A BRADYRHIZOBIUM JAPONICUM USDA 110 RSH (RELA/SPOT HOMOLOG) MUTANT NODULATES SOYBEAN IN THE PRESENCE OF COMBINED NITROGEN, BUT IS ALTERED IN N2-FIXATION.

PÉREZ GIMÉNEZ, JULIETA; ESTRADA NAVARRETE, GEORGINA; COVELLI, J.M.; BARRAZA CELIZ, AARÓN; LÓPEZ, M.F.; QUELAS, J. I.; SÁNCHEZ, FEDERICO; LODEIRO, A. R.

Munich

Conferencia; 10th European Nitrogen Fixation Congress (ENFC); 2012

Complex regulatory networks allow bacterial adaptation to diverse environmental challenges,among them nutrient stress, which is coped by the stringent response. This response isregulated by the intracellular (p)ppGpp level, which is determined by guanosinetetraphosphate synthase/hydrolase activities encoded in relA/spoT or rsh genes. In B.japonicum USDA 110, bll5065 is the unique ORF encoding an Rsh homolog. To study its rolein free-living and symbiotic rhizobia, we obtained the mutant LP 5065, in which bll5065 wasinterrupted by a Km-resistance cassette. This mutant had similar growth kinetics to the wildtype,but died faster in C- and N-free mineral solution.The stringent response may be triggered in the symbiotic interaction with soybean roots,because rhizobia face acid and nutrient stresses inside plant cells. We monitored transcriptaccumulation of some plant defense response-related genes in soybean roots by qRT-PCR.By 6-24 h after inoculation with LP 5065, transcript levels of these genes were lower than inroots inoculated with the wild-type, suggesting that release of pathogenesis-associatedfactors in response to the plant environment was diminished. However, LP 5065 developednormal nodules, smaller and in lower number than the wild-type. By contrast to Rhizobiumetli, LP 5065 bacteroids had similar contents of polyhydroxybutirate granules as the wildtype.Strikingly, LP 5065 was able to develope similarly normal nodules in the presence of 10or 20 mM NH4NO3, a condition in which the wild-type only formed empty nodules. Theseresults indicate that soybean plant defense responses could be related to inhibition ofnodulation by combined N.The mutant LP 5065 was affected in N2 fixation, as indicated by the 25 % lower ureidecontents in soybean leaves, and 44 % reduction in shoot dry weight of plants cultivatedwithout any combined N source. Therefore, the stringent response seems to be triggered inB. japonicum bacteroids, and required for normal N2-fixation activity.