Legumes not only establish beneficial symbiosis with rhizobia but are also colonized by other endophytic bacteria, which are believed to play a significant role in plant fitness. However, the molecular mechanisms through which non-rhizobial bacteria respond to host-derived signals remain poorly understood compared to those of the well-studied rhizobia–legume symbiosis. In this study, the responses of two non-rhizobial endophytic bacteria, Pseudomonas sp. Q1 and Kosakonia sp. MH5, to chickpea root exudates were analyzed using high-throughput proteomics. Proteomic analysis revealed the differential expression of proteins involved in metabolism, cell envelope biosynthesis, stress response, oxidative stress defense, chemotaxis, nitrogen metabolism, type IV secretion systems, and transmembrane transport.

A notable finding was the upregulation of the gacS gene (2,754 bp) in the proteome of Pseudomonas sp. Q1 following exposure to chickpea root exudates. This gene encodes GacS, a hybrid histidine kinase and a critical component of the Gac two-component signal transduction system. To explore the functional role of GacS, a gacS knockout mutant of Pseudomonas sp. Q1 was generated. The mutant ∆gacS strain showed no significant differences in swarming or swimming motility, biofilm formation, or phosphate solubilization compared to the wild-type strain. However, siderophore production in the mutant was reduced by approximately 8.8%, while the biosynthesis of antimicrobial metabolites remained unaffected. Additionally, under iron deficient conditions, subclover plants inoculated with the mutant ∆gacS exhibited significantly lower growth compared to those inoculated with the wild-type strain.

These findings indicate that GacS in Pseudomonas sp. Q1 is only partially involved in regulating key plant-beneficial traits, suggesting that other two-component signal transduction systems may compensate for GacS function in this strain. Further studies are needed to fully understand the role of GacS in plant–bacteria interactions. This research enhances our understanding of the molecular dialogue between chickpea and its endophytic bacteria and identifies potential genetic targets to improve plant–microbe interactions.