Multispecies biofilms are important models for studying the evolution of microbial interactions. Co-cultivation of Xanthomonas retroflexus (XR) and Paenibacillus amylolyticus (PA) systemically leads to the appearance of an XR wrinkled mutant (XRW), increasing biofilm production. The nature of this new interaction and the role of each partner remain unclear. We tested the involvement of secreted molecular cues in this interaction by exposing XR and XRW to PA or its supernatant and analysing the response using RNA-seq, colony-forming unit (CFU) estimates, biofilm quantification, and microscopy. Compared to wild type, the mutations in XRW altered its gene expression and increased its CFU number. These changes matched the reported effects for one of the mutated genes: a response regulator part of a two-component system involved in environmental sensing. When XRW was co-cultured with PA or its supernatant, the mutations effects on XRW gene expression were masked, except for genes involved in sedentary lifestyle, being consistent with the higher biofilm production. It appears that the higher biofilm production was the result of the interaction between the genetic context (mutations) and the biotic environment (PA signals). Regulatory genes involved in environmental sensing need to be considered to shed further light on microbial interactions.