Osmotic stress activates nif and fix genes and induces the Rhizobium tropici CIAT 899 Nod factor production via NodD2 by up-regulation of the nodA2 operon and the nodA3 gene

Abstract

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899.

Fig 1. Percentage of genes from each Gene Ontology category differentially expressed with 400 mM mannitol.

The percentage of genes shown corresponds to those genes present in the Gene Ontology database. Asteriks (*) indicate those categories with higher percentages.

Fig 2. Genes and processes differentially regulated with 400 mM mannitol in R. tropici CIAT 899.

Red labelled and black labelled gene names and arrows indicate down-regulation and up-regulation, respectively.

Fig 3

A. β-galactosidase activity of R. tropici CIAT 899 carrying plasmid pMP240 under different mannitol concentrations (55–500 mM). B. β-galactosidase activity of R. tropici CIAT 899 and its nodD mutant derivative strains carrying plasmid pMP240 under 400 mM mannitol. Black, blue, red, green, gray and white bars represent β-galactosidase activity levels of the wild-type, nodD1 mutant, nodD2 mutant, nodD3 mutant, nodD4 mutant and nodD5 mutant strains, respectively. Expression data were individually compared with the expression without inducing molecules of the wild-type strain using the Mann-Whitney non-parametrical test. The asterisks (*) indicate a significant difference at the level α = 5%. C. Thin-layer chromatography analysis of Nod factors produced by R. tropici CIAT 899 and the nodD2 mutant grown under control and 400 mM mannitol conditions. D. Biological activity assay of the NF purified from the wild-type and the nodD2 mutant strain applied to common bean roots. The number of nodule primordia was individually compared to those primordia formed by the wild-type strain using the Mann-Whitney non-parametrical test. The asterisks (*) indicate a significant difference at the level α = 5%.