Transcriptomic Insights into Defense-Associated Responses of Theobroma cacao During Interaction with Bacillus subtilis and Moniliophthora roreri

Cacao (Theobroma cacao L.) production is severely constrained by frosty pod rot, caused by Moniliophthora roreri. Beneficial bacteria such as Bacillus subtilis represent a promising biological-control strategy, but their influence on cacao transcriptional responses during pathogen exposure remains poorly understood. Here, we used Oxford Nanopore long-read RNA sequencing to characterize cacao pod transcriptional responses to B. subtilis, M. roreri, and their combined application under field conditions, while accounting for cacao genetic background. Differential-expression analysis was performed using reference-based gene counts and an FDR-controlled statistical framework. At the confirmatory level, nine unique genes showed significant differential expression across the planned contrasts. These included genes encoding an RBP45C-like RNA-binding protein, a mitochondrial small heat-shock protein, a subunit shared by RNA polymerases II/IV/V, a LEA14-like stress-associated protein, an expansin-like protein, and a GTL1-like trihelix transcription factor, linking treatment-associated responses to RNA regulation, mitochondrial proteostasis, transcriptional control, cellular stress protection, and cell-wall remodeling. Pathogen exposure alone was predominantly associated with reduced expression among the confirmatory genes, whereas several of these expression patterns changed in the opposite direction when the combined treatment was compared with pathogen exposure alone. A similar reciprocal pattern was observed for a smaller subset of genes when the combined treatment was compared with B. subtilis alone. However, no gene met the confirmatory differential-expression criterion when the combined treatment was compared directly with the untreated control. These findings indicate that B. subtilis does not uniformly increase defense-associated gene expression in cacao but is associated with a microbial-context-dependent reorganization of the transcriptional response to M. roreri. The FDR-supported genes identified here provide focused candidates for independent validation and future mechanistic studies of beneficial bacterium–cacao–pathogen interactions.