr-4c – lacking the n-terminal region to mimic a class ii pr- <dig> – showed rnase, chitinase and antifungal activity
<cit> .
however, some pr- <dig> only present rnase and antifungal activities
<cit>  or rnase and dnase activity but no chitinase property
<cit> .
these characteristics may also be observed in multigene family members; in wheat, four class ii pr- <dig>  were isolated, and all of them showed antifungal activity
<cit> .
moreover the recombinant protein wheatwin <dig> showed rnase activity, and a site-directed mutagenesis experiment revealed that the reduction of rnase activity was correlated to the loss of wheatwin <dig> antifungal properties – analyzed on mycelial growth
<cit> .
despite the importance of the pr proteins in plant defense, only few members of this family, tcpr- <dig>  tcpr <dig> and tcpr- <dig>  have been previously fully characterized in theobroma cacao <cit> .
two tcpr- <dig>  named tcpr-1f and tcpr-1g, were identified and analyzed at phylogenetic and expression levels.
the tcpr-1g gene was up-regulated during the biotrophic stage of the infection of susceptible cacao genotype by the basidiomycete moniliophthora perniciosa <cit>  – causal agent of the witches’ broom disease, one the most devastating diseases of cacao trees
<cit> .
the tcpr <dig> gene encodes an osmotin-like protein identified in leaves and roots from cacao ‘comun’  sumitted to drought, indicating its involvement in tolerance to osmotic stress
<cit> .
in the case of tcpr- <dig>  this gene was related to defense responses in the final stages of the witches’ broom disease.
the corresponding tcpr- <dig> protein also presents rnase enzymatic activity and antifungal action against m. perniciosa and saccharomyces cerevisiae <cit> .
here, we described another pr protein, the tcpr-4b, which was identified from a cacao pod-m. perniciosa interaction cdna library  and from a cacao meristem-m. perniciosa interaction library
<cit> .
first in silico and semi-quantitative rt-pcr analysis showed that pr4-b genes from cacao were differentially expressed between resistant and susceptible cacao varieties submitted to m. perniciosa infection
<cit> .
the characterization of the tcpr-4b and the cacao pr- <dig> family has been facilitated by the recent publication of the t. cacao genome
<cit> .
using bioinformatics, molecular biology and biochemistry, we showed that tcpr-4b belonged to a small multigene family and was differentially expressed in resistant and susceptible cacao genotypes infected vs non infected by m. perniciosa.
moreover, to our knowledge, this is the first report of a plant pr- <dig> that presents both rnase, dnase and antifungal properties but no chitinase activity.
this work also describes for the first time a complete pr- <dig> gene family in cacao, an important crop that may be used as plant model of fruit tree according to recent studies
<cit> .
finally, our paper shows that tcpr-4b may be a good candidate for biotechnological or molecular genetics control strategies of the witches’ broom disease in cacao.
RESULTS
sequence analysis of tcpr-4b
the tcpr-4b gene identified from t. cacao-m. perniciosa interaction library presents an orf of  <dig> nucleotides encoding a protein of  <dig> amino acid residues .
a hydrophobic region was observed at the n-terminal of the protein which may correspond to a transmembrane n-terminal helix or a signal peptide
<cit> .
a detailed analysis using the tmhmma server  <dig>  did not reveal any transmembrane helix and the signalp  <dig>  server predicted a signal peptide with a putative cleavage site between the a <dig> and q <dig> .
the tcpr-4b protein with signal peptide has a putative molecular mass of  <dig>  kda and a putative pi of  <dig> , while the protein without signal peptide has a molecular mass of  <dig>  kda and a putative pi of  <dig> .
only one phosphorylation site  was observed .
no glycosylation and acetylation site was found in the tcpr-4b sequence .
the tcpr-4b contains the functional barwin domain  .
the tcpr-4b amino acid sequence presents six conserved cystein residues  characteristic of the barwin domain and responsible for disulfide bond formation .
tcpr-4b does neither present chitin-binding domain nor c-terminal conserved domain – which corresponds to a signal for protein addressing to the vacuole
<cit>  .
the analysis of the tcpr-4b domains by comparison with other class i and pr-4s from various mono- and dicotyledonous species, showed that tcpr4-b presents a high identity with class ii pr-4s from other plant species  such as malus domestica , nicotiana tabacum , solanum lycopersicum , capsicum annuum , capsicum chinense , triticum aestivum  and lycoris radiate .
the blastn analysis of tcpr-4b against the cocoagenbd from t. cacao, detected a single gene located on the chromosome  <dig> and showing 100% of identity with tcpr-4b.
the complete tcpr-4b gene sequence  was 802 bp in length and contained  <dig> exons of  <dig> and 258 bp, and  <dig> intron of 82 bp .
tblastn search in the cocoagenbd also revealed the presence of  <dig> more pr- <dig> present in the t. cacao genome .
four of them  showed  <dig> barwin domains, two of them  contained one barwin and one chitin-binding domains, and one protein sequence  showed a unique barwin domain .
only one pr- <dig> protein from cacao  presented a transmembrane helix at the n-terminal end of the protein while the others sequences, including tcpr-4b, presented a signal peptide .
except for tg05_g <dig> and tcpr-4b, the pr- <dig> proteins from cacao contained a vacuolar signal .
the pr- <dig> proteins from t. cacao presented  <dig>  to  <dig> % of identity with tcpr-4b .
five of the  <dig> cacao pr- <dig> genes  were located on the chromosome  <dig>  one on the chromosome  <dig>  and the last one  did not present a defined localization .
the analysis of the chromosome  <dig> structure showed that the  <dig> pr- <dig> genes were organized in tandem in the  strand of the chromosome in a region corresponding approximately to  <dig> %  of the total chromosome length .
nd: non determined; sp: signal peptide; chtbd: chitin-binding domain; vs: vacuolar signal.
aprotein modified by genetic engineering.
phylogeny of t. cacao pr-4
the class i pr- <dig> proteins were grouped in a unique clade, and except for tc00g_ <dig>  all the proteins had a vacuolar signal .
the class ii proteins from dicotyledons as well as monocotyledons had no vacuolar signal, except for the cacao proteins that contain  <dig> barwin domains; these last ones  formed a separated clade.
the tcpr-4b protein had a higher identity with the class i sequences tg05_g <dig>  and tg05_g <dig>  .
tcpr-4b is also closer to tg05_g <dig> in relation to the genic organization; one and no introns were identified for tcpr-4b and tg05_g <dig>  respectively .
in the phylogenetic analysis, the protein tcpr-4b is closer to class i than from class ii proteins with two barwin domains .
molecular modeling of tcpr-4b
the alignment of the amino acid sequence of tcpr-4b with the barley barwin protein  presented  <dig> % of identity and 89% of similarity , and a rmsd of  <dig>  Å. identity above 50% and e-value below  <dig> - <dig> indicate that the barwin protein is a good model to be used as a template.
the molecular modeling of tcpr-4b  showed i)  <dig> α-helices, α <dig> , α <dig>  and α <dig> ; ii)  <dig> β-sheets, β <dig> , β <dig> , β <dig>  and β <dig> ; and iii)  <dig> loops l <dig> , l <dig> , l <dig> , l <dig> , l <dig> , l <dig> , l <dig>  and l <dig>  .
the validation analysis  of the tcpr-4b model presented  <dig> % of residues in most favored regions,  <dig> % in additional allowed regions and only 1% in disallowed regions indicating that 99% of amino acid residues are located in favored regions .
in addition, psipred validation revealed reasonable secondary structure, and anolea showed good energy values.
the conservation of the cysteins along the pr- <dig> polypeptides  highlights the importance of these residues to maintain the tridimensional structure of the protein
<cit> .
in the tcpr-4b, the cystein residues formed three disulfide bonds c49-c <dig>  c70-c <dig> and c84-c <dig> .
the phosphorylation site t <dig> is located in a β-sheet, on the molecule superficies, i.e.
in a region accessible to addition of a phosphate group .
in pr-4s, the conserved h <dig>  h <dig>  d <dig> and r <dig> are considered to be critical for antifungal and ribonuclease activity
<cit> .
in tcpr-4b the conserved residues h <dig>  h <dig>  d <dig> and r <dig> were observed; h <dig> and d <dig> are located in loops – d <dig> being more internal than h <dig> located at the surface of the protein .
the amino acids h <dig> and r <dig> formed a β-sheet at the molecular surface, i.e.
in an accessible region with moderate flexibility .
the molecular modeling of tcpr-4b also revealed the presence of a conserved region 35-41/peqnnwd located in a loop exposed on the molecule surface, i.e.
in accessible region with high flexibility .
on the contrary, the region 109-111/ldl, which also formed a loop, was located in the internal part of the protein .
expression of tcpr-4b in resistant and susceptible theobroma cacao genotypes
the expression of the tcpr-4b gene was analyzed in two cacao genotypes, tsh <dig>  and catongo  infected or not  with m. perniciosa .
for both genotypes and for all the harvesting point, the pcr amplification occurred at the same and unique melting temperature showing that only the tcpr-4b gene was amplified .
in both genotypes, the tcpr-4b expression was low at  <dig> hai, increased at  <dig> hai  and then decreased at  <dig> hai .
at  <dig> hai, the tcpr-4b expression was about  <dig> times higher in tsh <dig> than in catongo.
from  <dig> to  <dig> dai, tcpr-4b was repressed in catongo and overexpressed in tsh <dig> in comparison to the control; the expression in tsh <dig> was about  <dig>   <dig> and  <dig> times higher than the one observed in catongo at  <dig>   <dig> and  <dig> dai, respectively .
from  <dig> to  <dig> dai, tcpr-4b was superexpressed in both genotypes in comparison to the control .
except at  <dig> dai, the expression was higher in tsh <dig> than in catongo .
at  <dig> dai, the tcpr-4b expression was about  <dig>  higher in catongo than in tsh <dig>
production of the recombinant tcpr-4b protein
the tcpr-4b gene was cloned in pet28a plasmid and was successfully expressed in escherichia coli rosetta  while no visible band was observed in the controls  .
the induction of the protein expression was higher at 18°c than at 37°c .
for this reason, the conditions of  <dig>  m of iptg and 18°c  were established as the best conditions of tcpr-4b induction.
the recombinant protein had a molecular mass of 15 kda, close to the one predicted .
the tcpr-4b was successfully purified with a talon resin metal-affinity column .
ribonuclease and ca+ <dig> and mg+ <dig> dependent deoxyribonuclease activities
the nuclease function of the purified recombinant tcpr-4b was analyzed by incubation of the protein with rna or dna and visualization of the nucleic acid degradation patterns on agarose gels .
tomato rna degradation  was observed after 30 min of incubation with tcpr-4b; the degradation was more important when the amount of the purified recombinant protein increased .
the rna degradation pattern in the presence of 25 μg of tcpr-4b was similar to the positive control degradation obtained in samples incubated with commercial rnase a .
the negative controls showed no degradation  or very slight degradation smear .
moreover, no degradation was observed in presence of 15 μg of tcpr-4b plus rnase inhibitor , corroborating that the recombinant tcpr-4b had a ribonuclease activity.
plasmidial as well as genomic dna degradation was observed after overnight incubation with tcpr-4b plus 10 mm mgcl <dig>  or 1 mm of cacl <dig> .
no degradation was observed in presence of tcpr-4b without mgcl <dig> or cacl <dig> .
the degradation was more important when the amount of tcpr-4b increased .
the negative controls  showed no degradation .
samples of tcpr-4b plus mgcl <dig> incubated with 10 mm of edta , showed no dnase activity .
no rna or dna degradation was observed using the extract of bacteria containing the pet <dig> vector without insert , avoiding the possible action of some bacterial component on the obtained results .
chitinase activity
the chitinase activity of tcpr-4b was tested in two different conditions of ph using the buffera/ph  <dig>  and buffer b/ph  <dig>  .
regardless of the buffer used, tcpr-4b did not show any activity in comparison to the blank , while the protein extract from cacao meristems presented a chitinase activity between  <dig>  to  <dig>  u/h depending of the buffer and protein concentration used .
moreover, the tcpr-4b amount  did not affect the results .
antifungal activity of tcpr-4b
to test the antifungal activity of tcpr-4b on m. perniciosa, increasing concentrations of the recombinant protein  were incubated with dikaryotic broken hyphae of m. perniciosa.
the reduction of m. perniciosa survival was observed in all tested concentrations of tcpr-4b with a decrease of survival correlated to the increase of the protein concentration .
in presence of 5 μg/ml of tcpr-4b the fungus survival did not present any difference in comparison to control .
at  <dig>   <dig> and 40 μg/ml of tcpr-4b, the survival decreased significantly and reached  <dig> % in presence of 40 μg/ml of protein.
the ros production in living m. perniciosa hyphae treated with tcpr-4b was evaluated by incubation with dhe which selectively stains the mitochondrial superoxide .
the hyphae treated with tcpr-4b presented a bright red fluorescence  with more specific intense fluorescence in some foci .
the control  presented few or no fluorescence in comparison to the hyphae treated with tcpr-4b .
the action of tcpr-4b  on m. perniciosa survival was tested in presence  of rnase inhibitor .
in presence of tcpr-4b plus rnase inhibitor the survival was high  and statistically different from the survival in presence of tcpr-4b only .
the m. perniciosa survival in presence of tcpr-4b plus rnase inhibitor was similar as the fungus survival in presence of the controls .
the m. perniciosa survival in presence of tcpr-4b with or without mgcl <dig> was similar and low .
discussion
in this article, we characterized a pathogenesis-related protein 4b from theobroma cacao  previously identified in cacao-m. perniciosa interaction cdna libraries.
the tcpr-4b protein presented a barwin domain – highly conserved among the pr-4s – but no chitin-binding domain .
in comparison to the other six pr-4s from cacao, the tcpr-4b contains only one barwin domain and no vacuolar signal .
but the sequence analysis revealed that tcpr-4b contains a signal peptide, suggesting that it is an extracellular protein targeted to the apoplasm .
tcpr-4b showed high identity with class ii pr-4s from other plant species .
the enzymatic tests carried out with the recombinant tcpr-4b revealed that this protein presented both dnase and rnase activities .
to our knowledge, this is the second report of pr- <dig> with these two functions .
the tcpr-4b dnase activity is dependent of bivalent ions, mg2+ and ca2+ since chelation or absence of these ions prevent any dnase activity.
in some studies, such as in the case of capsicum chinense class ii pr- <dig> , the dnase catalytic function is independent of mg2+, but addition of these ions reinforced the dnase activity
<cit> .
on the contrary, the rnase activity of tcpr-4b was independent of bivalent ions, suggesting probable different catalytic sites for the two nuclease actions.
as observed in other works
<cit> , the rnase activity of tcpr-4b was inhibited by heating and in the presence of rnase inhibitor  which is able to annul the activity of type a, b and c rnases.
however, some exceptions were reported, for example, the ccpr- <dig> seemed to have a rnase activity mechanism different from those of the type a, b and c rnases
<cit> .
moreover, for class i pr-4 s, as fapr <dig>  the presence of the chitin-binding domain may contribute to the thermal stability of the protein avoiding the lack of rnase activity when the protein was submitted to heating
<cit> .
the antifungal activity of tcpr-4b was verified in vitro on m. perniciosa hyphae  and was associated to the increase of mitochondrial o2- production detected by dhe .
the high production of o2- should have altered the mitochondrial complex leading to oxidative stress, damage of dna, lack of cellular functions, apoptosis or necrosis, and consequently inhibition of fungal growth.
the action mechanism of the pr- <dig> probably involved the arg <dig>  his <dig>  asp <dig>  his <dig> residues and the 15-paqnnwd- <dig> region that are considered crucial for rnase and antifungal activities
<cit> .
the structural model of tcpr-4b showed that the his <dig>  his <dig>  arg <dig> residues and the region 35-peqnnwd- <dig> are present in accessible regions of the protein; only the asp <dig> amino acid had a more internal localization .
because of the accessibility of most of these residues in a highly flexible molecular structure, they may correspond, in tcpr-4b, to the interaction points with target molecules from m. perniciosa, and thus may be considered as good candidate for directed-site mutation studies.
the antifungal activity is common in different pr families, regardless of the specific enzymatic function of each of them
<cit> .
in the case of the pr- <dig> family, the presence or absence of chitin-binding domain is not correlated with the antifungal action; class i
<cit>  as well as class ii
<cit>  pr- <dig> proteins presented antifungal activity .
moreover, the presence of this domain is not associated to the chitinolytic function of the pr- <dig> proteins  as observed in the case of athel
<cit>  and fapr-4c
<cit> .
the fapr-4c protein, a truncated form of a class i protein obtained to mimic a class ii pr- <dig>  presented the same chitinases, rnase and antifungal activity as the original non truncated fapr-4
<cit> .
moreover, the chitinase function was demonstrated for a very few number of pr-4s ; here the tcpr-4b neither presented chitin-binding domain nor chitinase activity , but presented antifungal action against m. perniciosa .
these data indicate that the classification of pr- <dig> as chitinases is inadequate and could be questioned, as previously suggested
<cit> .
the doubts about the pr- <dig> protein function in relation to the presence/absence of the chitin-binding domain raises questions about the evolutionary origin of this domain.
there is no consensus as to whether the chitin-binding domain was incorporated in class ii proteins that contained a unique barwin domain, or if the chitin-binding domain was present in all sequences and was lost later on.
phylogenetic analysis suggested that the class i genes evolved after the incorporation of the n-terminal domain in class ii genes
<cit> .
but other authors highlighted the hypothesis that the class ii derived from class i after the lack of the hevein domain
<cit> .
here, the formation of a single clade only with most related pr <dig> sequences of t cacao, with duplicate barwin domain and vacuolar signal, indicates a possible single origin for these molecules .
nevertheless, we find other t cacao pr <dig> grouped in class i, along with sequences of other fungal species.
this last fact may indicate two evolutionary situations: pr <dig> class ii emerged from a common class i ancestral, which lost the ability chitin binding, or a class ii ancestral gave rise to two proteins of class i, tc <dig> g <dig> tc <dig> g <dig>  which acquired the ability to bind to chitin.
considering that the interaction between m. perniciosa and t. cacao, during the various stages of growth of this fungus, is intense and evolutionarily ancient, it is possible that class ii molecules in t cacao have given rise to class i.
moreover, we showed that the antifungal activity of tcpr-4b is directly dependent of its rnase activity; the survival of m. perniciosa colonies was higher in presence of tcpr-4b plus rnase inhibitor than in presence of tcpr-4b only .
experiment involving the tcpr-4b with or without mgcl <dig>  did not show any difference, suggesting that the antifungal activity does not depend of the dnase activity of the protein .
the association between the antifungal and rnase activity has been proposed for the pr- <dig> family.
directed site-mutations of the wheatwin  <dig> protein  on the conserved amino acids h <dig> and h <dig>  considered as crucial for rnase activity, reduced the wheatwin  <dig> capacity to degrade rna; in the double mutant h11l/h113l the reduction was higher than in the simple mutants
<cit> .
the absence of rnase actitivy due to the mutation insertion was correlated with the reduction of antifungal potential
<cit> .
the mdpr- <dig> protein also presented a correlated reduction of the rnase and the antifungal activities after treatment with a rnase inhibitor
<cit> .
these evidences suggested that the antifungal activity depends on the rnase activity.
moreover, in the case of the wheatwin  <dig> protein, it has been shown that the protein is able to enter to the cytoplasm of the fungal cell and act as rnase without cell membrane destruction
<cit> .
pr proteins are known to be involved in plant responses to infection by pathogens, and some pr- <dig> are more specifically associated to responses to fungal pathogens
<cit> .
here, we analyzed the expression of the tcpr-4b gene in resistant and susceptible cacao genotypes infected or not with m. perniciosa.
at the early stages of the infection, the global tcpr-4b expression pattern was similar between resistant and susceptible infected plants even if a significant difference was observed at  <dig> hai between the two genotypes .
this early expression may be considered as the first plant reaction against the pathogen in both genotypes, working as an induced defense mechanism against m. perniciosa basidiospore germination, and hyphae penetration and progression.
the apoplastic addressing of the tcpr-4b , together with its nuclease activities , suggest that these two functions may be related to the antifungal activity of the protein  mainly during the biotrophic phase of the disease, when m. perniciosa grown intercellularly
<cit> .
the higher intensity of the tcpr-4b expression in the resistant tsh <dig> plants  may contribute to the reduction of m. perniciosa hyphae penetration and progression in this genotype
<cit> .
in susceptible cacao meristems, m. perniciosa hyphae were detected in the intercellular space of the sub-epidermis layer  <dig> hai while in the resistant genotype no or few fungal penetration was observed
<cit> .
for most of the pathosystems, the response of plant resistance or susceptibility is more associated to differences of time/velocity and magnitude/intensity of some gene expression than to difference in gene set composition
<cit> .
for example, during the arabidopsis-colletotrichum higginsianum interaction, the pr- <dig> expression was detected in both resistant and susceptible inoculated plants.
in both genotypes, the expression increased through the time course of the disease, but the highest pr- <dig> expression was observed at  <dig> hai in the resistant genotype and only  <dig> dai in the susceptible one
<cit> .
this result was in accordance with literature data showing that, generally, a higher pr protein accumulation and/or an earlier gene expression was observed in the incompatible interaction rather than in the compatible one
<cit> .
after the initial stage, in the resistant cacao plants, the tcpr-4b expression was observed in all the samples with a relatively high level  while a gene repression was observed from  <dig> to  <dig> dai in susceptible plants  followed by an increase of expression from  <dig> to  <dig> dai .
in the resistant cacao genotype, even if no macroscopic symptom were observed, some metabolic alterations have been related after inoculation of the plants by m. perniciosa <cit> .
among them, h2o <dig> production associated to calcium oxalate crystal  dissolution as well as expression of genes involved in ca2+ dependent-signalization were observed at the initial stages after inoculation
<cit> .
moreover, a germin-like oxalate oxidase – responsible for coc dissolution – was expressed constantly in the resistant cacao genotype, making a high ca2+ amount available a long time after the plant has been in contact with the pathogen
<cit> .
in the susceptible plants, cocs were accumulated in cortex during the biotrophic phase  and dissolved during the transition to the necrotrophic phase
<cit> .
moreover, the necrophic phase is characterized in the susceptible plant by a nuclear dna degradation associated to programmed cell death
<cit> .
considering that the tcpr-4b dnase activity is ca2+-dependent, the high and almost constant expression of the corresponding gene in the tsh <dig> genotype from  <dig> to  <dig> dai, could reflect the role of tcpr-4b in a protection mechanism against further foreign invading pathogens, as suggested for pr- <dig> from other plants
<cit> .
in the catongo plants, the tcpr-4b expression coincided with the coc dissolution during biotrophic/necrotrophic phase transition as well as to the nuclear dna degradation and ladder formation during the necrotrophic phase
<cit>  suggesting that in this genotype, the tcpr-4b may act as one of the element of the pcd process.
the increase of nuclease activity, some of them ca2+ and mg2+ dependent have been identified in plants during pcd processes
<cit> .
for example, in wheat seeds the highest accumulation of pr- <dig> occurred in period concomitant with pcd of the endosperm
<cit> .
in conclusion, in cacao, the coc dissolution which occurred in both resistant and susceptible genotypes led to extracellular ca2+ availability that may be used by the tcpr-4b for its acting as dnase in resistance mechanisms or in pcd during the infection process.
this may be reinforced by the fact that the calcium availability is higher in the apoplasm in normal cells, while an influx of calcium in the intracellular area occurred during pcd
<cit> .
CONCLUSIONS
we reported the first evidence of a pr- <dig> with a dual dnase and rnase activity together with an antifungal activity.
to our knowledge, this is the first report of pr- <dig> showing these three functions simultaneously, but no chitinase activity .
moreover, we showed that the antifungal activity of tcpr-4b is directly related to the rnase function – and not to the dnase one .
these functions could be related to the role of the tcpr- <dig> during the cacao-m. perniciosa interaction, contributing to the maintenance of the symptom reduction in the resistant genotype and to the pcd mechanism in susceptible one.
thus, the tcpr-4b could be considered as a good candidate for biotechnological applications in order to detain witches’ broom disease.

 the production and accumulation of pathogenesis-related proteins in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. the pr proteins are defined as plant proteins induced in pathological situations not necessary implied with a direct interaction with the pathogen. however, several characterized pr proteins showed enzymatic activities related to antimicrobial properties. among the pr protein families already described, at least present enzymatic activity such glucanases, osmotins and thaumatins, protease inhibitors, lysozymes, peroxidase, ribonucleases and chitinases. focusing on pr- proteins, several studies showed the involvement of this family in plant defense responses regulated by signal molecules, such as salicylic acid, abscissic acid, jasmonate and ethylene. in rice, maize and wheat, the pr- gene expression was induced in the first hours after the contact with the pathogenic fungus and/or the fungal elicitors. moreover, it has been shown that the activation of wheat pr- genes depends on both sa and ja dependent metabolic pathways. in dicotyledones, the induction of the pr- gene expression was also observed in the initial stages of disease in the case of red pepper infected by the pepper mild mottle virus or tobacco infected by the tabacco mosaic virus, as well as in response to ethephon – an et-releasing compound in chinese cabbage. in apple, the mdpr- gene expression was associated to the plant defense response against botryosphaeria dothidea, through signalization pathways dependent of sa and ja, as well as to several physiological functions such as flower formation. the presence of a c-terminal conserved barwin domain – first identified in proteins obtained from barley seeds, and containing conserved cystein residues forming disulfide bridges – corresponds to the characteristic structure of the pr- proteins. moreover, most of the pr- proteins have a signal peptide and some of them show transmembrane structure in the n-terminal region. some pr- proteins also present a c-terminal extension domain involved in protein targeting to the vacuole. it has been shown that the barley barwin protein is able to slightly interact with the oligosaccharide β- tetramer of n-acetylglucosamine, an analog of chitin. the pr- protein classification is based on the presence or absence of the chitin ligation domain also known as hevein-like domain – from the name of hevein, a small antifungal molecule present in hevea brasilienses latex. the pr- proteins could show chitinase, rnase and/or dnase activities as well as antifungal properties; these functions could be related one to another and have been detected in both class i and ii proteins. the class i tobacco pr- protein ntcbp showed antifungal activity against trichoderma viride and fusarium solani by causing germinative tube lyses and fungal growth inhibition. in fig, the class i fapr- protein as well as its truncated form fapr-4c – lacking the n-terminal region to mimic a class ii pr- – showed rnase, chitinase and antifungal activity. however, some pr- only present rnase and antifungal activities or rnase and dnase activity but no chitinase property. these characteristics may also be observed in multigene family members; in wheat, four class ii pr- were isolated, and all of them showed antifungal activity. moreover the recombinant protein wheatwin showed rnase activity, and a site-directed mutagenesis experiment revealed that the reduction of rnase activity was correlated to the loss of wheatwin antifungal properties – analyzed on mycelial growth. despite the importance of the pr proteins in plant defense, only few members of this family, tcpr- tcpr and tcpr- have been previously fully characterized in theobroma cacao. two tcpr- named tcpr-1f and tcpr-1g, were identified and analyzed at phylogenetic and expression levels. the tcpr-1g gene was up-regulated during the biotrophic stage of the infection of susceptible cacao genotype by the basidiomycete moniliophthora perniciosa – causal agent of the witches’ broom disease, one the most devastating diseases of cacao trees. the tcpr gene encodes an osmotin-like protein identified in leaves and roots from cacao ‘comun’ sumitted to drought, indicating its involvement in tolerance to osmotic stress. in the case of tcpr- this gene was related to defense responses in the final stages of the witches’ broom disease. the corresponding tcpr- protein also presents rnase enzymatic activity and antifungal action against m. perniciosa and saccharomyces cerevisiae. here, we described another pr protein, the tcpr-4b, which was identified from a cacao pod-m. perniciosa interaction cdna library and from a cacao meristem-m. perniciosa interaction library. first in silico and semi-quantitative rt-pcr analysis showed that pr4-b genes from cacao were differentially expressed between resistant and susceptible cacao varieties submitted to m. perniciosa infection. the characterization of the tcpr-4b and the cacao pr- family has been facilitated by the recent publication of the t. cacao genome. using bioinformatics, molecular biology and biochemistry, we showed that tcpr-4b belonged to a small multigene family and was differentially expressed in resistant and susceptible cacao genotypes infected vs non infected by m. perniciosa. moreover, to our knowledge, this is the first report of a plant pr- that presents both rnase, dnase and antifungal properties but no chitinase activity. this work also describes for the first time a complete pr- gene family in cacao, an important crop that may be used as plant model of fruit tree according to recent studies. finally, our paper shows that tcpr-4b may be a good candidate for biotechnological or molecular genetics control strategies of the witches’ broom disease in cacao.  sequence analysis of tcpr-4b the tcpr-4b gene identified from t. cacao-m. perniciosa interaction library presents an orf of nucleotides encoding a protein of amino acid residues. a hydrophobic region was observed at the n-terminal of the protein which may correspond to a transmembrane n-terminal helix or a signal peptide. a detailed analysis using the tmhmma server did not reveal any transmembrane helix and the signalp server predicted a signal peptide with a putative cleavage site between the a and q. the tcpr-4b protein with signal peptide has a putative molecular mass of  kda and a putative pi of, while the protein without signal peptide has a molecular mass of  kda and a putative pi of. only one phosphorylation site was observed. no glycosylation and acetylation site was found in the tcpr-4b sequence. the tcpr-4b contains the functional barwin domain. the tcpr-4b amino acid sequence presents six conserved cystein residues characteristic of the barwin domain and responsible for disulfide bond formation. tcpr-4b does neither present chitin-binding domain nor c-terminal conserved domain – which corresponds to a signal for protein addressing to the vacuole. the analysis of the tcpr-4b domains by comparison with other class i and pr-4s from various mono- and dicotyledonous species, showed that tcpr4-b presents a high identity with class ii pr-4s from other plant species such as malus domestica, nicotiana tabacum, solanum lycopersicum, capsicum annuum, capsicum chinense, triticum aestivum and lycoris radiate. the blastn analysis of tcpr-4b against the cocoagenbd from t. cacao, detected a single gene located on the chromosome and showing 100% of identity with tcpr-4b. the complete tcpr-4b gene sequence was 802 bp in length and contained exons of and 258 bp, and intron of 82 bp. tblastn search in the cocoagenbd also revealed the presence of more pr- present in the t. cacao genome. four of them showed barwin domains, two of them contained one barwin and one chitin-binding domains, and one protein sequence showed a unique barwin domain. only one pr- protein from cacao presented a transmembrane helix at the n-terminal end of the protein while the others sequences, including tcpr-4b, presented a signal peptide. except for tg05_g and tcpr-4b, the pr- proteins from cacao contained a vacuolar signal. the pr- proteins from t. cacao presented to % of identity with tcpr-4b. five of the cacao pr- genes were located on the chromosome one on the chromosome and the last one did not present a defined localization. the analysis of the chromosome structure showed that the pr- genes were organized in tandem in the strand of the chromosome in a region corresponding approximately to % of the total chromosome length. nd: non determined; sp: signal peptide; chtbd: chitin-binding domain; vs: vacuolar signal. aprotein modified by genetic engineering. phylogeny of t. cacao pr-4 the class i pr- proteins were grouped in a unique clade, and except for tc00g_ all the proteins had a vacuolar signal. the class ii proteins from dicotyledons as well as monocotyledons had no vacuolar signal, except for the cacao proteins that contain barwin domains; these last ones formed a separated clade. the tcpr-4b protein had a higher identity with the class i sequences tg05_g and tg05_g. tcpr-4b is also closer to tg05_g in relation to the genic organization; one and no introns were identified for tcpr-4b and tg05_g respectively. in the phylogenetic analysis, the protein tcpr-4b is closer to class i than from class ii proteins with two barwin domains. molecular modeling of tcpr-4b the alignment of the amino acid sequence of tcpr-4b with the barley barwin protein presented % of identity and 89% of similarity, and a rmsd of  Å. identity above 50% and e-value below - indicate that the barwin protein is a good model to be used as a template. the molecular modeling of tcpr-4b showed i) α-helices, α, α and α; ii) β-sheets, β, β, β and β; and iii) loops l, l, l, l, l, l, l and l. the validation analysis of the tcpr-4b model presented % of residues in most favored regions, % in additional allowed regions and only 1% in disallowed regions indicating that 99% of amino acid residues are located in favored regions. in addition, psipred validation revealed reasonable secondary structure, and anolea showed good energy values. the conservation of the cysteins along the pr- polypeptides highlights the importance of these residues to maintain the tridimensional structure of the protein. in the tcpr-4b, the cystein residues formed three disulfide bonds c49-c c70-c and c84-c. the phosphorylation site t is located in a β-sheet, on the molecule superficies, i.e. in a region accessible to addition of a phosphate group. in pr-4s, the conserved h h d and r are considered to be critical for antifungal and ribonuclease activity. in tcpr-4b the conserved residues h h d and r were observed; h and d are located in loops – d being more internal than h located at the surface of the protein. the amino acids h and r formed a β-sheet at the molecular surface, i.e. in an accessible region with moderate flexibility. the molecular modeling of tcpr-4b also revealed the presence of a conserved region 35-41/peqnnwd located in a loop exposed on the molecule surface, i.e. in accessible region with high flexibility. on the contrary, the region 109-111/ldl, which also formed a loop, was located in the internal part of the protein. expression of tcpr-4b in resistant and susceptible theobroma cacao genotypes the expression of the tcpr-4b gene was analyzed in two cacao genotypes, tsh and catongo infected or not with m. perniciosa. for both genotypes and for all the harvesting point, the pcr amplification occurred at the same and unique melting temperature showing that only the tcpr-4b gene was amplified. in both genotypes, the tcpr-4b expression was low at hai, increased at hai and then decreased at hai. at hai, the tcpr-4b expression was about times higher in tsh than in catongo. from to dai, tcpr-4b was repressed in catongo and overexpressed in tsh in comparison to the control; the expression in tsh was about and times higher than the one observed in catongo at and dai, respectively. from to dai, tcpr-4b was superexpressed in both genotypes in comparison to the control. except at dai, the expression was higher in tsh than in catongo. at dai, the tcpr-4b expression was about higher in catongo than in tsh production of the recombinant tcpr-4b protein the tcpr-4b gene was cloned in pet28a plasmid and was successfully expressed in escherichia coli rosetta while no visible band was observed in the controls. the induction of the protein expression was higher at 18°c than at 37°c. for this reason, the conditions of  m of iptg and 18°c were established as the best conditions of tcpr-4b induction. the recombinant protein had a molecular mass of 15 kda, close to the one predicted. the tcpr-4b was successfully purified with a talon resin metal-affinity column. ribonuclease and ca+ and mg+ dependent deoxyribonuclease activities the nuclease function of the purified recombinant tcpr-4b was analyzed by incubation of the protein with rna or dna and visualization of the nucleic acid degradation patterns on agarose gels. tomato rna degradation was observed after 30 min of incubation with tcpr-4b; the degradation was more important when the amount of the purified recombinant protein increased. the rna degradation pattern in the presence of 25 μg of tcpr-4b was similar to the positive control degradation obtained in samples incubated with commercial rnase a. the negative controls showed no degradation or very slight degradation smear. moreover, no degradation was observed in presence of 15 μg of tcpr-4b plus rnase inhibitor, corroborating that the recombinant tcpr-4b had a ribonuclease activity. plasmidial as well as genomic dna degradation was observed after overnight incubation with tcpr-4b plus 10 mm mgcl or 1 mm of cacl. no degradation was observed in presence of tcpr-4b without mgcl or cacl. the degradation was more important when the amount of tcpr-4b increased. the negative controls showed no degradation. samples of tcpr-4b plus mgcl incubated with 10 mm of edta, showed no dnase activity. no rna or dna degradation was observed using the extract of bacteria containing the pet vector without insert, avoiding the possible action of some bacterial component on the obtained results. chitinase activity the chitinase activity of tcpr-4b was tested in two different conditions of ph using the buffera/ph  and buffer b/ph . regardless of the buffer used, tcpr-4b did not show any activity in comparison to the blank, while the protein extract from cacao meristems presented a chitinase activity between to u/h depending of the buffer and protein concentration used. moreover, the tcpr-4b amount did not affect the results. antifungal activity of tcpr-4b to test the antifungal activity of tcpr-4b on m. perniciosa, increasing concentrations of the recombinant protein were incubated with dikaryotic broken hyphae of m. perniciosa. the reduction of m. perniciosa survival was observed in all tested concentrations of tcpr-4b with a decrease of survival correlated to the increase of the protein concentration. in presence of 5 μg/ml of tcpr-4b the fungus survival did not present any difference in comparison to control. at and 40 μg/ml of tcpr-4b, the survival decreased significantly and reached % in presence of 40 μg/ml of protein. the ros production in living m. perniciosa hyphae treated with tcpr-4b was evaluated by incubation with dhe which selectively stains the mitochondrial superoxide. the hyphae treated with tcpr-4b presented a bright red fluorescence with more specific intense fluorescence in some foci. the control presented few or no fluorescence in comparison to the hyphae treated with tcpr-4b. the action of tcpr-4b on m. perniciosa survival was tested in presence of rnase inhibitor. in presence of tcpr-4b plus rnase inhibitor the survival was high and statistically different from the survival in presence of tcpr-4b only. the m. perniciosa survival in presence of tcpr-4b plus rnase inhibitor was similar as the fungus survival in presence of the controls. the m. perniciosa survival in presence of tcpr-4b with or without mgcl was similar and low. discussion in this article, we characterized a pathogenesis-related protein 4b from theobroma cacao previously identified in cacao-m. perniciosa interaction cdna libraries. the tcpr-4b protein presented a barwin domain – highly conserved among the pr-4s – but no chitin-binding domain. in comparison to the other six pr-4s from cacao, the tcpr-4b contains only one barwin domain and no vacuolar signal. but the sequence analysis revealed that tcpr-4b contains a signal peptide, suggesting that it is an extracellular protein targeted to the apoplasm. tcpr-4b showed high identity with class ii pr-4s from other plant species. the enzymatic tests carried out with the recombinant tcpr-4b revealed that this protein presented both dnase and rnase activities. to our knowledge, this is the second report of pr- with these two functions. the tcpr-4b dnase activity is dependent of bivalent ions, mg2+ and ca2+ since chelation or absence of these ions prevent any dnase activity. in some studies, such as in the case of capsicum chinense class ii pr-, the dnase catalytic function is independent of mg2+, but addition of these ions reinforced the dnase activity. on the contrary, the rnase activity of tcpr-4b was independent of bivalent ions, suggesting probable different catalytic sites for the two nuclease actions. as observed in other works, the rnase activity of tcpr-4b was inhibited by heating and in the presence of rnase inhibitor which is able to annul the activity of type a, b and c rnases. however, some exceptions were reported, for example, the ccpr- seemed to have a rnase activity mechanism different from those of the type a, b and c rnases. moreover, for class i pr-4 s, as fapr the presence of the chitin-binding domain may contribute to the thermal stability of the protein avoiding the lack of rnase activity when the protein was submitted to heating. the antifungal activity of tcpr-4b was verified in vitro on m. perniciosa hyphae and was associated to the increase of mitochondrial o2- production detected by dhe. the high production of o2- should have altered the mitochondrial complex leading to oxidative stress, damage of dna, lack of cellular functions, apoptosis or necrosis, and consequently inhibition of fungal growth. the action mechanism of the pr- probably involved the arg his asp his residues and the 15-paqnnwd- region that are considered crucial for rnase and antifungal activities. the structural model of tcpr-4b showed that the his his arg residues and the region 35-peqnnwd- are present in accessible regions of the protein; only the asp amino acid had a more internal localization. because of the accessibility of most of these residues in a highly flexible molecular structure, they may correspond, in tcpr-4b, to the interaction points with target molecules from m. perniciosa, and thus may be considered as good candidate for directed-site mutation studies. the antifungal activity is common in different pr families, regardless of the specific enzymatic function of each of them. in the case of the pr- family, the presence or absence of chitin-binding domain is not correlated with the antifungal action; class i as well as class ii pr- proteins presented antifungal activity. moreover, the presence of this domain is not associated to the chitinolytic function of the pr- proteins as observed in the case of athel and fapr-4c. the fapr-4c protein, a truncated form of a class i protein obtained to mimic a class ii pr- presented the same chitinases, rnase and antifungal activity as the original non truncated fapr-4. moreover, the chitinase function was demonstrated for a very few number of pr-4s; here the tcpr-4b neither presented chitin-binding domain nor chitinase activity, but presented antifungal action against m. perniciosa. these data indicate that the classification of pr- as chitinases is inadequate and could be questioned, as previously suggested. the doubts about the pr- protein function in relation to the presence/absence of the chitin-binding domain raises questions about the evolutionary origin of this domain. there is no consensus as to whether the chitin-binding domain was incorporated in class ii proteins that contained a unique barwin domain, or if the chitin-binding domain was present in all sequences and was lost later on. phylogenetic analysis suggested that the class i genes evolved after the incorporation of the n-terminal domain in class ii genes. but other authors highlighted the hypothesis that the class ii derived from class i after the lack of the hevein domain. here, the formation of a single clade only with most related pr sequences of t cacao, with duplicate barwin domain and vacuolar signal, indicates a possible single origin for these molecules. nevertheless, we find other t cacao pr grouped in class i, along with sequences of other fungal species. this last fact may indicate two evolutionary situations: pr class ii emerged from a common class i ancestral, which lost the ability chitin binding, or a class ii ancestral gave rise to two proteins of class i, tc g tc g which acquired the ability to bind to chitin. considering that the interaction between m. perniciosa and t. cacao, during the various stages of growth of this fungus, is intense and evolutionarily ancient, it is possible that class ii molecules in t cacao have given rise to class i. moreover, we showed that the antifungal activity of tcpr-4b is directly dependent of its rnase activity; the survival of m. perniciosa colonies was higher in presence of tcpr-4b plus rnase inhibitor than in presence of tcpr-4b only. experiment involving the tcpr-4b with or without mgcl did not show any difference, suggesting that the antifungal activity does not depend of the dnase activity of the protein. the association between the antifungal and rnase activity has been proposed for the pr- family. directed site-mutations of the wheatwin protein on the conserved amino acids h and h considered as crucial for rnase activity, reduced the wheatwin capacity to degrade rna; in the double mutant h11l/h113l the reduction was higher than in the simple mutants. the absence of rnase actitivy due to the mutation insertion was correlated with the reduction of antifungal potential. the mdpr- protein also presented a correlated reduction of the rnase and the antifungal activities after treatment with a rnase inhibitor. these evidences suggested that the antifungal activity depends on the rnase activity. moreover, in the case of the wheatwin protein, it has been shown that the protein is able to enter to the cytoplasm of the fungal cell and act as rnase without cell membrane destruction. pr proteins are known to be involved in plant responses to infection by pathogens, and some pr- are more specifically associated to responses to fungal pathogens. here, we analyzed the expression of the tcpr-4b gene in resistant and susceptible cacao genotypes infected or not with m. perniciosa. at the early stages of the infection, the global tcpr-4b expression pattern was similar between resistant and susceptible infected plants even if a significant difference was observed at hai between the two genotypes. this early expression may be considered as the first plant reaction against the pathogen in both genotypes, working as an induced defense mechanism against m. perniciosa basidiospore germination, and hyphae penetration and progression. the apoplastic addressing of the tcpr-4b, together with its nuclease activities, suggest that these two functions may be related to the antifungal activity of the protein mainly during the biotrophic phase of the disease, when m. perniciosa grown intercellularly. the higher intensity of the tcpr-4b expression in the resistant tsh plants may contribute to the reduction of m. perniciosa hyphae penetration and progression in this genotype. in susceptible cacao meristems, m. perniciosa hyphae were detected in the intercellular space of the sub-epidermis layer hai while in the resistant genotype no or few fungal penetration was observed. for most of the pathosystems, the response of plant resistance or susceptibility is more associated to differences of time/velocity and magnitude/intensity of some gene expression than to difference in gene set composition. for example, during the arabidopsis-colletotrichum higginsianum interaction, the pr- expression was detected in both resistant and susceptible inoculated plants. in both genotypes, the expression increased through the time course of the disease, but the highest pr- expression was observed at hai in the resistant genotype and only dai in the susceptible one. this result was in accordance with literature data showing that, generally, a higher pr protein accumulation and/or an earlier gene expression was observed in the incompatible interaction rather than in the compatible one. after the initial stage, in the resistant cacao plants, the tcpr-4b expression was observed in all the samples with a relatively high level while a gene repression was observed from to dai in susceptible plants followed by an increase of expression from to dai. in the resistant cacao genotype, even if no macroscopic symptom were observed, some metabolic alterations have been related after inoculation of the plants by m. perniciosa. among them, h2o production associated to calcium oxalate crystal dissolution as well as expression of genes involved in ca2+ dependent-signalization were observed at the initial stages after inoculation. moreover, a germin-like oxalate oxidase – responsible for coc dissolution – was expressed constantly in the resistant cacao genotype, making a high ca2+ amount available a long time after the plant has been in contact with the pathogen. in the susceptible plants, cocs were accumulated in cortex during the biotrophic phase and dissolved during the transition to the necrotrophic phase. moreover, the necrophic phase is characterized in the susceptible plant by a nuclear dna degradation associated to programmed cell death. considering that the tcpr-4b dnase activity is ca2+-dependent, the high and almost constant expression of the corresponding gene in the tsh genotype from to dai, could reflect the role of tcpr-4b in a protection mechanism against further foreign invading pathogens, as suggested for pr- from other plants. in the catongo plants, the tcpr-4b expression coincided with the coc dissolution during biotrophic/necrotrophic phase transition as well as to the nuclear dna degradation and ladder formation during the necrotrophic phase suggesting that in this genotype, the tcpr-4b may act as one of the element of the pcd process. the increase of nuclease activity, some of them ca2+ and mg2+ dependent have been identified in plants during pcd processes. for example, in wheat seeds the highest accumulation of pr- occurred in period concomitant with pcd of the endosperm. in conclusion, in cacao, the coc dissolution which occurred in both resistant and susceptible genotypes led to extracellular ca2+ availability that may be used by the tcpr-4b for its acting as dnase in resistance mechanisms or in pcd during the infection process. this may be reinforced by the fact that the calcium availability is higher in the apoplasm in normal cells, while an influx of calcium in the intracellular area occurred during pcd.  we reported the first evidence of a pr- with a dual dnase and rnase activity together with an antifungal activity. to our knowledge, this is the first report of pr- showing these three functions simultaneously, but no chitinase activity. moreover, we showed that the antifungal activity of tcpr-4b is directly related to the rnase function – and not to the dnase one. these functions could be related to the role of the tcpr- during the cacao-m. perniciosa interaction, contributing to the maintenance of the symptom reduction in the resistant genotype and to the pcd mechanism in susceptible one. thus, the tcpr-4b could be considered as a good candidate for biotechnological applications in order to detain witches’ broom disease. 
after the initial stage, the tcpr-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection. the production and accumulation of pathogenesis-related proteins in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. tcpr-4b presents a barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. to our knowledge, this is the first report of a pr- showing simultaneously rnase, dnase and antifungal properties, but no chitinase activity. moreover, tcpr-4b presented antifungal action against m. perniciosa, and the reduction of m. perniciosa survival was related to ros production in fungal hyphae. in cacao, tcpr-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the pcd mechanism, in resistant and susceptible cacao genotypes, respectively. the recombinant tcpr-4b protein showed rnase, and bivalent ions dependent-dnase activity, but no chitinase activity. in the cacao genome, tcpr-4b belonged to a small multigene family organized mainly on chromosome tcpr-4b rt-qpcr analysis in resistant and susceptible cacao plants infected by m. perniciosa showed an increase of expression at 48 hours after infection in both cacao genotypes. moreover, we showed that the antifungal activity of tcpr-4b is directly related to rnase function.
 the production and accumulation of pathogenesis-related proteins in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. a pathogenesis-related protein cdna was identified from a cacao-moniliophthora perniciosa interaction cdna library and named tcpr-4b.  tcpr-4b presents a barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. molecular modeling of tcpr-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. in the cacao genome, tcpr-4b belonged to a small multigene family organized mainly on chromosome tcpr-4b rt-qpcr analysis in resistant and susceptible cacao plants infected by m. perniciosa showed an increase of expression at 48 hours after infection in both cacao genotypes. after the initial stage, the tcpr-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection. the recombinant tcpr-4b protein showed rnase, and bivalent ions dependent-dnase activity, but no chitinase activity. moreover, tcpr-4b presented antifungal action against m. perniciosa, and the reduction of m. perniciosa survival was related to ros production in fungal hyphae.  to our knowledge, this is the first report of a pr- showing simultaneously rnase, dnase and antifungal properties, but no chitinase activity. moreover, we showed that the antifungal activity of tcpr-4b is directly related to rnase function. in cacao, tcpr-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the pcd mechanism, in resistant and susceptible cacao genotypes, respectively. nucleasesgene expressionros productionmolecular modeling 