A Pseudomonas fluorescens AND-gate biosensor for protein expression at plant root proximity

Nico van DonkAntoine RaynalEnrique Asin-Garcia^*

• Bioprocess Engineering Group, Wageningen University & Research, Wageningen, Netherlands

By 2050, global population growth will significantly increase food demand, placing additional pressure on agriculture, a sector already vulnerable to climate change. Traditional approaches like fertilizers and pesticides have helped boost yields but are increasingly seen as unsustainable. As bioengineering becomes more accessible, engineered soil microorganisms are emerging as promising alternatives. However, their application in the rhizosphere is often limited by poor survivability and the high metabolic cost of expressing heterologous genes without appropriate regulation. To address this, we developed a microbial whole-cell biosensor that activates gene expression only under favorable conditions: in close proximity to plant roots and at high bacterial population densities. We engineered the pSal/nahR system in our host Pseudomonas fluorescens SBW25 to respond to salicylic acid, a key root exudate. In parallel, we implemented a quorum sensing system based on LuxI and the luxpR/LuxR pair to monitor cell density. Both inputs were integrated using a toehold switch-based AND gate, triggering expression only when both conditions were met. This strategy minimizes metabolic burden and offers a tightly controlled system for expression at target locations. While further validation in rhizosphere-like conditions is required, our results provide a foundation for safer open-environment applications of microorganisms, making this biosensor a versatile tool for future agricultural biotechnology. Graphical abstract. A Pseudomonas fluorescens AND-gate biosensor. P. fluorescens SBW25 was engineered to detect two signals: root exudates indicating proximity to plant roots, and quorum sensing molecules indicating a sufficient bacterial population. These inputs were integrated using a genetic AND gate, creating a biosensor that activates gene expression only under optimal conditions for root-associated delivery. The biosensor can be used to control various target genes, making it broadly applicable across multiple agricultural applications.