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Witches’ broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches’ brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (β-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies.

In plants, serpins are a superfamily of serine and cysteine protease inhibitors involved in stress and defense mechanisms, with potential for controlling agricultural pests, making them important biotechnological tools. The objective of this study was to characterize a serpin from Theobroma cacao, called TcSERPIN, to identify its endogenous targets and determine its function and biotechnological potential. TcSERPIN has 390 amino acid residues and shows conservation of the main active site, RCL. Cis-elements related to light, stress, hormones, anaerobic induction, cell cycle regulation and defense have been identified in the gene’s regulatory region. TcSERPIN transcripts are accumulated in different tissues of Theobroma cacao. Furthermore, in plants infected with Moniliophtora perniciosa and Phytophthora palmivora, the expression of TcSERPIN was positively regulated. The protein spectrum, rTcSERPIN, reveals a typical β-sheet pattern and is thermostable at pH 8, but loses its structure with temperature increases above 66°C at pH 7. At the molar ratios of 0.65 and 0.49, rTcSERPIN inhibited 55 and 28% of the activity of papain from Carica papaya and trypsin from Sus scrofa, respectively. The protease trap containing immobilized rTcSERPIN captured endogenous defense proteins from cocoa extracts that are related to metabolic pathways, stress and defense. The evaluation of the biotechnological potential against geohelminth larvae showed that rTcSERPIN and rTcCYS4 (Theobroma cacao cystatin 4) reduced the movement of larvae after 24 hours. The results of this work show that TcSERPIN has ideal biochemical characteristics for biotechnological applications, as well as potential for studies of resistance to phytopathogens of agricultural crops.

IntroductionThe Family of pathogenesis-related proteins 10 (PR-10) is widely distributed in the plant kingdom. PR-10 are multifunctional proteins, constitutively expressed in all plant tissues, playing a role in growth and development or being induced in stress situations. Several studies have investigated the preponderant role of PR-10 in plant defense against biotic stresses; however, little is known about the mechanisms of action of these proteins. This is the first systematic review conducted to gather information on the subject and to reveal the possible mechanisms of action that PR-10 perform.MethodsTherefore, three databases were used for the article search: PubMed, Web of Science, and Scopus. To avoid bias, a protocol with inclusion and exclusion criteria was prepared. In total, 216 articles related to the proposed objective of this study were selected.ResultsThe participation of PR-10 was revealed in the plant’s defense against several stressor agents such as viruses, bacteria, fungi, oomycetes, nematodes and insects, and studies involving fungi and bacteria were predominant in the selected articles. Studies with combined techniques showed a compilation of relevant information about PR-10 in biotic stress that collaborate with the understanding of the mechanisms of action of these molecules. The up-regulation of PR-10 was predominant under different conditions of biotic stress, in addition to being more expressive in resistant varieties both at the transcriptional and translational level.DiscussionBiological models that have been proposed reveal an intrinsic network of molecular interactions involving the modes of action of PR-10. These include hormonal pathways, transcription factors, physical interactions with effector proteins or pattern recognition receptors and other molecules involved with the plant’s defense system.ConclusionThe molecular networks involving PR-10 reveal how the plant’s defense response is mediated, either to trigger susceptibility or, based on data systematized in this review, more frequently, to have plant resistance to the disease.

The zinc/iron-regulated transporter-like protein (ZIP) gene family first identified in plants is highly distributed in the plant kingdom. This family has previously been reported to transport several essential and non-essential cationic elements, including those toxic to many economically important crops such as cacao (Theobroma cacao L.). In this article, we present a detailed study on physicochemical properties, evolution, duplication, gene structure, promoter region and TcZIP family three-dimensional protein structure. A total of 11 TcZIP genes have been identified to encode proteins from 309 to 435 aa, with localization in the plasma membrane and chloroplast, containing 6-9 putative domains (TM). Interspecies phylogenetic analysis subdivided the ZIP proteins into four groups. Segmental duplication events significantly contributed to the expansion of TcZIP genes. These genes underwent high pressure of purifying selection. The three-dimensional structure of the proteins showed that α helix conformations are predominant with several pocket sites, containing the metal binding site, with the residues leucine (LEU), alanine (ALA), glycine (GLY), serine (SER), lysine (LYS) and histidine (HIS) the most predicted. Regarding the analysis of the protein-protein interaction and enrichment of the gene ontology, four biological processes were assigned, the most important being the cation transport. These new discoveries expand the knowledge about the function, evolution, protein structures and interaction of ZIP family proteins in cacao and contribute to develop cacao genotypes enriched with important mineral nutrients as well as genotypes that bioaccumulate or exclude toxic metals.

IntroductionTheobroma cacao, the cocoa tree, is a target for pathogens, such as fungi from the genera Phytophthora, Moniliophthora, Colletotrichum, Ceratocystis, among others. Some cacao pathogens are restricted to specific regions of the world, such as the Cacao swollen shoot virus (CSSV) in West African countries, while others are expanding geographically, such as Moniliophthora roreri in the Americas. M. roreri is one of the most threatening cacao pathogens since it directly attacks the cacao pods driving a significant reduction in production, and therefore economic losses. Despite its importance, the knowledge about the microenvironment of this pathogen and the cocoa pods is still poorly characterized.MethodsHerein we performed RNA sequencing of spores in differential stages of culture in a medium supplemented with cacao pod extract and mycelium collected of the susceptible variety ICT 7121 naturally infected by the pathogen to evaluate the diversity and transcriptional activity of microorganisms associated with the in vitro sporulation of M. roreri.ResultsOur data revealed a great variety of fungi and bacteria associated with M. roreri, with an exceptional diversity of individuals from the genus Trichoderma sp. Interestingly, the dynamics of microorganisms from different kingdoms varied proportionally, suggesting they are somehow affected by M. roreri culture time. We also identified three sequences similar to viral genomes from the Narnaviridae family, posteriorly confirmed by phylogenetic analysis as members of the genus Narnavirus. Screening of M. roreri public datasets indicated the virus sequences circulating in samples from Ecuador, suggesting a wide spread of these elements. Of note, we did not identify traces of the viral sequences in the M. roreri genome or DNA sequencing, restricting the possibility of these sequences representing endogenized elements.DiscussionTo the best of our knowledge, this is the first report of viruses infecting the fungus of the genus Moniliophthora and only the third description of viruses that are able to parasite elements from the Marasmiaceae family.

Agroforestry systems (AFS) for cocoa production combine traditional land-use practices with local biodiversity conservation, resulting in both ecological and agricultural benefits. The cacao-cabruca AFS model is widely implemented in regions of the Brazilian Atlantic Forest. Carpotroche brasiliensis (Raddi) A. Gray (Achariaceae) is a tree found in cabruca landscapes that is often used for reforestation and biotechnological applications. Despite its importance, we still lack information about viruses circulating in C. brasiliensis, particularly considering the possibility of spillover that could affect cocoa production. In our study, we analyzed the Carpotroche brasiliensis virome from Atlantic Forest and cacao-cabruca AFS regions using metatranscriptomics from several vegetative and reproductive organs. Our results revealed a diverse virome detecting near-complete or partial coding sequences of single- and double-stranded DNA and RNA viruses classified into at least six families (Botourmiaviridae, Bromoviridae, Caulimoviridae, Genomoviridae, Mitoviridae, and Rhabdoviridae) plus unclassified elements. We described with high confidence the near-complete and the partial genomes of two tentative novel viruses: Carpotroche-associated ilarvirus and Carpotroche-associated genomovirus, respectively. Interestingly, we also described sequences likely derived from a rhabdovirus, which could represent a novel member of the genus Gammanucleorhabdovirus. We observed higher viral diversity in cacao-cabruca AFS and reproductive organs of C. brasiliensis with preferential tropism to fruits, which could directly affect production. Altogether, our results provide data to better understand the virome in this unexplored agroecological interface, such as cacao-cabruca AFS and forest ecosystem, providing information on the aspects of virus–plant interactions.

The leaf surface combines biochemical substances and pre-existing morphological structures, as well as the presence of microorganisms. This dynamic environment constitutes a plant's initial defense, as well as the first contact of phytopathogens during invasion. Spore germination starts on the phylloplane and is a fundamental process for fungal development, and hence the establishment of disease. In this review, we address the phylloplane's innate defense mechanisms and biochemical reactions involved in the early stage of phytopathogenic fungal development. The focus is present the pre-infection molecular and biochemical processes of the interaction between Theobroma cacao and Moniliophthora perniciosa, showing how the defense mechanisms of the phylloplane can act to inhibit proteins involved at the beginning of fungal spore germination. We conclude that the phylloplane of the cocoa resistant genotype to M. perniciosa has performed chemical compounds, pre-existing morphological structures and the presence of microorganisms that participate in the pre-infection defense of the plant. Also, the inhibition of proteins involved in the germination mechanism of M. perniciosa basidiospores by chemical and structural compounds present in the cocoa phylloplane may decrease the disease index. Therefore, understanding how the phylloplane defense acts in the fungal spore germination process is essential to develop pre-infection control strategies for cacao plants against witches' broom.

Species of the genus Phytophthora cause black-pod rot, which is the disease responsible for the largest losses in cocoa production in the world. The species Phytophthora palmivora affects cacao tree cultivation in all producing countries. However, proteomic level studies of the Theobroma cacao–P. palmivora interaction are incipient. Thus, the aim of this study was to analyze this pathosystem through comparative proteomics and systems biology analyses. The proteins were extracted from leaves of T. cacao PA 150 (resistant) and SIC 23 (susceptible) clones 48 h after inoculation with P. palmivora using inoculation with sterile distilled water as controls. There were differences in the protein profile between the control and inoculated treatments of both clones. Thirty-seven distinct proteins were identified on 88 spots of the PA 150 treatments, and 39 distinct proteins were identified on 120 spots of the SIC 23 treatments. The metabolisms of ATP, carbohydrates, and nitrogen compounds had higher percentages of proteins with increased accumulation after inoculation in both clones. Systems biology analysis demonstrated that the networks contain a higher number of proteins in the clusters corresponding to processes of photosynthesis and glucose metabolism, suggesting that they are the most affected by the infection. In addition, lipoxygenase (LOX), 2-methylene-furan-3-one reductase-like, and co-chaperonin CPN20 proteins and a probable CC-NBS-LRR protein may be involved in resistance to black-pod disease caused by P. palmivora.

The Trichoderma genus comprises several species of fungi whose diversity of secondary metabolites represents a source of potential molecules with medical application. Because of increased pathogen resistance and demand for lower production costs, the search for new pharmacologically active molecules effective against pathogens has become more intense. This is particularly evident in the case of American cutaneous leishmaniasis due to the high toxicity of current treatments, parenteral administration, and increasing rate of refractory cases. We have previously shown that a fungus from genus Trichoderma can be used for treating cerebral malaria in mouse models and inhibit biofilm formation. Here, we evaluated the effect of the ethanolic extract of Trichoderma asperelloides (Ext-Ta) and its fractions on promastigotes and amastigotes of Leishmania amazonensis, a major causative agent of cutaneous leishmaniasis in the New World. Ext-Ta displayed leishmanicidal action on L. amazonensis parasites, and its pharmacological activity was associated with the low-molecular-weight fraction (LMWF) of Ext-Ta. Ultrastructural analysis demonstrated morphological alterations in the mitochondria and the flagellar pocket of promastigotes, with increased lipid body and acidocalcisome formation, microtubule disorganization of the cytoplasm, and intense vacuolization of the cytoplasm when amastigotes were present. We suggest the antiparasitic activity of Trichoderma fungi as a promising tool for developing chemotherapeutic leishmanicidal agents.

Toxoplasma gondii infection can cause abortions or congenital infection for a vast number of domestic animals and humans, leading to economic loss in veterinary sciences, as well as severe consequences for immunocompromised patients. Bidens pilosa Linné has been used in ethnopharmacology for treatment of diseases, as malaria, diabetes and hepatitis, in addition to its use as antioxidant, antiallergic, anti-inflammatory, and antiviral. The components of this plant have never been studied before for treatment of toxoplasmosis, and the conventional drugs currently used to treat this disease have high degree of toxicity. Thus, the aim of this study was to evaluate the effect of B. pilosa against T. gondii, by analyzing a total extract of this plant in parallel with a fraction obtained by precipitation in acetone. Also, it was assessed if the acetonic fraction could present lectinic activity, followed by its identification by mass spectrometry. It was observed with the experimental models designed that both total extract and acetonic fraction of B. pilosa were able to control T. gondii infection by in vitro and in vivo experiments, in addition to their low toxicity to host cells. Both total extract and acetonic fraction of this plant display capacity to impair replication of T. gondii tachyzoites. Interesting, the B. pilosa acetonic fraction treatment for 10 days after infection decreases significantly the number of T. gondii brain cyst in comparison with controls. The protein isolated from B. pilosa acetonic fraction was characterized as a novel lectin identified as maturase K. Taken together, these findings open new perspectives to treat patients infected by T. gondii. Future studies will be necessary to investigate the precise mechanism underlying the control of T. gondii infection to impair the replication of this parasite in the host cells after treatment with B. pilosa maturase K.

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.