Abstract
Aims
Bradyrhizobium diazoefficiens is a soil bacterium capable of establishing nitrogen-fixing symbiosis with soybean plants, an important crop for food production worldwide. This interaction is under the control of several regulatory factors that affect the bacterial efficiency to nodulate legume plants and fix nitrogen within root nodules. To date, the function of the NtrYX two-component system in B. diazoefficiens remains unclear. In this work, we investigated the role of NtrY in the symbiotic interaction of B. diazoefficiens with soybean plants.
Methods
We constructed a non-polar mutant of the sensor protein NtrY of the system, and then analysed the symbiosis of this mutant with soybean plants.
Results
We found that the ntrY mutant was defective in plant dry weight, nitrogen content and nitrogenase activity. Haem c-staining of cbb3 oxidase components showed a clear reduction in the expression of this terminal oxidase in ntrY bacteroids with respect to wild type bacteroids. Such defect in cbb3 expression correlated with a decreased respiratory capacity of the bacteroids produced by the ntrY mutant.
Conclusions
These results suggest that the role of B. diazoefficiens NtrYX in symbiotic nitrogen fixation might be a consequence of its involvement in cbb3 oxidase expression in bacteroids.
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Abbreviations
- CFU:
-
Colony formation units
- HK:
-
Histidine kinase
- Lb:
-
leghaemoglobin
- LB:
-
Luria–Bertani medium
- NDWP:
-
Nodule dry weight per plant
- NNP:
-
Nodule number per plant
- OD500:
-
Optical density-500 nm
- PPF:
-
Photosynthetic Photon Flux
- PSY:
-
Peptone–salts–yeast extract
- RR:
-
Response regulator
- SDWP:
-
Shoot dry weight per plant
- TCS:
-
Two component system
- TMPD:
-
N,N,N′,N′-tetramethyl-p-phenylenediamine
- WT:
-
Wild-type
- YEM:
-
Yeast-extract-mannitol medium
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Acknowledgements
We are grateful to Paula Giménez, Silvana Tongiani and Abel Bortolameotti (members of CPA CONICET at IBBM) and Rubén Bustos (UNLP). We also thank Susana Jurado and Roxana Peralta (Servicio Central de Microscopía, Facultad de Ciencias Veterinarias, UNLP, La Plata, Argentina) for excellent assistance with electron microscopy. We are also grateful to Germán Tortosa (EEZ, CSIC, Granada, Spain) for excellent technical assistance and Cristina Sanchez for fixN primers design. Thanks are also due to A Di Maggio for careful copyediting of the manuscript and Gabriel Robles-Luna for his help in image editing.
Funding
This work was supported by the Agencia Nacional de Promoción de la Investigación Científica y Tecnológica (ANPCyT; project PICT 2013–2864), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secyt-UNLP, Argentina. MJD received financial support from the European Regional Development Fund (ERDF), cofinanced grants AGL2013–45087-R and AGL2017–85676-R from the Ministerio de Economía y Competitividad (Spain) and PE2012-AGR1968 from the Junta de Andalucía. MFL was supported by fellowships from CONICET and by a travel grant from Secyt-UNLP. VAH was supported by a fellowship from CONICET. SLG is researcher at CONICET.
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Fig. S1
Construction of a B. diazoefficiens ntrY deletion mutant. (A) Scheme of the strategy followed to obtain the ntrY deletion mutant (LP4489) without alteration of the reading frame. (B) Verification of the deletional mutation by PCR using specific primers on genomic DNA of wild-type (USDA 110) or ntrY mutant (LP 4489) strains. Primers 4489check FW and RV hybridize to genomic regions outside the mutation site generating products of 2678 pb on the wild-type strain and 413 pb if the deletion was successful. 1, PCR product using USDA110 DNA as template; 2, PCR product using LP4489 DNA as template. A DNA ladder (M) was used to verify the size of the PCR fragments obtained. Arrows depict positions and orientation of the PCR primers used for mutagenesis and checking (see Table S1 and Material and methods). (PNG 318 kb)
Fig. S2
Haem-staining relative band intensity analysis. Image analysis of free-living samples was performed using the ImageJ gel plot tool. Results show the relative intensity of FixP-FixO/c1 (A) or CycM (B) bands in the ntrY mutant compared with the USDA110 wild-type strain as the mean and standard error of three independent gels. (PNG 678 kb)
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López, M.F., Hegel, V.A., Torres, M.J. et al. The Bradyrhizobium diazoefficiens two-component system NtrYX has a key role in symbiotic nitrogen fixation of soybean plants and cbb3 oxidase expression in bacteroids. Plant Soil 440, 167–183 (2019). https://doi.org/10.1007/s11104-019-04067-0
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DOI: https://doi.org/10.1007/s11104-019-04067-0