Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure.Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia.Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process.Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction.

Nitric oxide (NO) is crucial in animal- and plant-pathogen interactions, during which it participates in host defense response and resistance. Indications for the presence of NO during the symbiotic interaction between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti have been reported but the role of NO in symbiosis is far from being elucidated. Our objective was to understand the role or roles played by NO in symbiosis. As a first step toward this goal, we analyzed the bacterial response to NO in culture, using a transcriptomic approach. We identified approximately 100 bacterial genes whose expression is upregulated in the presence of NO. Surprisingly, most of these genes are regulated by the two-component system FixLJ, known to control the majority of rhizobial genes expressed in planta in mature nodules, or the NO-dedicated regulator NnrR. Among the genes responding to NO is hmp, encoding a putative flavohemoglobin. We report that an hmp mutant displays a higher sensitivity toward NO in culture and leads to a reduced nitrogen fixation efficiency in planta. Because flavohemoglobins are known to detoxify NO in numerous bacterial species, this result is the first indication of the importance of the bacterial NO response in symbiosis.

SummaryNitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis.Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored.An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence.Together, our results strongly suggest that NO is a signal in developmental as well as stressinduced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.