Optimizing rhizobacterial genetic circuit designs for agricultural sustainability

Facilitating interactions between a plant's roots and its external environment is key to tackling various impending food, energy, and sustainability related challenges. For example, plants with modified root architecture can reduce atmospheric carbon dioxide levels, or even increase crop yields to sustain the growing human population.

One way to do this is by constructing a 'genetic circuit' within plant cells. A genetic circuit is a collection of biological components encoding an RNA or a protein, which allows individual cells to perform specific functions. Inside plant cells, it might sense environmental conditions, interpret cues, and exhibit desired phenotypes. However, designing these circuits in plants remains a challenge.

While additional research is required to design plant-based circuits, bacterial circuits have seen enormous progress. Several components are available to design bacterial circuits, which are then used to facilitate complex cellular functions. This design extends to plant roots, which are the site of critical plant-bacteria interactions. Rhizobacteria – free-living bacteria that colonize plant roots – significantly impact plant health, nutrient uptake, and soil chemistry. Hence, their genetic circuit design can be used to engineer plants with desirable qualities.

To this end, a team of researchers including Professor José R. Dinneny, and his postdoctoral student Dr Christopher M. Dundas from Stanford University reviewed the genetic components and best practices for designing rhizobacterial circuits. Their findings, which were published in BioDesign Research, primarily focused on the sensors, actuators, and chassis species that are used to regulate plant microbiome processes.

"Learning about approaches to design genetic circuits can help scientists engineer plant-rhizosphere interactions in an effective way," said Dr Dundas while discussing the motivation behind this review.

First, the team explored tools that can facilitate the successful construction of genetic circuits in rhizobacteria. In particular, bioinformatic tools, orthogonal gene expression machinery, and genome mining are being used to predict functional promoter sequences and ribosome-binding site (RBS) sequences, to engineer transcription and translation in rhizobacteria.

Next-generation genome engineering tools are also being employed to reduce rhizobacteria's dependence on host replication machinery and selection. Furthermore, several toolkits have been developed for the construction of broad-host-range plasmids required for rhizobacterial transformation.

Next, the team discussed 'rhizobacterial chassis', which facilitates effective colonization of the root tissues, and in turn enables the circuit to function optimally. Creating an ideal chassis can be achieved by targeting certain genes that regulate colonization-related traits of rhizobacteria, such as chemotaxis, root attachment, extent of colonization, biofilm formation, and the ability to dodge the plant immune system.

Moreover, selecting a competent rhizobacterial species is necessary to avoid unwanted effects associated with excessive bacterial growth in the roots.

Plant root exudates, which rhizobacteria are exposed to routinely, are attractive sensing targets for tracking plant health. The article sheds light on the advantages due to which small molecule-responsive transcriptional regulators such as sugars, nitrogen compounds, secondary metabolites, and phytohormones are preferred for the development of plant health biosensors, or sensor circuits.

Sensor circuits in turn, help to drive the expression of multiple genes and downstream pathways.

Lastly, the article provides an overview of 'rhizobacterial actuators' or actuator circuits that drive the desired phenotypes in colonized plants. Actuator design can be improved by fine-tuning biosynthetic gene expression, which in turn can improve nutrient uptake, biotic/abiotic stress tolerance, and growth of the plant.

"The strategies that we presented can help re-wire genetic circuits to improve plant health and productivity via the design-build-test-learn cycle. As new technologies emerge, it will be exciting to see how different bacterial research areas intersect with rhizobacterial sensors and actuators," remarked Dr Dundas.

How can these findings benefit related emerging technologies? "The intersection of material science with synthetic biology is gaining a lot of traction. Our findings contain useful insights for the development of functionalized living materials, which can be used to colonize plant roots for a variety of applications," explained Dr Dundas in response.

Although rhizobacterial genetic circuits have enormous potential to reshape agricultural sustainability, it is critical to address the technical, regulatory, and ethical constraints surrounding this technology. Moreover, their deployment in varying climatic conditions also needs to be explored. Nonetheless, researchers are optimistic about expanding these circuits to address global food security and sustainability challenges.