Resistant and susceptible cacao genotypes exhibit defense gene polymorphism and unique early responses to Phytophthora megakarya inoculation.

KEY MESSAGE: Key genes potentially involved in cacao disease resistance were identified by transcriptomic analysis of important cacao cultivars. Defense gene polymorphisms were identified which could contribute to pathogen recognition capacity. Cacao suffers significant annual losses to the water mold Phytophthora spp. (Oomycetes). In West Africa, P. megakarya poses a major threat to farmer livelihood and the stability of cocoa production. As part of a long-term goal to define key disease resistance genes in cacao, here we use a transcriptomic analysis of the disease-resistant cacao clone SCA6 and the susceptible clone NA32 to characterize basal differences in gene expression, early responses to infection, and polymorphisms in defense genes. Gene expression measurements by RNA-seq along a time course revealed the strongest transcriptomic response 24 h after inoculation in the resistant genotype. We observed strong regulation of several pathogenesis-related genes, pattern recognition receptors, and resistance genes, which could be critical for the ability of SCA6 to combat infection. These classes of genes also showed differences in basal expression between the two genotypes prior to infection, suggesting that prophylactic expression of defense-associated genes could contribute to SCA6's broad-spectrum disease resistance. Finally, we analyzed polymorphism in a set of defense-associated receptors, identifying coding variants between SCA6 and NA32 which could contribute to unique capacities for pathogen recognition. This work is an important step toward characterizing genetic differences underlying a successful defense response in cacao.

Witches' broom resistant genotype CCN51 shows greater diversity of symbiont bacteria in its phylloplane than susceptible genotype catongo.

BACKGROUND: Theobroma cacao L. (cacao) is a perennial tropical tree, endemic to rainforests of the Amazon Basin. Large populations of bacteria live on leaf surfaces and these phylloplane microorganisms can have important effects on plant health. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated studies of the phylloplane microbiome. In this study, we characterized the bacterial microbiome of the phylloplane of the catongo genotype (susceptible to witch's broom) and CCN51 (resistant). Bacterial microbiome was determined by sequencing the V3-V4 region of the bacterial 16S rRNA gene. RESULTS: After the pre-processing, a total of 1.7 million reads were considered. In total, 106 genera of bacteria were characterized. Proteobacteria was the predominant phylum in both genotypes. The exclusive genera of Catongo showed activity in the protection against UV radiation and in the transport of substrates. CCN51 presented genus that act in the biological control and inhibition in several taxonomic groups. Genotype CCN51 presented greater diversity of microorganisms in comparison to the Catongo genotype and the total community was different between both. Scanning electron microscopy analysis of leaves revealed that on the phylloplane, many bacterial occur in large aggregates in several regions of the surface and isolated nearby to the stomata. CONCLUSIONS: We describe for the first time the phylloplane bacterial communities of T. cacao. The Genotype CCN51, resistant to the witch's broom, has a greater diversity of bacterial microbioma in comparison to Catongo and a greater amount of exclusive microorganisms in the phylloplane with antagonistic action against phytopathogens.

TcNPR3 from Theobroma cacao functions as a repressor of the pathogen defense response.

BACKGROUND: Arabidopsis thaliana (Arabidopsis) NON-EXPRESSOR OF PR1 (NPR1) is a transcription coactivator that plays a central role in regulating the transcriptional response to plant pathogens. Developing flowers of homozygous npr3 mutants are dramatically more resistant to infection by the pathogenic bacterium Pseudomonas syringae, suggesting a role of NPR3 as a repressor of NPR1-mediated defense response with a novel role in flower development. RESULTS: We report here the characterization of a putative NPR3 gene from the tropical tree species Theobroma cacao (TcNPR3). Like in Arabidopsis, TcNPR3 was constitutively expressed across a wide range of tissue types and developmental stages but with some differences in relative levels compared to Arabidopsis. To test the function of TcNPR3, we performed transgenic complementation analysis by introducing a constitutively expressing putative TcNPR3 transgene into an Arabidopsis npr3 mutant. TcNPR3 expressing Arabidopsis plants were partially restored to the WT pathogen phenotype (immature flowers susceptible to bacterial infection). To test TcNPR3 function directly in cacao tissues, a synthetic microRNA targeting TcNPR3 mRNA was transiently expressed in cacao leaves using an Agrobacterium-infiltration method. TcNPR3 knock down leaf tissues were dramatically more resistance to infection with Phytophthora capsici in a leaf bioassay, showing smaller lesion sizes and reduced pathogen replication. CONCLUSIONS: We conclude that TcNPR3 functions similar to the Arabidopsis NPR3 gene in the regulation of the cacao defense response. Since TcNPR3 did not show a perfect complementation of the Arabidopsis NPR3 mutation, the possibility remains that other functions of TcNPR3 remain to be found. This novel knowledge can contribute to the breeding of resistant cacao varieties against pathogens through molecular markers based approaches or biotechnological strategies.

Chocolate consumption modulates cytokine production in healthy individuals.

Epidemiological studies suggest that chocolate increases the incidence and severity of acne. Here we demonstrate that chocolate consumption primes human blood mononuclear cells from volunteers to release more interleukin-1ß (IL-1ß) and IL-10 upon stimulation with Propionibacterium acne or Staphylcoccus aureus, the two microorganisms involved in the pathogenesis of acne. In contrast, production of the Th17-derived cytokine IL-22 was inhibited by chocolate. Modulation of inflammation could represent an important mechanism through which chocolate consumption influences acne.

Quantitative risk assessment relating to adventitious presence of allergens in food: a probabilistic model applied to peanut in chocolate.

Peanut allergy is a public health concern, owing to the high prevalence in France and the severity of the reactions. Despite peanut-containing product avoidance diets, a risk may exist due to the adventitious presence of peanut allergens in a wide range of food products. Peanut is not mentioned in their ingredients list, but precautionary labeling is often present. A method of quantifying the risk of allergic reactions following the consumption of such products is developed, taking the example of peanut in chocolate tablets. The occurrence of adventitious peanut proteins in chocolate and the dose-response relationship are estimated with a Bayesian approach using available published data. The consumption pattern is described by the French individual consumption survey INCA2. Risk simulations are performed using second-order Monte Carlo simulations, which separately propagates variability and uncertainty of the model input variables. Peanut allergens occur in approximately 36% of the chocolates, leading to a mean exposure level of 0.2 mg of peanut proteins per eating occasion. The estimated risk of reaction averages 0.57% per eating occasion for peanut-allergic adults. The 95% values of the risk stand between 0 and 3.61%, which illustrates the risk variability. The uncertainty, represented by the 95% credible intervals, is concentrated around these risk estimates. Children have similar results. The conclusion is that adventitious peanut allergens induce a risk of reaction for a part of the French peanut-allergic population. The method developed can be generalized to assess the risk due to the consumption of every foodstuff potentially contaminated by allergens.

Cocoa: antioxidant and immunomodulator.

Cocoa, a product consumed since 600 BC, is now a subject of increasing interest because of its antioxidant properties, which are mainly attributed to the content of flavonoids such as ( - )-epicatechin, catechin and procyanidins. Moreover, recent findings suggest a regulatory effect of cocoa on the immune cells implicated in innate and acquired immunity. Cocoa exerts regulatory activity on the secretion of inflammatory mediators from macrophages and other leucocytes in vitro. In addition, emerging data from in vivo studies support an immunomodulating effect. Long-term cocoa intake in rats affects both intestinal and systemic immune function. Studies in this line suggest that high-dose cocoa intake in young rats favours the T helper 1 (Th1) response and increases intestinal gammadelta T lymphocyte count, whereas the antibody-secreting response decreases. The mechanisms involved in this activity are uncertain; nonetheless, because redox-sensitive pathways control immune cell function, the action of cocoa flavonoids on modulating cell signalling and gene expression deserves investigation.

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

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

Suppression of Plant Immunity by Fungal Chitinase-like Effectors.

Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.

Ecological implications of anti-pathogen effects of tropical fungal endophytes and mycorrhizae.

We discuss studies of foliar endophytic fungi (FEF) and arbuscular mycorrhizal fungi (AMF) associated with Theobroma cacao in Panama. Direct, experimentally controlled comparisons of endophyte free (E-) and endophyte containing (E+) plant tissues in T. cacao show that foliar endophytes (FEF) that commonly occur in healthy host leaves enhance host defenses against foliar damage due to the pathogen (Phytophthora palmivora). Similarly, root inoculations with commonly occurring AMF also reduce foliar damage due to the same pathogen. These results suggest that endophytic fungi can play a potentially important mutualistic role by augmenting host defensive responses against pathogens. There are two broad classes of potential mechanisms by which endophytes could contribute to host protection: (1) inducing or increasing the expression of intrinsic host defense mechanisms and (2) providing additional sources of defense, extrinsic to those of the host (e.g., endophyte-based chemical antibiosis). The degree to which either of these mechanisms predominates holds distinct consequences for the evolutionary ecology of host-endophyte-pathogen relationships. More generally, the growing recognition that plants are composed of a mosaic of plant and fungal tissues holds a series of implications for the study of plant defense, physiology, and genetics.

Characterization of the legumains encoded by the genome of Theobroma cacao L.

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa.

Tracing tree nut allergens in chocolate: A comparison of DNA extraction protocols.

The present work aimed at comparing different DNA extraction methods, from chocolate matrices, for the effective application in molecular techniques to detect tree nut allergens. For this study, DNA from almond or hazelnut model chocolates was extracted using seven selected protocols: the in-house methods of CTAB-PVP (cetyltrimethylammonium bromide-polyvinylpyrrolidone), Wizard with and without RNase, Wizard-PVP with and without RNase, and the Wizard Magnetic and Nucleospin kits. The extracts were assessed for their suitability for amplification by qualitative PCR and real-time PCR. From the evaluated protocols, Nucleospin presented the best results for almond and hazelnut amplification, achieving a limit of detection of 0.005% (w/w) with high PCR efficiency, linearity and range of amplification. These results highlight the importance of the DNA extraction protocol in the case of food allergens from complex matrices, such as chocolate, in which sensitivity is a key parameter.

Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao.

Previous work has implicated glycerol-3-phosphate (G3P) as a mobile inducer of systemic immunity in plants. We tested the hypothesis that the exogenous application of glycerol as a foliar spray might enhance the disease resistance of Theobroma cacao through the modulation of endogenous G3P levels. We found that exogenous application of glycerol to cacao leaves over a period of 4 days increased the endogenous level of G3P and decreased the level of oleic acid (18:1). Reactive oxygen species (ROS) were produced (a marker of defence activation) and the expression of many pathogenesis-related genes was induced. Notably, the effects of glycerol application on G3P and 18:1 fatty acid content, and gene expression levels, in cacao leaves were dosage dependent. A 100 mm glycerol spray application was sufficient to stimulate the defence response without causing any observable damage, and resulted in a significantly decreased lesion formation by the cacao pathogen Phytophthora capsici; however, a 500 mm glycerol treatment led to chlorosis and cell death. The effects of glycerol treatment on the level of 18:1 and ROS were constrained to the locally treated leaves without affecting distal tissues. The mechanism of the glycerol-mediated defence response in cacao and its potential use as part of a sustainable farming system are discussed.

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: dark chocolate.

Immunodetection of allergens in dark chocolate is complicated by interference from the chocolate components. The objectives of this study were to establish reference materials for detecting multiple allergens in dark chocolate and to determine the accuracy and precision of allergen detection by enzyme-linked immunosorbent assay (ELISA) before and after chocolate processing. Defatted peanut flour, whole egg powder, and spray-dried milk were added to melted chocolate at seven incurred levels and tempered for 4 h. Allergen concentrations were measured using commercial ELISA kits. Tempering decreased the detection of casein and ß-lactoglobulin (BLG), but had no significant effect on the detection of peanut and egg. Total coefficients of variation were higher in tempered than untempered chocolate for casein and BLG, but total and analytical CVs were comparable for peanut and egg. These findings indicate that processing has a greater effect on recovery and variability of casein and BLG than peanut and egg detection in a dark chocolate matrix.

Evaluation of the allergenicity potential of TcPR-10 protein from Theobroma cacao.

BACKGROUND: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. METHODOLOGY/PRINCIPAL FINDINGS: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. CONCLUSIONS/SIGNIFICANCE: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties.

Spleen lymphocyte function modulated by a cocoa-enriched diet.

Previous studies have shown the down-regulating in vitro effect of cocoa flavonoids on lymphocyte and macrophage activation. In the present paper, we report the capacity of a long-term rich cocoa diet to modulate macrophage cytokine secretion and lymphocyte function in young rats. Weaned rats received natural cocoa (4% or 10% food intake), containing 32 mg flavonoids/g, for 3 weeks. Spleen immune function was then evaluated through the analysis of lymphocyte composition, their proliferative response and their ability to secrete cytokines and Ig. In addition, the status of activated peritoneal macrophages was established through tumour necrosis factor (TNF)-alpha secretion. The richest cocoa diet (10%) caused a reduction of TNF-alpha secretion by peritoneal macrophages showing anti-inflammatory activity. Similarly, although a 10% cocoa diet increased lymphocyte proliferation rate, it down-regulated T helper 2 (Th2)-related cytokines and decreased Ig secretion. These changes were accompanied by an increase in spleen B cell proportion and a decrease in Th cell percentage. In summary, these results demonstrate the functional activity of a cocoa-high dosage in down-regulating the immune response that might be beneficial in hypersensitivity and autoimmunity.

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate.

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 microg g-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.

Effect of Theobroma cacao flavonoids on immune activation of a lymphoid cell line.

We analysed the effect of (-)-epicatechin and cocoa extract on the activation of a lymphoid cell line. Particularly the expression of IL-2 receptor alpha (IL-2Ralpha or CD25) and, the secretion of IL-2 and IL-4 were established after flavonoid treatment. Two media culture conditions (1 and 10 % of fetal calf serum supplementation) and the different moments of flavonoid addition (simultaneously or 2 h before cell-activation) were compared. IL-2Ralpha (CD25) expression on activated cells was significantly reduced by epicatechin and cocoa extract in a dose-dependent manner, achieving the highest inhibition of about 50 % when flavonoids were added 2 h before stimulation. IL-2 secretion was also inhibited by the presence of both epicatechin and cocoa extract, displaying 60 and 75 % of inhibition, respectively. Cocoa flavonoids were also able to enhance 3-4.5-fold IL-4 release. In summary, cocoa extract down-modulated T lymphocyte activation and therefore the acquired immune response. This fact could be important in some states of the immune system hyperactivity such as autoimmune or chronic inflammatory diseases.

Cacao, actualización de sus propiedades beneficiosas para la salud humana / Cocoa, an update of its beneficial properties for human health

Las semillas de cacao (Theobroma cacao L., Esterculiáceas) y sus derivados, además de su valor nutritivo, tienen un gran interés por sus propiedades farmacológicas. En su composición destacan tres grupos de principios: bases nitrogenadas, lípidos y compuestos fenólicos, principalmente catequinas y sus oligómeros, las proantocianidinas (de dímeros a decámeros). Los polifenoles del cacao poseen actividad antioxidante, reducen la oxidación del colesterol-LDL, incrementan los niveles de colesterol-HDL, reducen la glucemia basal y postprandial, incrementan la secreción de insulina y mejoran la sensibilidad a la misma. Todos estos efectos metabólicos, junto a la inhibición de la lipoxigenasa, incremento del óxido nítrico y la disminución de la agregación plaquetaria, le confieren propiedades protectoras cardiacas y vasculares. Además disminuye la síntesis de mediadores proinflamatorios e incrementa la de los antiinflamatorios e inhibe factores de transcripción implicados en la inflamación y carcinogénesis, lo que le confiere propiedades antiinflamatorias y quimiopreventivas del cáncer. También se han descrito un efecto neuroprotector (AU)

In addition to its nutritional value, Cocoa seeds (Theobroma cacao L., Sterculiaceae) and its derivatives also have a great interest due to its pharmacological properties. Three groups of constituents stand out: nitrogen bases, lipids and polyphenols, mainly catechins and their oligomers proanthocianidins (dimers to decamers). The phenolic compounds in cocoa have anti-oxidant properties, reduce LDL-cholesterol oxidation, increase HDL-cholesterol levels, reduce the basal and postprandial glycaemia, and increase the secretion of insulin, improving the sensibility to it. These metabolic effects, together with the inhibition of lipoxygenase, the increase of nitric oxide and the reduction of platelet aggregation, confer cocoa good vascular and cardiac protective properties. Moreover, these polyphenols decrease the synthesis of proinflammatory mediators and increase that of anti-inflammatory mediators. Furthermore, they inhibit transcription factors implicated in inflammation and carcinogenesis, conferring anti-inflammatory and cancer chemopreventive properties to cocoa. Its neuroprotective effects have also been reported (AU)