Research report: Genetic code expansion enables plant-directed control of bacterial activity

Engineered plants can use a noncanonical amino acid to switch on selected bacterial genes, according to a laboratory proof-of-concept study in Arabidopsis, tomato, poplar and two strains of the soil bacterium Bacillus subtilis. The system joins plant biosynthesis of O-methyl-L-tyrosine, abbreviated OMY, to genetic code expansion in bacteria. In this arrangement, an engineered bacterial protein-synthesis system reads OMY as a signal: an orthogonal aminoacyl-tRNA synthetase charges a suppressor tRNA, and the tRNA inserts OMY at engineered amber stop codons, allowing translation to continue and produce functional proteins.

The results establish a communication system under controlled laboratory conditions, not yet a field-ready method for regulating microbes in agricultural soils. The researchers first installed the genetic code expansion machinery in B. subtilis PY79, a laboratory model strain. They used an engineered Methanocaldococcus jannaschii tyrosyl-tRNA synthetase and its corresponding suppressor tRNA, together with a fluorescent reporter containing an in-frame amber stop codon.

Without OMY, translation was expected to stop at that codon; with OMY, the reporter should be completed and become fluorescent. Exogenous OMY produced dose-dependent activation, including twofold induction at 1 micromolar and fivefold induction at 10 micromolar, although incomplete termination created substantial background signal. Adding a second amber codon reduced background almost 55-fold and yielded a 3,100-fold difference between conditions with and without OMY.

The number and position of stop codons therefore emerged as an important design variable rather than a universal setting. The system was not limited to a fluorescent marker. The researchers inserted an amber codon into the sfp gene, which encodes the enzyme required to activate the surfactin synthetase machinery.

Without OMY, the engineered B. subtilis strain produced no detectable surfactin; with 500 micromolar OMY, it produced approximately 0.18 grams per litre. OMY itself did not alter bacterial growth under the tested conditions. These experiments demonstrate that a plant-derived chemical can control bacterial protein production and a microbially synthesized compound.

They do not yet show that the platform can improve crop yield, control plant pathogens or safely deploy engineered organisms in an open agricultural environment. The result also remained dependent on the engineered translation machinery and the placement of amber codons in the targeted bacterial genes. On the plant side, the researchers engineered expression of the enzyme MfnG, which enables OMY biosynthesis.

Transient expression in Nicotiana benthamiana leaves produced a metabolite with the expected mass-to-charge ratio that coeluted with an OMY standard; no corresponding signal appeared in control leaves. They then generated stable Arabidopsis lines expressing an sfGFP–MfnG fusion under the constitutive UBQ10 promoter. Hydroponically grown transgenic seedlings released an average of 3.1 micromolar OMY into the surrounding medium, whereas none was detected from wild-type plants.

No optimization of metabolic flux, transport or tyrosine supplementation was required to reach this concentration in the laboratory setup. The finding establishes that a plant can manufacture and release the chemical signal under the tested growth conditions, without demonstrating how much would be available in a mature crop or a field rhizosphere. When the OMY-producing Arabidopsis plants were placed on agar with bacteria carrying an OMY-dependent NanoLuc reporter, luminescence appeared specifically around the transgenic roots.

In mixed plates containing transformed and wild-type seedlings, the signal remained concentrated around the OMY-producing plants, demonstrating genotype-specific activation rather than a general effect of seedling placement. OMY-independent bacteria also generated somewhat stronger signals near roots, which the researchers attributed to higher local bacterial density. The results nevertheless support spatial selectivity: chemical production by one plant could regulate nearby bacterial gene expression while producing a smaller signal around neighboring wild-type plants.

The researchers did not directly measure OMY concentration or bacterial cell density at the roots, so the relative contributions of the chemical gradient and local accumulation of cells remain unresolved. Constitutive OMY production carried a biological cost. Arabidopsis lines overexpressing MfnG had shorter primary roots, reduced lateral-root density, impaired gravitropism, delayed germination and a more rapid decline in seed viability with age.

Lateral-root density was the number of emerged lateral roots normalized by total primary-root length, rather than a direct count of lateral roots. Exogenous OMY inhibited wild-type root growth in a concentration-dependent manner, with a half-maximal inhibitory concentration of approximately 94–101 micromolar. The investigators found no evidence that the effect resulted from depletion of aromatic amino acid precursors or elevated misincorporation of OMY into native proteins.

Aromatic amino acid supplementation did not rescue the phenotype, and proteomics detected no significant increase in O-methylated tyrosine residues. The mechanism of toxicity therefore remained unresolved, although the severity of the measured effects was reduced when OMY production was restricted in space or time. The team next restricted when and where the signal was made.

A root-cap-specific promoter produced OMY in approximately one-fifth of root cells, while an estradiol-inducible system allowed production to be triggered by treatment. Both conditional designs produced less severe growth abnormalities than constitutive expression and brought root growth, lateral-root density, gravitropism and germination close to wild-type values. Root-cap-specific plants released 0.73 micromolar OMY in the tested hydroponic medium and activated the bacterial reporter in cocultivation, although their signal was weaker than that from constitutively expressing plants.

More sensitive flow-cytometry measurements detected reporter induction at OMY concentrations as low as 0.05 and 0.5 micromolar. The experiments therefore showed that spatially restricted production could preserve a detectable response without requiring the larger accumulation measured under constitutive expression. The inducible Arabidopsis system also produced a strong response after activation.

Plants treated with 20 micromolar estradiol generated approximately 18.7 times more maximum bacterial luminescence than uninduced plants. The peak-to-baseline ratio did not differ significantly between treated and untreated groups because the baseline did not decay equally between plant positions, showing why maximum intensity and spatial contrast must be interpreted separately. Collectively, the conditional experiments demonstrate a way to reduce the plant fitness costs while retaining measurable bacterial control.

They do not establish that either promoter would provide useful control in mature crops exposed to natural environmental and microbial variation. In particular, the estradiol system depends on an external chemical trigger, while the root-cap promoter is linked to a specific tissue and developmental context. To move beyond the model laboratory strain, the researchers engineered B. subtilis UD1022, a wild isolate known to colonize Arabidopsis roots.

Its response to OMY was dose-dependent, but its optimal reporter configuration differed from that of PY79: a single amber codon produced the best fold change in UD1022, whereas two codons were most effective in PY79. In autoclaved soil supplied with 500 micromolar OMY, UD1022 reporter fluorescence increased 41-fold by day three. When OMY-producing and wild-type Arabidopsis grew in the same sterilized soil, bacteria exposed to the engineered plants had a median fluorescence of 802 arbitrary fluorescence units, compared with 530 for wild-type plants, and the fluorescent bacterial fraction rose from 9.8% to 36.1%.

This experiment showed transfer of plant-derived OMY in soil, but its sterilized, single-strain setting excluded interactions with a natural microbial community. The platform was also tested in agriculturally relevant plants through a rapid hairy-root transformation method. Composite tomato plants carrying MfnG in transgenic hairy roots and composite poplar plants with the same construct produced luminescence from OMY-dependent B. subtilis reporters specifically at fluorescent-marker-positive roots.

These results show that OMY biosynthesis and bacterial activation can extend beyond Arabidopsis to tomato and poplar, but the shoots in the composite plants remained wild type, and the work did not produce stable, whole-plant transgenic crops. Field use will require testing in nonsterile soils, where OMY may be degraded or transformed by resident organisms, engineered bacteria may face competition, and unintended activation of native organisms must be assessed. The study’s central achievement is therefore a cross-kingdom proof of concept: plants can synthesize a chemical signal that selectively activates engineered bacterial genes, while tissue-specific and inducible production can limit the plant-level costs.

Subject of Research: Genetic code expansion for plant-directed control of engineered soil bacteria

Article Title: Genetic code expansion enables plant-directed control of bacterial activity

Article References: Zhong, V., Jones, M. A., Cabales, A., Gevorgyan, A., Inckemann, R., Johnson, A. A., Karunadasa, S. S., Forti, A. M., Xu, S.-L., Kunjapur, A. M., & Brophy, J. A. N. (2026). Genetic code expansion enables plant-directed control of bacterial activity. Nature Biotechnology. https://doi.org/10.1038/s41587-026-03360-5

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03360-5

Keywords: genetic code expansion, noncanonical amino acids, plant-microbe signaling, synthetic biology, Bacillus subtilis, Arabidopsis, plant biotechnology, soil bacteria, OMY, surfactin, Genetic, code