The Rhizobium tropici CIAT 899 NodD2 protein regulates the production of Nod factors under salt stress in a flavonoid-independent manner

Pablo del Cerro,Manuel Megías,Francisco Javier Ollero,Francisco Pérez-Montaño,Antonio Gil-Serrano,Francisco Javier López-Baena,Mariangela Hungria

doi:10.1038/srep46712

Abstract

In the symbiotic associations between rhizobia and legumes, NodD promotes the expression of the nodulation genes in the presence of appropriate flavonoids. This set of genes is implied in the synthesis of Nodulation factors, which are responsible for launching the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. This strain produces Nodulation factors under abiotic stress such as acidity or high concentration of salt. Genome sequencing of CIAT 899 allowed the identification of five nodD genes. Whereas NodD1 is essential to nodulate Leucaena leucocephala, Lotus japonicus and Macroptilium atropurpureum, symbiosis with P. vulgaris and Lotus burtii decreased the nodule number but did not abolish the symbiotic process when NodD1 is absent. Nodulation factor synthesis under salt stress is not regulated by NodD1. Here we confirmed that NodD2 is responsible for the activation of the CIAT 899 symbiotic genes under salt stress. We have demonstrated that NodD1 and NodD2 control the synthesis of the Nod factor necessary for a successful symbiosis with P. vulgaris and L. burtii. This is the first time that NodD is directly implied in the activation of the symbiotic genes under an abiotic stress.

Highlights

Flavonoids are phenylpropanoid metabolites exuded from plant roots, often in response to elicitors generated by microorganisms present in the rhizosphere[1]
Guasch-Vidal et al.[27] demonstrated that even in the absence of inducer flavonoids, CIAT 899 was able to induce the synthesis of Nod factors (NF) under salt stress conditions in a nodD1-independent manner
To determine which of these five nodD genes induces the synthesis of NF under salt stress condition, plasmid pMP240 harbouring the nodA promoter of R. leguminosarum bv. viciae fused to the lacZ gene was transferred by conjugation to the CIAT 899 wild-type strain and to the nodD mutant derivative strains (Table S1). β-galactosidase assays showed similar activation levels in the presence of NaCl (300 mM) in all strains (Figure S2), with the exception of the strain affected in nodD2, in which the up-regulation of the nodA gene was only detected in cultures supplemented with apigenin but not in the presence of salt (Fig. 1A)

Summary

Introduction

Flavonoids are phenylpropanoid metabolites exuded from plant roots, often in response to elicitors generated by microorganisms present in the rhizosphere[1]. The mutation in the nodD2 gene provoked a significant decrease in nodule number in P. vulgaris, suggesting the putative implication of NodD2 in the symbiotic process[21,22] Main features of this broad host-range rhizobial strain include its high tolerance to environmental stresses such as high temperature, acidity and salinity[18,19,23], and its capacity to produce a large variety of NF24. A transcriptomic analysis of CIAT 899 grown in the presence of apigenin (a CIAT 899 inducer flavonoid) or salt showed similarities in gene activation patterns for the nod genes, and for other important symbiotic genes located downstream of their respective NB: nodA1BCSUIJH (NB1), nodA2hsnTnodFE (NB2), nodM (NB3), y4wEF (NB4) and two genes with still unknown functions (NB5) For this reason, the salt-dependent production of these symbiotic molecules could suggest a strategy of CIAT 899 to ensure nodulation under salt stress conditions[28]. We have demonstrated that both NodD1 and NodD2 control the entire NF synthesis in CIAT 899 and are necessary for a successful symbiosis with P. vulgaris and L. burtii

Methods

Results

Conclusion