A Bradyrhizobium diazoefficiens rsh mutant nodulates soybean in the presence of high concentrations of combined nitrogenA Bradyrhizobium diazoefficiens rsh mutant nodulates soybean in the presence of high concentrations of combined nitrogen

TORRES TEJERIZO, GONZALO A.; ITURRALDE, ESTEBAN TOMÁS; PÉREZ GIMÉNEZ, JULIETA; LODEIRO, ANÍBAL R.

Córdoba

Congreso; XXXII Reunión Argentina XVI Congreso Latinoamericano de Fisiología Vegetal; 2018

Sociedad Argentina de Fisiología Vegetal

Bacteria respond to stress using various transcriptionally regulated systems, being the stringent response among the most important. This response is triggered by accumulation of the second messenger (p)ppGpp generated and degraded by RelA/SpoT proteins respectively. In Bradyrhizobium diazoefficiens, the homologue to RelA/SpoT is a bifunctional enzyme called Rsh. To study its role in free-living and symbiotic rhizobia, we obtained a Δrsh mutant which had similar growth kinetics to the wild-type, but died faster in C- and N-free mineral solution, indicating that the expression of rsh was required for bacteria adaptation to nutrient-deprivation stress. Moreover, although the Δrsh mutant was motile and expressed both of the B. diazoefficiens flagellar systems, it produced swimming halos with a different rings pattern than the wild-type, and was defective in swarming. The Δrsh mutant was able to nodulate soybean but fixed less nitrogen compared to the wild type. When the Δrsh mutant was co-inoculated with the wild-type, only 25% of the nodules were occupied by the mutant. Strikingly, the Δrsh mutant nodulated normally in the presence of 10mM NH4NO3, a condition in which the wild-type only formed empty nodules. These results indicate that Δrsh skips the soybean plant inhibition of nodulation by combined N. The detection of combined N and nodules quantity in soybean is exerted by related CLE peptides, named GmNIC for the combined N response, and GmRIC1 and GmRIC2 for the nodules quantity response. We observed that, although the accumulation of GmNIC transcript in response to combined N did not respond to the mutation in the bacteria, the Δrsh mutant induced an accumulation of GmRIC1 and GmRIC2 in N-free cultivated plant roots, which was absent in those cultivated with NH4NO3. Therefore, some pathways of the autoregulation of nodulation in soybean might have a link with the stringent response of the inoculated rhizobia and concomitantly, with the determinants that the bacteria are expressing in this condition.