A three-way transcriptomic crosstalk interaction in a biocontrol agent (Bacillus velezensis), a fungal pathogen (Colletotrichum gloeosporioides), and a walnut host (Juglans regia L.).

Walnut anthracnose, caused by Colletotrichum gloeosporioides, is a globally significant disease. Bacillus velezensis demonstrates strong inhibitory effects against C. gloeosporioides. Current research primarily focuses on interactions between two species, leaving the molecular mechanisms of three-way interactions among walnut, pathogen, and biocontrol agent unexplored. To elucidate the molecular mechanisms in the "Walnut-C. gloeosporioides-B. velezensis" system, we employed three-way RNA-Seq to obtain transcriptome data for each species. We identified 111,674, 799, and 79 differentially expressed genes (DEGs) in walnut, C. gloeosporioides, and B. velezensis respectively. By integrating WGCNA and PPI network analyses, we discovered 3 key modules and 10 hub genes in C. gloeosporioides, and 2 key modules and 2 hub genes in B. velezensis. Functional enrichment analyses (GO and KEGG) revealed that walnut DEGs were enriched in secondary metabolism, disease-resistance proteins, plant hormone signal transduction, and pathogenesis-related proteins; C. gloeosporioides DEGs were associated with binding, antigen processing and presentation, and regulation of actin cytoskeleton; B. velezensis DEGs were linked to transmembrane transport, amino acid biosynthesis, carbon metabolism, and TCA cycle. These results demonstrate that the transcriptional responses of the three species mutually influence each other within the three-way system. This study addresses knowledge gaps in the biocontrol-pathogen-host interaction and provides a theoretical foundation for developing environmentally sustainable strategies to control walnut anthracnose.