Dandara Bispo Oliveira^1,2, Fatima Cerqueira Alvim ¹, Luciel Fernandes ², Dahyana Brito ², Jean-Philippe², Stefan Royaert², Fabiana Cavalcanti¹

Theobroma cacao L. is a plant of high economic importance, whose productivity is significantly impacted by diseases such as black pod rot, caused by species of the genus Phytophthora. Recently, a new species, Phytophthora theobromicola, has been identified as highly aggressive in Brazilian plantations, especially in the state of Bahia. To understand the molecular mechanisms related to cacao resistance to Phytophthora, we used proteomic tools to analyze representative proteins from the fruit peel, as this is the main site of pathogen infection. The primary objective was to identify differentially abundant proteins in cacao genotypes with contrasting resistance to black pod rot: TSH 1188 (moderately resistant) and CCN 51 (resistant). For this, mature fruits were inoculated with P. theobromicola, and peel samples were collected 96 hours after inoculation for protein extraction and proteomic analysis. Due to the presence of mucilage, lignin, and phenolic compounds in the collected samples, it was necessary to optimize the protein extraction protocol—adapted from methods used for leaf samples—with additional steps of sonication and successive washes to remove impurities. The efficiency of extraction was validated by resolving the proteins through polyacrylamide gel electrophoresis (SDS-PAGE). Sample quantification using the Bradford method revealed a significant increase in protein yield using the optimized protocol (average of 3.8 µg/mg) compared to the leaf-adapted method (average of 1.9 µg/mg). Sample quality was confirmed through mass spectrometry analysis, which resulted in the identification of over 1,500 proteins in injected samples of the CCN 51 genotype alone. Bioinformatic analysis allowed the identification of proteins involved in signaling pathways, stress response, and plant defense—processes expected to occur in the resistant cacao genotype. Furthermore, the functional analysis of the proteins revealed a strong association with resistance mechanisms. The protein–protein interaction (PPI) network analysis showed connections between key proteins involved in immunity and regulation of biotic stress. This work reinforces the potential of the fruit peel as a model tissue in cacao proteomic studies and demonstrates the effectiveness of the developed protocol. Moreover, it provides novel data on the T. cacao × P. theobromicola interaction, a pathogen species that is still poorly characterized.

Agradecimentos: Agradeço a Universidade Estadual de Santa Cruz e a Mars - Centro de Ciência do Cacau pelo incentivo ao projeto