Cocoa Apoplastome Contains Defense Proteins Against Pathogens.

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches' broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao-M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, ß-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase (P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease.

State of the Art of the Molecular Biology of the Interaction between Cocoa and Witches' Broom Disease: A Systematic Review.

Significant scientific advances to elucidate the Moniliophthora perniciosa pathosystem have been achieved in recent years, but the molecular biology of this pathogen-host interaction is still a field with many unanswered questions. In order to present insights at the molecular level, we present the first systematic review on the theme. All told, 1118 studies were extracted from public databases. Of these, 109 were eligible for the review, based on the inclusion and exclusion criteria. The results indicated that understanding the transition from the biotrophic-necrotrophic phase of the fungus is crucial for control of the disease. Proteins with strong biotechnological potential or that can be targets for pathosystem intervention were identified, but studies regarding possible applications are still limited. The studies identified revealed important genes in the M. perniciosa-host interaction and efficient molecular markers in the search for genetic variability and sources of resistance, with Theobroma cacao being the most common host. An arsenal of effectors already identified and not explored in the pathosystem were highlighted. This systematic review contributes to the understanding of the pathosystem at the molecular level, offering new insights and proposing different paths for the development of new strategies to control witches' broom disease.

BASIDIN as a New Protein Effector of the Phytopathogen Causing Witche's Broom Disease in Cocoa.

The fungus Moniliophthora perniciosa secretes protein effectors that manipulate the physiology of the host plant, but few effectors of this fungus have had their functions confirmed. We performed functional characterization of a promising candidate effector of M. perniciosa. The inoculation of rBASIDIN at 4 µmol L-1 in the mesophyll of leaflets of Solanum lycopersicum caused symptoms of shriveling within 6 h without the presence of necrosis. However, when sprayed on the plant at a concentration of 11 µmol L-1, it caused wilting symptoms only 2 h after application, followed by necrosis and cell death at 48 h. rBASIDIN applied to Theobroma cacao leaves at the same concentration caused milder symptoms. rBASIDIN caused hydrogen peroxide production in leaf tissue, damaging the leaf membrane and negatively affecting the photosynthetic rate of Solanum lycopersicum plants. Phylogenetic analysis indicated that BASIDIN has orthologs in other phytopathogenic basidiomycetes. Analysis of the transcripts revealed that BASIDIN and its orthologs are expressed in different fungal species, suggesting that this protein is differentially regulated in these basidiomycetes. Therefore, the results of applying BASIDIN allow the inference that it is an effector of the fungus M. perniciosa, with a strong potential to interfere in the defense system of the host plant.

TcSERPIN, an inhibitor that interacts with cocoa defense proteins and has biotechnological potential against human pathogens.

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 ß-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.

Apoplastomes of contrasting cacao genotypes to witches' broom disease reveals differential accumulation of PR proteins.

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 (ß-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.

Plant Serpins: Potential Inhibitors of Serine and Cysteine Proteases with Multiple Functions.

Plant serpins are a superfamily of protein inhibitors that have been continuously studied in different species and have great biotechnological potential. However, despite ongoing studies with these inhibitors, the biological role of this family in the plant kingdom has not yet been fully clarified. In order to obtain new insights into the potential of plant serpins, this study presents the first systematic review of the topic, whose main objective was to scrutinize the published literature to increase knowledge about this superfamily. Using keywords and the eligibility criteria defined in the protocol, we selected studies from the Scopus, PubMed, and Web of Science databases. According to the eligible studies, serpins inhibit different serine and non-serine proteases from plants, animals, and pathogens, and their expression is affected by biotic and abiotic stresses. Moreover, serpins like AtSerpin1, OSP-LRS, MtSer6, AtSRP4, AtSRP5, and MtPiI4, act in resistance and are involved in stress-induced cell death in the plant. Also, the system biology analysis demonstrates that serpins are related to proteolysis control, cell regulation, pollen development, catabolism, and protein dephosphorylation. The information systematized here contributes to the design of new studies of plant serpins, especially those aimed at exploring their biotechnological potential.

Pathogenesis-related protein 10 in resistance to biotic stress: progress in elucidating functions, regulation and modes of action.

Introduction: The 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. Methods: Therefore, 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. Results: The 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. Discussion: Biological 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. Conclusion: The 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.

TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10-7 mol L-1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and ß-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL-1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.