Synthetic microbe-to-plant communication channels

Abstract

Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a "sender device" in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a "receiver device" in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia.

Fig. 1. Design of bacteria-to-plant communication.

a Bacteria receive a signal in the soil (grey diamonds) that induces the release of the communication signal (orange circles) to be sensed by regulatory proteins in the plant cell. b The communication channel is modular. To change the signals to which the plant responds, it simply can be grown with different bacteria engineered to connect different sensors (A or B) to the synthesis of the chemical used for communication. The bacterium can also integrate these signals using genetic circuits; an OR gate is shown. c The plant pC-HSL receiver device. The genetic part DNA sequences are provided in Supplementary Data 1. d The modifications to the prokaryotic RpaR regulator (orange) are shown to make it functional in plants. e Phenotypic comparison of A. thaliana wild-type to that carrying the pC-HSL receiver (A. thaliana 315_14_5). The plants were induced for 24 h in MS media in the hydroponic system. f Phenotypic comparison of wild-type A. thaliana with that carrying the pC-HSL receiver (A. thaliana 315_14_5_1) grown in soil. The data points represent replicates performed with different plants (n = 6 for height, fresh weight, dry weight, number of rosette leaves, number of primary shoots and primary root length; n = 13–14 for primary root length and lateral root density) and the bars represent the means of these points. Statistically significant differences were determined using two-tailed Student’s t test (ns, not significant P > 0.05). Source data are provided as a Source Data file.

Fig. 2. The A. thaliana pC-HSL receiver.

a Fluorescence microscopy images of the induction of the pC-HSL receiver expressing GFP (green) and stained with propidium iodide (PI, red). A. thaliana 315_14_5_1 was induced with 1 µM pC-HSL for 24 h in a hydroponic system (Methods). Images are representative of experiments performed on three different days with different plants (Supplementary Fig. 7). b Response function of the A. thaliana pC-HSL receiver. Each color represents experiments repeated on 6 different days with different plants (A. thaliana 315_14_5). All the data were fit to Eq. 1 (parameters in Supplementary Table 1). Raw images used to calculate the MPI are provided in Supplementary Fig. 12. c Orthogonality of the pC-HSL receiver. A. thaliana 315_14_5 was induced with 100 µM of each inducer (p-coumarate, OHC14-HSL, OC6-HSL, OC12-HSL and pC-HSL) for 24 h in a hydroponic system (Methods). The  points represent replicates performed with different plants (A. thaliana 315_14_5) on different days (n = 6 for pC-HSL and uninduced, n = 3 for other HSLs, and n = 2 for p-coumarate) and the bars represent the means of these points. d Microscopy images of the induction of the pC-HSL receiver in soil. A. thaliana 315_14_5_1 was grown and induced by watering the plants with 100 µM pC-HSL in sterile soil (Methods). Images are representative of experiments performed on three different days with different plants. e Induction of the A. thaliana pC-HSL receiver in soil. The bars represent the mean fluorescence from three plants grown on different days (A. thaliana 315_14_5). Raw images used to calculate the MPI are provided in Supplementary Fig. 16. There is a 13-fold upregulation between induction with 0 µM of pC-HSL and 100 µM of pC-HSL. Statistical significance was determined using two-tailed Student’s t test (***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant P > 0.05). Source data are provided as a Source Data file.

Fig. 3. Bacteria-to-plant communication to A. thaliana in hydroponics and in soil.

a The constitutive production of pC-HSL by P. putida and K. pneumoniae was first used to induce the receiver in plants. b Phenotypic comparison of A. thaliana 315_14_5_1 (Supplementary Table 3) grown in hydroponics with and without wild-type P. putida or wild-type K. pneumoniae (Methods). c The induction of the pC-HSL receiver in A. thaliana (A. thaliana 315_14_5_1) by P. putida or by K. pneumoniae constitutively producing pC-HSL (pTT337, Supplementary Fig. 35) in hydroponics (Methods) is shown. The data were extracted from the images in Supplementary Fig. 18. The points were obtained for n = 3 plants on different days and the bars represent the means of these points. d Induction of the A. thaliana pC-HSL receiver in plant roots in hydroponics (Methods). The induction by wild-type P. putida (left) and K. pneumoniae (right) was compared to when P. putida and K. pneumoniae constitutively produce pC-HSL (pTT337). Images are representative of experiments performed on three different days with different plants (A. thaliana 315_14_5_1). e Induction of the A. thaliana pC-HSL receiver by P. putida constitutively producing pC-HSL (pTT337) in sterile and non-sterile soil (Methods). P. putida was introduced either by seed inoculation or through watering (Methods). The data were extracted from the images in Supplementary Fig. 23. The points were obtained for n = 3 plants (A. thaliana 315_14_5_1) on different days and the bars represent the means of these points. f Microscopy images of the induction of the A. thaliana pC-HSL receiver by P. putida from panel e. Statistical significance was determined using two-tailed Student’s t test (***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant P > 0.05). Source data are provided as a Source Data file.

Fig. 4. Bacterial sensing and communication to A. thaliana and potato.

a.A. thaliana responds to P. putida engineered to relay pC-HSL upon sensing inducers IPTG or aTc (pTT409 and pTT410, Supplementary Fig. 35) in hydroponics (Methods). The data were extracted from the images in Supplementary Fig. 28. The points were obtained for n = 3 plants (A. thaliana 315_14_5_1) on different days and the bars represent the means. Microscopy images match the blue-circled replicate. b.P. putida and K. pneumoniae were engineered to detect arsenic (pTT417, Supplementary Fig. 37) and communicate the output to the A. thaliana pC-HSL receiver in hydroponics (Methods). The data were extracted from the images in Supplementary Fig. 29. The points were obtained for n = 3 plants (A. thaliana 315_14_5_1,) on different days and the bars represent the means. Microscopy images match the red-circled replicate. c A. thaliana 315_14_5_1 was co-cultured with P. putida sTT659 (Supplementary Table 4) engineered with an OR gate (pTT434, Supplementary Fig. 38), producing pC-HSL in response to either aTc or IPTG. The data were extracted from the images in Supplementary Fig. 30. Growth conditions and replicates were the same as in part a. P values for each of the induced state compared to the uninduced state are: +IPTG/-aTc: 0.02,-IPTG/+ aTc: 0.05, + IPTG/+ aTc: 0.03. Microscopy images match the blue-circled replicate. d A. thaliana 315 co-cultured with two strains of P. putida, each producing pC-HSL in response to a different signal. Strains, growth conditions, and replicates were the same as in part a. The data were extracted from the images in Supplementary Fig. 31. P values for each of the induced state compared to the uninduced state are: +IPTG/-aTc: 0.008,-IPTG/+ aTc: 0.010, + IPTG/+ aTc: 0.0007. Microscopy images match the yellow-circled replicate. Statistical significance was determined using two-tailed Student’s t test (***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant P > 0.05). Source data are provided as a Source Data file.