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. 2022 Apr 19;119(16):e2117465119.
doi: 10.1073/pnas.2117465119. Epub 2022 Apr 11.

Engineered plant control of associative nitrogen fixation

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Engineered plant control of associative nitrogen fixation

Timothy L Haskett et al. Proc Natl Acad Sci U S A. .

Abstract

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

Keywords: barley; nitrogen fixation; rhizobium; rhizopine; symbiosis.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Coexpression of the myo-inositol uptake genes intBC enhances rhizopine uptake and sensitivity of the transcriptional response. (A) Genetic schematics (not to scale) for the second-generation rhizopine receiver plasmid pSIR02 and a near identical plasmid lacking intBC. (B) PmocB promoter activity (RFU, defined as GFP fluorescence/ODλ600 nm) was measured in populations of Ac carrying pSIR01 or pSIR02. (C) Flow-cytometry analysis of AcCherry (pSIR02) cells induced with 10 μM SI or not induced after 18-h incubation (n = 4). Cells exhibiting GFP fluorescence above 20,000 a.u. were considered as GFP+. Supplementary statistics supporting flow cytometry are provided in Dataset S1. (D) The intracellular contents of thrice washed Ac cells grown in the presence of 100 μM SI was bio-assayed for SI using S. meliloti carrying pOPS0046 and pOPS0385 as a sensor. Error bars represent one SEM (n = 3). Independent two-tailed Student’s t tests were used to compare means. Exact values for P > 0.05 are given, ***P < 0.001.
Fig. 2.
Fig. 2.
Rhizopine-inducible expression on RhiP barley roots and in the rhizosphere. (A) Schematic representation of the experimental design to monitor PmocB expression on barley roots and in the rhizosphere. AcCherry cells are marked with a constitutively expressed mCherry gene allowing tracking. The strain additionally carries plasmid pSIR02. (B) Maximum-intensity projections showing mCherry and GFP fluorescence by AcCherry (pSIR02) cells colonizing the rhizoplane of wild-type and T2 RhiP barley at 9 dpi. Projections are representative of 15 images acquired from 3 whole lateral roots excised from 5 plants per treatment. (C) Flow-cytometry analysis of AcCherry (pSIR02) cells recovered from the rhizoplane and endosphere (RA) or rhizosphere (RS) of wild-type (blue), T1 RhiP (green), and T2 RhiP (brown) barley plants at 9 dpi (n = 5). Cells exhibiting mCherry fluorescence above 5,000 a.u. were analyzed. GFP+ cells are defined as those exhibiting GFP fluorescence above the mean 99th percentile of the populations colonizing wild-type barley. Supplementary statistics supporting flow cytometry are provided in Dataset S5. (D) In situ PmocB promoter activity (median GFP fluorescence intensity) was measured for R2 GFP+ bacteria in C. Error bars represent one SEM. Independent two-tailed Student’s t tests were used to compare means. Exact values for P > 0.05 are given, *P < 0.05, **P < 0.01.
Fig. 3.
Fig. 3.
In vitro rhizopine control of N2-fixation. (A) Genetic schematics (not to scale) for nifA-rpoN and nifAL94Q/D95Q rhizopine controller plasmids. (B) PnifH promoter activity (RFU, defined as mCherry fluorescence/ODλ600 nm) was measured in populations of Ac and AcΔnifA (n = 3) carrying rhizopine controller plasmids and the reporter pOPS1218 grown in N2-fixing conditions (N-free UMS supplemented with 20 mM succinate with a starting headspace of 3% O2). SI and NH4Cl was added to a final concentration of 10 μM and 10 mM, respectively, where indicated. (C) The same conditions were used to assess specific nitrogenase activity in Ac and AcΔnifA carrying rhizopine controller plasmids. Error bars represent one SEM (n = 3). Independent two-tailed Student’s t tests were used to compare means. Exact values for P > 0.05 are given, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Fig. 4.
Fig. 4.
In situ plant-dependent control of associative N2-fixation (A) Schematic representation of the experimental design to monitor rhizopine-dependent PnifH expression on barley roots. AcCherry cells were marked with a constitutively expressed mCherry gene allowing tracking. The strain additionally carries plasmid pSIN03. (B) Maximum-intensity projections showing mCherry and GFP fluorescence by AcCherryΔnifA (pSIN03) cells colonizing the rhizoplane of wild-type and T2 RhiP barley at 9 dpi after 24-h exposure to 1% O2 in the headspace. Projections are representative of 15 images acquired from 3 whole lateral roots excised from 5 plants per treatment. (C) Flow-cytometry analysis of AcCherry (pSIN03) cells recovered from the rhizopine and endosphere (RA) or rhizosphere (RS) of wild-type (blue), T1 RhiP (green), and T2 RhiP (brown) barley plants at 9 dpi after 24-h exposure to 1% O2 in the headspace (n = 5). Cells exhibiting mCherry fluorescence above 1,500 a.u. were analyzed. GFP+ cells are defined as those exhibiting GFP fluorescence above the mean 99th percentile of the populations colonizing wild-type barley. Supplementary statistics supporting flow-cytometry are provided in Dataset S7. (D) In situ PnifH promoter activity (median GFP fluorescence intensity) was measured for R2 GFP+ bacteria in C. (E) In situ ARAs performed at 9 dpi after 48-h exposure to 1% O2 in the headspace. Error bars represent one SEM. Independent two-tailed Student’s t tests with Holm–Bonferroni adjusted P values were used to compare means. Exact values for P > 0.05 are given, **P < 0.01.

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