Biofilm formation by Pseudomonas putida KT2440 contributes to improve tomato drought stress resilience and priming for enhanced gene regulation

Pseudomonas putida KT2440 is a plant growth-promoting rhizobacterium (PGPR), known to enhance tolerance to pathogen infection, but its role in drought stress mitigation remains largely unexplored. This study aimed to assess whether inoculation with KT2440 improves tomato tolerance to drought. Inoculation with the KT2440 wild type (WT) significantly improved ecophysiological drought stress responses by increasing leaf water potential and photosynthetic rate. It also resulted in an impact on the holobiont cell physiology through modulation of the activity signature of key enzymes of carbohydrate (e.g., PGM and vacInv) and antioxidant (e.g., GR, MDHAR, and cwPOX) metabolism under drought conditions. To functionally assess the role of biofilm formation in drought response, biofilm-deficient mutants KT2440 Alg, with only one gene cluster for the exopolysaccharide alginate deleted, and KT2440 Q, with four exopolysaccharide gene clusters (alg, bcs, pea and peb) deleted, were used. Inoculation with these two mutants led to reduced drought resilience, with partial or complete loss of protective effects in the Alg and Q mutants, respectively. This was reflected in lowered leaf water potential, photosynthetic rate, and reduced antioxidant and carbohydrate metabolism enzyme activities compared to inoculation with the corresponding wild type. Global RNA sequencing revealed that under drought conditions 360 % more genes were differentially regulated in the presence of KT2440 WT compared to the mock inoculated control, whereas this value decreased again to only 140 % more differentially regulated genes after recovery from the drought stress. Thus, KT2440 specifically primes the plant for a much more pronounced transcriptional response only during the impact of drought, thus providing resilience protection on demand. This priming for enhanced abiotic stress responsiveness was partially dependent on the ability to form biofilm. Both under well-watered and drought stress the number of differentially regulated genes was strongly reduced in plants inoculated with KT2440 Q compared to WT. Gene ontology and expression analyses showed significant upregulation of pathways involved in photosynthesis, phytohormone signaling, antioxidant metabolism, and drought resilience in KT2440-inoculated plants. Although KT2440 WT showed higher biofilm formation compared to the Alg and Q mutants, the strains did not differ in their ability for root colonization. These findings provide novel insights into the contribution of biofilm formation to PGPR-mediated drought tolerance and protection on demand via priming for enhanced transcriptional regulation under stress, supporting the potential of KT2440 for environmentally friendly mitigating of drought stress responses in crops.