NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis

PLOS ONE, Dec 2019

The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.

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NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis

April NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis Pablo del Cerro 0 1 Amanda A. P. Rolla-Santos 1 Rocío Valderrama-Fernández 1 Antonio Gil- Serrano 1 Ramón A. Bellogín 0 1 Douglas Fabiano Gomes 1 Francisco Pérez-Montaño 0 1 Manuel Megías 0 1 Mariangela Hungría 1 Francisco Javier Ollero 0 1 0 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Sevilla , Spain, 2 Embrapa Soja, Cx. Postal 231, 86001-970, Londrina, Paraná, Brazil , 3 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. Sevilla , Spain 1 Editor: Francisco Martinez-Abarca, Estacion Experimental del Zaidin-CSIC , SPAIN The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wildtype strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species. - OPEN ACCESS Data Availability Statement: All relevant data are in the body of the manuscript and in the supporting information. Funding: This work was supported by the Project AGL2012-38831 of the Ministerio de Economía y Competitividad (Spanish Government, www.mineco. gob.es), CNPq (National Council for Scientific and Technological Development, http://cnpq.br/, Science without Borders 400205/2012-5). AAPR-S acknowledges a postdoctoral fellowship from CNPq (National Council for Scientific and Technological Development, http://cnpq.br/, Science without Borders 400205/2012-5). MH a research fellow from CNPq (NPq (National Council for Scientific and Technological Development, http://cnpq.br/). FPM a postdoctoral fellowship from VPP (University of Seville, https://investigacion.us.es/secretariadoinvestigacion/plan-propio). PdC a FPU fellowship from Ministerio de Economía y Competitividad (FPU14/00160, Spanish Government, www.mineco. gob.es). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Introduction The establishment of the symbiosis between rhizobia and their specific host legumes involves highly complex events that culminate in the formation of nodules and in the establishment of the nitrogen fixation process. Nodulation requires a molecular dialogue between the bacteria and the host plants. The dialogue begins with the exudation of molecules from the legume roots, mostly flavonoids, which are recognized by the specific rhizobium, inducing the transcription of a set of nodulation genes [ 1, 2 ]. These genes are responsible for the biosynthesis and secretion of Nod factors (NFs), identified as lipochitooligosaccharides, required for launching the nodulation and the nitrogen-fixation process [ 3–7 ]. A large number of bacterial transcriptional regulators actuate nodulation, the most important of which are the NodD proteins, which belong to the LysR-type transcriptional-regulator family. The nodD genes are constitutively expressed and their cognate proteins are responsible for the recognition of suitable flavonoids, which start the transcription of the nodulation genes [ 8, 9 ]. Another important group of transcriptional regulators is the family of metal-sensing regulatory proteins. In this case, a specific metal-ion union regulates the protein’s allosteric conformation and modulates the expression of several target ge (...truncated)


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Pablo del Cerro, Amanda A. P. Rolla-Santos, Rocío Valderrama-Fernández, Antonio Gil-Serrano, Ramón A. Bellogín, Douglas Fabiano Gomes, Francisco Pérez-Montaño, Manuel Megías, Mariangela Hungría, Francisco Javier Ollero. NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis, PLOS ONE, 2016, Volume 11, Issue 4, DOI: 10.1371/journal.pone.0154029