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Most species of the legume family are able to form symbiotic associations with nitrogen-fixing soil bacteria, commonly referred to as rhizobia. These associations result in the development of a specialized organ of the root system, called the nodule, in which the bacteria invade the symbiotic plant cells. The early steps of the nodulation process have been studied extensively in the model legumes Medicago truncatula and Lotus japonicus. The very early steps of the plant–microbe interaction are highly similar in these two models; the genes and molecules involved in the reciprocal recognition of the two symbiotic partners are largely conserved (Oldroyd et al., 2011). The interaction starts with the exudation of flavonoids by the plant roots. These molecules are secondary metabolites recognized by the bacteria. In response, the rhizobia synthesize nodulation factors, called Nod factors, which are perceived in a host-specific manner by the plant to initiate the symbiotic process. At the root surface, root hairs curl to surround attached bacteria. These entrapped bacteria induce invagination of the plasma membrane and the formation of a progressing structure, called the infection thread, in which bacteria develop towards the root cortex. Nod factors trigger cortical cell dedifferentiation and the initiation of a nodule primordium. Infection threads reaching the primordium release bacteria within the plant cells. At this stage, nodule formation diverges between Medicago and Lotus. In Medicago, distal primordium cells will not become infected with rhizobia and will develop into an apical nodule meristem (Timmers et al., 1999). Persistence of the meristem results in continuously growing, indeterminate nodules. By contrast, the primordia formed on Lotus plants will not form a meristem, and therefore these nodules are of the determinate type.
Persistence of the apical meristem in indeterminate nodules of Medicago results in nodules harboring different cell types organized in zones (Vasse et al., 1990). The apical meristematic zone, or zone I, contains constantly dividing plant cells that ensure nodule growth (Timmers et al., 1999). In the infection zone, zone II, bacteria are released from the infection threads and invade the plant cells. The rhizobia and their surrounding plant-derived membrane form the symbiosomes. Within the symbiosomes, the bacteria undergo a morphological terminal differentiation involving cell elongation and genome amplification, a process which is governed by numerous plant nodule-specific cysteine-rich peptides, the so-called NCR peptides, which are addressed to the bacterium-containing compartments (Van de Velde et al., 2010). In the interzone II–III, bacteria fully differentiate into nitrogen-fixing bacteroids. In the fixation zone III, the bacteroids reduce atmospheric nitrogen and transfer the resulting ammonium to the plant cells (Vasse et al., 1990). Zone IV, also called the senescence zone, is formed in older nodules and is characterized by the degeneration of both the host cells and their bacteroids. A recent study has indicated that leaf senescence markers, such as cysteine proteinases, are induced in this zone (Van de Velde et al., 2006). The proximal zone V is characterized by the presence of saprophytic rhizobia which proliferate in the remains of the cells after their senescence (Timmers et al., 2000).
In M. truncatula, several genes involved in the first steps of the interaction, Nod factor signaling and the immediate downstream events have been described (Oldroyd et al., 2011). Far less is known in this model organism about the genes involved in the subsequent steps of the symbiotic process, once bacteria have been released into the plant cell cytoplasm. This contrasts with the knowledge acquired on other legume organisms, especially on L. japonicus, in which genes such as LjIGN1, FEN1 and SEN1 have been cloned and described (Kumagai et al., 2007; Hakoyama et al., 2009, 2012). In Lotus, bacteroids do not undergo terminal differentiation; thus, the study of Medicago mutants altered in these symbiotic steps is also of particular interest to understand the role of terminal differentiation. Starker et al. (2006) identified eight M. truncatula mutants altered in their capacity to support nitrogen fixation, that are, Fix− mutants (see also Mitra & Long, 2004). These mutants clustered into seven complementation groups and the corresponding genes were designated as dnf1 to dnf7, where dnf stands for does not fix nitrogen. Microarray experiments were performed on dnf1, 2 and 7, and the expression of nodulation markers was studied in all of them (Mitra & Long, 2004; Starker et al., 2006). Of the seven mutated genes involved in nitrogen fixation, only DNF1 has been cloned so far (Wang et al., 2010). This gene encodes a signal peptidase subunit required to target NCR peptides to the symbiosomes (Van de Velde et al., 2010).
In recent years, ambitious projects have been developed to produce genomic resources useful for the study of the M. truncatula–Sinorhizobium meliloti symbiosis. Amongst these are extensive transcriptomic data (El Yahyaoui et al., 2004; Van de Velde et al., 2006; Benedito et al., 2008; Maunoury et al., 2010; Moreau et al., 2011), large insertional mutant collections (Tadege et al., 2008; Iantcheva et al., 2009; Cheng et al., 2011), developed thanks to the establishment of mutagenesis protocols (d'Erfurth et al., 2003; Rakocevic et al., 2009), and the recently published M. truncatula genome sequence (Young et al., 2011).
We made use of these resources to identify a symbiotic plant gene, DNF2, and we demonstrate its involvement in the nitrogen fixation process. Phenotypes of knockout mutants were examined, and we showed that this gene is required for symbiosome persistence, to prevent plant defense reactions and to avoid early nodule senescence.
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- Materials and Methods
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Here, we have described a new symbiotic gene of M. truncatula, DNF2 (MTR_4g085800), that is required for nitrogen fixation, but not for nodule development. Three R108 insertion mutant lines harboring different mutant alleles of this gene were isolated and shown to be allelic to the previously described dnf2 Jemalong deletion mutant (Mitra & Long, 2004; Starker et al., 2006).
The Jemalong dnf2 mutant displays no acetylene reduction activity. In addition, this mutant does not support the expression of the bacterial gene, nifH, which is required for nitrogen fixation, suggesting that the maintenance of bacteroids may be affected (Starker et al., 2006). Consistent with the observation that DNF2 is maximally expressed at the onset of nitrogen fixation, our work revealed no differences between dnf2 and WT until bacteria were released from infection threads. At this stage, and in contrast with WT, rhizobia in dnf2 nodules do not differentiate into N2-fixing, fully elongated bacteroids. The electron microscopy study showed that, in the mutant, the peribacteroid space was larger and the bacterial cells remained small, confirming that they were not differentiated (Fig. 5b). The enlarged peribacteroid space indicates that the peribacteroid membrane is continuously produced in the mutant despite the cessation of bacterial enlargement and differentiation, suggesting that the two processes are disconnected. We also observed that bacteroids lose their viability (Fig. 7b,d) after being released into dnf2 symbiotic cells, in contrast with WT (Fig. 7a,c), possibly because they are influenced by the toxicity of NCR peptides, as is the bacterial bacA mutant (Haag et al., 2011), or because they trigger active defense-like responses in the dnf2 mutant. Consistent with the loss of bacterial viability, bacteroids appear to be degraded more rapidly, and the nitrogen-fixing zone is replaced by a zone of empty cells containing cellular debris in dnf2 nodules (Fig. 3e–h).
The cysteine proteinase marker TC100436 is expressed prematurely in the dnf2 mutant (Fig. 4a), consistent with the early degeneration of infected cells observed in zone III* (Fig. 3). Interestingly, senescence markers, such as cysteine proteinases, are induced in the L. japonicus sen1 mutant (Suganuma et al., 2004), as well as in ineffective nodules induced by a nifH::Tn5 S. meliloti mutant on alfalfa (Barsch et al., 2006). This suggests that senescence reflects a general response of nonfunctional nodules (Maunoury et al., 2010).
The impaired differentiation and bacterial death are correlated with activation of plant defense-like reactions in dnf2 mutant nodules, as indicated by the expression of the PR10 defense gene (Fig. 4b) and the accumulation of phenolic compounds (Fig. 6). PR10 induction and necrotic nodules are triggered when inefficient symbiosis results from boron deprivation in Pisum sativum and Phaseolus vulgaris (Redondo-Nieto et al., 2007). However, this phenotype is not a typical feature of Fix− mutants. Indeed, in the R108 background, the majority of Fix− mutants described in Pislariu et al. (2012) do not accumulate these compounds. dnf2-1 has also been suggested to accumulate phenolic compounds near the infection zone (Pislariu & Dickstein, 2007); thus, defense-like reactions are not likely to be a specific trait of dnf2 in the R108 background. PR10 gene induction and the accumulation of phenolic compounds were not observed in R108 WT nodules (Figs 4b, 6), even at 32 dpi when late senescent nodules are present, as attested by the induction of the selected senescence marker (Fig. 4a). Furthermore, defense-like reactions were not described in the L. japonicus sen1 mutant (Suganuma et al., 2004) or in nodules induced by the nifH or nifA Fix− mutants (Barsch et al., 2006; Maunoury et al., 2010). This supports the idea that, during aborted symbiosis, early senescence is not necessarily associated with defense-like reactions. The DNF2 gene encodes a PI-PLCXD-containing protein that has homologs in many plant species, including nonlegumes and nonmycorrhized plants, with multiple homologs in each. Although some homologs of DNF2 obviously fulfill roles unrelated to symbiosis, the nodule-specific expression of DNF2 and the mutant phenotype indicate that the DNF2 PI-PLCXD-containing protein has been recruited to play a specific role in the rhizobium–legume symbiosis with M. truncatula. The expression profile of the DNF2 homolog, LjNUF in L. japonicus (Kinoshita et al., 2004), suggests that it may play a similar role in the determinate nodule symbiosis.
PI-PLCXD-containing proteins are distinct from bona fide eukaryotic phosphatidylinositol-specific phospholipase C proteins (PI-PLCs). PI-PLCs have a multidomain architecture consisting of various regulatory domains and of a PLC catalytic core, which is composed of X and Y regions, separated by a linker. By contrast, PI-PLCXD-containing proteins only contain an X domain (Heinz et al., 1998). A subgroup of PI-PLCXD-containing proteins, which includes DNF2, is found in eukaryotes and in bacteria. These proteins have a similar overall structure, consisting of an N-terminal signal peptide, indicating probable entry of the proteins in the secretory pathway, followed by the PI-PLCXD, and with a total size of 320–420 amino acids. However, the biochemical and biological functions of these proteins remain unclear and, to our knowledge, DNF2 is the first example for which a biological function can be implied based on the phenotype in knockout mutants.
Canonical PI-PLCs cleave phosphatidylinositol (PI) or phosphorylated phosphatidylinositol (PIP) derivatives to produce inositol phosphate and diacylglycerol. These compounds are second messengers or precursors of second messengers, and can participate in plant defense reactions (Canonne et al., 2011). It is difficult to reconcile this with the observed phenotype. As DNF2 does not present all the characteristic domains of eukaryotic PI-PLC, one could consider the possibility that DNF2 interacts with PI(P) without triggering cleavage. A speculation that could fit with the two observations would be that, by binding to PI(P), DNF2 prevents cleavage. This would result in the repression of defense reactions normally triggered by PI(P) degradation products. In eukaryotes, phosphatidylinositides play important roles in membrane trafficking (Michell, 2011). It is also known that some intracellular bacterial pathogens can produce PI(P)-binding proteins or PI(P)-modifying enzymes to disturb membrane trafficking and to persist inside animal cells (Weber et al., 2009). DNF2 might act in a similar way through phosphatidylinositides to avoid the premature lysis of the symbiosome. It will be interesting to determine whether DNF2 supports bacteroid persistence in plant cells by acting on PI(P), especially given the induction of plant defense-like reactions in the dnf2 mutants.
The localization of the DNF2 protein will probably contribute to an understanding of its molecular mechanism. Our study has revealed the existence of five mRNA splice variants for DNF2. All variants conserved the region coding for the PI-PLCXD. The most abundant transcript, corresponding to TC119183, encodes a signal peptide-containing protein, but additional DNF2 mRNAs are predicted to generate proteins lacking the signal peptide (Fig. 1a). Putative splice variants of the third exon that include the first intron of a Phaseolus vulgaris DNF2 homolog are produced in nodules of common bean (Ramirez et al., 2005; Fig. S5). Such a splicing event is not detectable amongst Arabidopsis expressed sequence tags (ESTs), suggesting that this phenomenon could be legume specific. The different amino acid sequences of the predicted DNF2 proteins, resulting from the alternative splicing of the primary DNF2 transcript, indicate that they may be addressed to different compartments of the cell. Some signal peptide-containing proteins that are expressed in symbiotic cells are known to be transported to the symbiosomes (Liu et al., 2006; Coque et al., 2008; Hohnjec et al., 2009; Van de Velde et al., 2010; Meckfessel et al., 2012). Future studies will be necessary to determine the subcellular location of the different peptides and their capacity to restore the WT phenotype.
Taken together, our results indicate that DNF2 is necessary for complete differentiation of bacteroids (Figs 5b, S6) and to maintain symbiosomes (Figs 3, 7). Bacterial mutants altered in the nitrogenase structural gene nifH or in micro-oxic respiration fixL undergo terminal differentiation resulting in fully elongated bacteroids (Maunoury et al., 2010). This indicates that the lack of elongation of bacteroids does not result from an expression defect of nif or fix genes in dnf2, and thus that dnf2 acts upstream of the establishment of micro-aerobic respiration.
In addition to the bacterial lack of differentiation, mobilization of plant defense-like reactions was observed in the DNF2 mutants (Figs 4b, 6). It is possible that these phenomena are linked and that incomplete bacteroid differentiation or a defect in symbiosome maintenance in the dnf2 mutants results from, or triggers, the induction of defense-like reactions. Arguments exist supporting both hypotheses. For instance, the undifferentiated bacA bacterial mutant does not trigger plant defense-like reactions. By contrast, only a fraction of the dnf2 mutant nodules are brownish, suggesting that plant defense-like reactions are only secondary effects of the DNF2 mutation. Future studies are now required to establish the sequence of events resulting from DNF2 mutations.
In summary, in this work, we have identified the DNF2 gene and have further defined its role in the symbiotic process as an actor in symbiosome maintenance. The challenge for future studies will be to determine the molecular function of DNF2 in order to explain its role in the persistence of the symbiosome inside the symbiotic cells.