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Plants are subjected to a continually changing environment, and must respond very quickly and efficiently to adjust to environmental fluctuations. It is now widely accepted that reactive oxygen species (ROS) are involved in signalling in response to biotic and abiotic stresses and in developmental processes (Apel & Hirt, 2004). The tight regulation of ROS homeostasis by a multifaceted network of ROS production and ROS scavenging enzymes creates a baseline from which ROS spikes may be generated and act as signals in various cellular processes (Mittler et al., 2004, 2011; Tognetti et al., 2012). ROS have been implicated in processes as diverse as root hair growth (Foreman et al., 2003; Jones et al., 2007; Monshausen et al., 2007), stomatal closure (Pei et al., 2000) and plant–microbe interactions (Apel & Hirt, 2004; Pauly et al., 2006; Nanda et al., 2010).
There is now compelling evidence that ROS play an important role in signalling processes during the establishment of legume–Rhizobium symbioses (Santos et al., 2001; Ramu et al., 2002; Rubio et al., 2004; Pauly et al., 2006; Jamet et al., 2007). This interaction involves a complex molecular dialogue between the host plant and the symbiont, leading to the root hair becoming infected with the bacterium and ultimately forming root nodules, in which nitrogen fixation takes place (Oldroyd & Downie, 2008). ROS, such as the superoxide anion () and hydrogen peroxide (H2O2), have been detected at early stages of the symbiotic interaction (for a review, see Pauly et al., 2006; Nanda et al., 2010). and H2O2 have been found in infection threads (ITs) (Santos et al., 2001; Jamet et al., 2007), which, in c. 75% of nodulating legumes (Sprent, 2007), are unique invasive invaginations of plant origin that allow the bacterium to invade the cortical cells (Oldroyd & Downie, 2008). The rate of continual ROS production in roots has been shown to decrease temporarily during the first few hours after Nod factor (NF) treatment (Shaw & Long, 2003; Lohar et al., 2007). Moreover, a Sinorhizobium meliloti nodC mutant with defective NF biosynthesis has been shown to induce higher levels of H2O2 accumulation (Bueno et al., 2001). By contrast, no production was observed when Medicago truncatula plants were inoculated with an S. meliloti nodD1ABC mutant unable to produce NFs (Ramu et al., 2002). Similarly, a transient increase in ROS levels has been detected at the tip of actively growing root hair cells within seconds of the addition of NF in Phaseolus vulgaris (Cardenas et al., 2008). Moreover, the inhibition of ROS production in M. truncatula roots inoculated with S. meliloti has been shown to prevent IT formation (Santos et al., 2001; Ramu et al., 2002; Peleg-Grossman et al., 2007). ROS thus seem to be critical for optimal symbiosis (Jamet et al., 2007; Marino et al., 2011).
NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), appear to play an important role in ROS production during the symbiotic process (Marino et al., 2011). They have been identified as a major source of the oxidative burst observed during plant–pathogen interactions (Torres & Dangl, 2005; Sagi & Fluhr, 2006; Mittler et al., 2011; Marino et al., 2012). In the S. meliloti–M. truncatula symbiosis, diphenylene iodonium (DPI) – an inhibitor of flavoproteins, such as NADPH oxidases – abolishes ROS production early in the interaction (Peleg-Grossman et al., 2007; Cardenas et al., 2008). Seven NADPH oxidase-encoding genes have been identified in the M. truncatula genome (Marino et al., 2011) and the expression patterns of these genes have been characterized (Lohar et al., 2007; Marino et al., 2011). It has been suggested that the decrease in ROS efflux observed 1 h after the treatment of M. truncatula roots with NF is associated with a transient decrease in MtRbohs gene expression (Lohar et al., 2007). Furthermore, the downregulation of MtRbohA expression in the nodule by RNA interference methods leads to a decrease in biological nitrogen fixation, indicating a probable role in symbiosis (Marino et al., 2011). Similar results have been reported recently for Phaseolus vulgaris, in which a NADPH oxidase gene has been shown to be crucial for successful rhizobial colonization and, probably, for the maintenance of the correct growth and shape of ITs (Montiel et al., 2012).
Despite widespread acceptance of the role of ROS in signalling, it remains unclear how ROS signals are perceived, transmitted and induce a specific response (Mittler et al., 2011). Given the crucial role of these molecules in signalling in plant cells, efforts have recently been made to identify the genes regulated by ROS. Genome-wide transcriptome analyses have proved to be useful for the assessment of the specificity of ROS signalling, with the analysis of gene expression profiles after the modification of ROS levels (Gadjev et al., 2006; Inzé et al., 2011). These transcriptome analyses have made it possible to determine the effect, not only of different ROS, but also of the accumulation of ROS in various subcellular compartments on gene expression. They have shown that most of the genes responding to a stimulus (stress, oxidative stress-causing agents, etc.) are expressed in only one specific set of experimental conditions (i.e. in response to one particular ROS species). This strongly suggests that gene expression levels are a hallmark of specific oxidative signals characterized by the identity of the ROS concerned and/or its site of production (Gadjev et al., 2006; Grennan, 2008). Interestingly, a meta-analysis of H2O2-induced gene expression in eukaryotes (including Arabidopsis) has provided evidence to suggest that the regulation of gene expression by ROS is strongly conserved across kingdoms (Vandenbroucke et al., 2008).
In this context, the aim of this study was to identify the genes targeted by H2O2 during the establishment of the M. truncatula–S. meliloti symbiosis. The use of a combination of pharmacological and transcriptomic approaches led to the identification of several genes potentially regulated by H2O2 content. We then analysed the expression profile of an H2O2-induced gene encoding a putative serine/threonine protein kinase. Using an artificial micro-RNA (amiRNA) strategy, we showed that this gene plays an important role in the establishment of the rhizobial symbiosis.
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By using DPI to inhibit NADPH oxidases, we identified genes putatively regulated by H2O2 or other ROS ( or hydroxyl radical) during the establishment of the M. truncatula–S. meliloti symbiotic interaction. A large number of genes were found to be regulated in opposite ways by rhizobial infection and DPI (Tables 1, S2). Despite the probable side effects of DPI (indeed, flavoproteins, such as dehydrogenases, monooxygenases and other oxygenases may be DPI targets), this suggests an important role for H2O2 and/or other ROS in gene regulation early in the establishment of symbiosis. ROS are unlikely to regulate any particular gene directly: the changes in gene expression observed here probably result from signalling pathways involving ROS receptors, redox-sensitive TFs and Ca2+ signals (Mittler et al., 2011). In this framework, several possible H2O2 target genes can be identified. Some (18 genes) are involved in hormone synthesis or signalling (Table S2), consistent with the role of phytohormones in the establishment and functioning of symbiosis (Oldroyd & Downie, 2008). This is the case, in particular, for genes involved in gibberellic acid (GA) (Mtr.156.1.s1_at; Mtr.25006.1.s1_at: ent-copalyl diphosphate synthase; Mtr.13370.1.S1_at: gibberellin 3-β-dioxygenase) or brassinosteroid (BR) (Mtr.25019.1.s1_at; Mtr.7754.1.s1_s_at: dihydroflavonol 4-reductase; Yuan et al., 2007) biosynthesis; these genes may be regulated by H2O2 during the establishment of symbiosis. Moreover, deregulation of the pea Ramosus1 homologue (Mtr.37123.1.s1_s_at; Foo et al., 2005) may link H2O2 with auxin in nodule development, as suggested for root development (De Tullio et al., 2010).
Genes involved in cell wall synthesis (potentially related to IT progression) constitute a second group of possible targets (19 genes). Examples include genes encoding expansins (Mtr.22752.1.s1_at, Mtr.41561.1.s1_at), polygalacturonases (Mtr.31710.1.s1_at; Mtr.1427.1.s1_at), pectate lyases (Mtr.38613.1.s1_at, Mtr.26489.1.s1_at) and a cellulase (Mtr.1496.1.s1_at). Moreover, several M. truncatula subtilase-encoding genes (serine proteases; Mtr.5721.1.s1_s_at, Mtr.45771.1.s1_at, Mtr.45770.1.s1_at) were found to be induced. The upregulation of these genes seems to be maintained during rhizobial symbiosis (Benedito et al., 2008). Subtilase induction has also been demonstrated in plant–fungus interactions (Takeda et al., 2009) and in actinorhizal symbioses (Ribeiro et al., 1995; Laplaze et al., 2000). Indeed, a role for subtilases has been suggested in the exchange of signals between the two symbiotic partners, or in cell wall modification during IT growth (for a review, see Schaller et al., 2012). Similarly, modification of the cellular redox balance by glutathione depletion during nodulation has been shown to affect both hormonal balance (effects principally on auxin and ethylene) and cell wall formation (Pucciariello et al., 2009).
TFs are also possible targets. The MtbHlh1 gene investigated here did not appear to be regulated by H2O2, but other bHLH-type TFs (Mtr.15416.1.s1_at, Mtr.24842.1.s1_at, Mtr.8357.1.s1_s_at) may display such regulation. Indeed, a bHLH (At1g10585) TF seems to be involved in the primary cellular stress responses mediated by high levels of H2O2 (Vanderauwera et al., 2005). Well-characterized TFs involved in the establishment of symbiosis are strongly affected by ROS depletion. This is the case, in particular, for NSP1/2 (Kalo et al., 2005; Smit et al., 2005), ERF (Middleton et al., 2007) and MtNIN (Marsh et al., 2007). Thus, genes encoding TFs regulated early in the interaction with S. meliloti may also be good candidates for regulation by H2O2.
Finally, 22 M. truncatula genes (of the 447 studied; Table S2) displayed sequence similarity to 19 Arabidopsis genes previously reported to be regulated by ROS (Table S4, Gadjev et al., 2006). These genes included those encoding jasmonic acid carboxyl methyltransferase (Mtr.407.1.S1_at), a TF (HAP2-1; Mtr.43750.1.S1_at) and a receptor kinase (Mtr.24165.1.S1_at), all of which may be of particular interest. The M. truncatula Hap2-1 gene encodes a CCAAT-binding TF involved in nodule development (Combier et al., 2006).
Moreover, the regulation of expression by H2O2 has been demonstrated for three genes. MtSrl1 encodes a member of the 2OG and Fe(II)-dependent oxygenase superfamily (2-OG dioxygenases). Gechev et al. (2005) identified several 2-OG dioxygenases putatively regulated by H2O2. Van Damme et al. (2008) identified a gene encoding 2-OG dioxygenase (Dmr6; downy mildew resistant 6) as displaying an increase in expression in compatible and incompatible oomycete interactions, 1 d after inoculation. Genome-wide expression analysis of Arabidopsis dmr6 mutants has revealed the enhanced expression of a subset of defence-associated genes, including dmr6 itself, suggesting that DMR6-mediated resistance results from the activation of plant defence responses during interactions with plant oomycetes (Van Damme et al., 2008). Similarly, the early induction of MtSrl1 expression by S. meliloti may downregulate plant defence reactions to facilitate the establishment of the symbiotic interaction.
ITs are produced during the M. truncatula–S. meliloti rhizobial symbiosis. These unique structures are associated with bacterial colonization in c. 75% of legume–Rhizobium symbioses (Sprent, 2007). They are invaginations of the plant cell membrane that grow towards the poles of the cell, with the cell wall matrix on the inside of the IT. Given the importance of the cytoskeleton in directing cell division and cell growth, IT development would be expected to require dynamic cytoskeleton rearrangements (Miyahara et al., 2010; Oldroyd et al., 2011). We found that a gene encoding an ABL-like protein (ABlL; Mtr.20281.1.S1_at; MtAblL) was regulated by S. meliloti infection and H2O2. The protein encoded by this gene is a subunit of the WAVE complex, which is involved in actin microfilament nucleation and branching (Szymanski, 2005). Loss-of-function mutations have recently been described in genes encoding proteins involved in actin rearrangements in leguminous plants (Yokota et al., 2009; Miyahara et al., 2010). Interestingly, the corresponding mutant plants have defects in the polar growth of root hairs and the formation and maintenance of ITs. The regulation of MtAblL by H2O2 may contribute to IT growth.
MtSpk1 appears to be induced by both NF and H2O2 treatment. Transcriptional fusion experiments confirmed that MtSpk1 was expressed early in S. meliloti infection (Fig. 4c,d). Moreover, this gene was clearly expressed in the infection zone (Fig. 4e). We used the HyPer protein probe (Belousov et al., 2006) for the imaging of H2O2 accumulation in nodules. The genetic nature of the mechanism used to produce the HyPer probe means that this specific fluorescent probe can be expressed anywhere in the cell. It has been successfully targeted to plant peroxisomes, for example (Costa et al., 2010). With this ratiometric fluorescent probe, we detected a doubling of H2O2 levels in the infection zone (Fig. 5) in which MtSpk1 was strongly expressed (Fig. 4e). This suggests that H2O2 may regulate MtSpk1 expression in vivo. The MtRbohB gene is a good candidate for involvement in this H2O2 production, as it has been shown to be expressed in the same zone (Marino et al., 2011).
Inactivation of the MtSpk1 gene resulted in significantly lower nodule numbers, with no effect on root development (Fig. 7), suggesting a role for MtSpk1 in the establishment of the symbiotic interaction. Several receptor kinases participate in the NF signalling cascade (Oldroyd & Downie, 2004), but only a few reports have highlighted the involvement of soluble kinases in the M. truncatula symbiotic interaction (Gargantini et al., 2006). The presence of a putative calmodulin recruitment motif in the N-terminal region of MtSPK1 suggests that this protein may be involved in cross-talk with the well-known calcium signalling process occurring during rhizobial symbiosis (Oldroyd et al., 2011). Alternatively, in silico analysis of the MtSPK1 sequence suggests that this protein may be located in the nucleus (Chou & Shen, 2010). Subcellular localization appeared to confirm this (Fig. S6), pointing to a role as a putative transcriptional regulator. This situation is reminiscent of that for the Arabidopsis OXI1 gene, which encodes a serine/threonine kinase. This gene is induced in response to diverse H2O2-generating stimuli, including plant–microbe interactions (Rentel et al., 2004). OXI1 has been shown to be an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses (Rentel et al., 2004). Thus, overall, our results suggest that MtSpk1 encodes a protein with an important role in the signalling processes associated with the establishment of rhizobial symbiosis. The results obtained here (Fig. 8) suggest that MtSpk1 may be involved in the differentiation of nodule cells, via MtHap2-1 (Combier et al., 2006), and in the correct formation of root nodules, via MtNin (Marsh et al., 2007), consistent with its localization in nodule primordia.
In conclusion, this work provides a first list of the genes putatively regulated by H2O2 during the establishment of symbiotic interactions. Moreover, to our knowledge, it also identifies the first gene involved in the early stages of symbiosis to be regulated by both H2O2 and NF. Further studies are required to determine the precise function of the protein encoded by this gene in the NF signalling cascade. Finally, the identification of molecular targets of MtSPK1 should make it possible to elucidate the role of this protein in plant–microsymbiont communication.