Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1

Abstract

Legumes associate with rhizobia to form nitrogen (N)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N-fixing bacteroids by producing defensin-like nodule-specific cysteine-rich peptides (NCRs) [3, 4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules [9]. Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S. meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S. meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions.

Symbiotic Outcome Modified by the Diversification from 7 to over 700 Nodule-Specific Cysteine-Rich Peptides

“…From the meristem (ZI) there are four distinct zones in indeterminate nodules whose ploidy level increases: the infection zone (ZII), the interzone (IZ), the nitrogen fixation zone (ZIII) and the senescence zone (ZIV). Zonal changes are also marked by specific expression of thioredoxin-s1 (Trx-s1) [9], the demethylase gene, DEMETER, and different levels of DNA methylation [10]. Transcriptomic analysis following laser-dissection capture of the different zones also suggest a varied and zone-specific expression of different NCRs [11,12].…”

“…For example, one plant-derived thioredoxin (Trx-s1) has been found to be induced in the nodule infection zone and to interact with several NCR peptides. While inactivation of Trx-s1 in the host plant negatively affects TBD, the ectopic expression of this enzyme in the endosymbiont partially enhances TBD in Trx-s1 inactivated plants [9]. It is likely that these redox enzymes play a crucial role in maintaining the appropriate cellular redox state including that of the multiple cysteine residues in NCR peptides, thus regulating their functions.…”

Glutathione Deficiency in Sinorhizobium meliloti Does Not Impair Bacteroid Differentiation But Induces Early Senescence in the Interaction With Medicago truncatula

“…Single events were recorded and analyzed with BD FACSDiva v6.1.3 (BD BioSciences) and Kaluza v1.2 software (Beckman Coulter, Villepinte, France), respectively. Experiments were performed with biological triplicates and the recording of more than 200,000 events for each sample to ensure the statistical robustness of each assay (Ribeiro et al, 2017).…”

“…The bacteroid differentiation is correlated to an increase in size and in DNA content of the bacteroid compared to the bacterium (Mergaert et al, 2006). These criteria could be used to differentiate bacteria from bacteroids using flow cytometry (Ribeiro et al, 2017). To test whether GSH deficiency in the bacteria impairs the bacteria differentiation, we have analyzed the bacteroid and bacterial content in nodules from control plants and SmgshB inoculated plants at 10 and 20 dpi using flow cytometry (Figure 2).…”

“…Moreover, the expression of two bacterial genes nifD (nitrogenase molybdenum-iron protein alpha chain) and nifH (nitrogenase iron protein), encoding nitrogenase subunits and expressed in nitrogen-fixing bacteroids, was analyzed in control and SmgshB nodules. Trx s1 is specifically expressed during the symbiotic interaction between M. truncatula and S. meliloti (Alkhalfioui et al, 2008) and its expression is localized in the nodule infection zone (Ribeiro et al, 2017). NCR001 is considered as a specific marker of the nitrogen fixation zone (Mergaert et al, 2003) like meliloti 2011 strain or SmgshB mutant strain after 10 or 20 dpi.…”