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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]