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Plants have evolved to interact with different microorganisms to establish different types of associations ranging from parasitic to mutualistic. The immune system of the plant has developed to recognize signal/pattern molecules to discriminate between deleterious and beneficial microorganisms (Zipfel & Felix, 2005). Arbuscular mycorrhiza (AM) fungi belong to the latter group and plants forming this association specifically recognize their symbiotic partners and consequently modify their genetic programs to accommodate them (Parniske, 2004). The molecular bases of this recognition process are starting to be understood. They point to common signaling pathways shared with other microbe–plant associations like the nodule symbiosis and to specific signaling pathways commonly elicited by all arbuscular mycorrhizal fungi (Catoira et al., 2000; Stracke et al., 2002; Liu et al., 2003, 2007; Hohnjec et al., 2005; Kistner et al., 2005; Gutjahr et al., 2008; Güther et al., 2009). As only a few plant families are nonmycorrhizal (Smith & Read, 1997), it is clear that most plants have maintained the necessary machinery to associate with these obligate symbionts for at least 450 million yr (Redecker et al., 2000). However, some plants such as the model Arabidopsis thaliana are missing some of the essential plant signaling elements required for mycorrhization (Lévy et al., 2004; Mitra et al., 2004; Kevei et al., 2005). Future research will disclose how many other components are missing or altered in nonmycorrhizal plants.
Similar to the rhizobial symbiosis, where the plant is the first to ‘talk’ to its bacterial partner with a cocktail of flavonoids that induces expression of the nod genes, in the arbuscular mycorrhizal symbiosis the fungus reacts to the production of strigolactones by the roots with hyphal branching (Akiyama et al., 2005). This phenomenon is known to occur upon recognition of compatible host root exudates (Giovannetti et al., 1993; Buee et al., 2000), although it is not sufficient to induce appressoria formation. However, in contrast to the nodule symbiosis, where the identification of the lipochitin–oligosaccharide signals (Nod factors; NF) has led to considerable advances in the dissection of the signal cascade that initiates the symbiotic program in the plant, it is not known how AM fungi ‘talk’ to their host plants. From genetic experiments using legume mutants impaired in nodule symbiosis we know that receptors for NF perception (LYK3, NFP in Medicago truncatula, NFR1 and NFR5 in Lotus japonicus) are not involved in perception of mycorrhizal signals (Wegel et al., 1998; Limpens & Bisseling, 2003;Madsen et al., 2003; Radutoiu et al., 2003). Therefore it is likely that AM fungal signals are of a different chemical nature. Nod factor perception culminates in the activation of at least four specific transcription factors that mediate all NF-induced transcriptional responses (Kalóet al., 2005; Smit et al., 2005; reviewed in Oldroyd & Downie, 2008). Although, these transcription factors are also dispensable for arbuscular mycorrhiza symbiosis (Catoira et al., 2000; Oldroyd & Long, 2003; Hirsch et al., 2009), at least seven of the intermediate signaling components are essential for establishment of the AM symbiosis (Catoira et al., 2000; Kistner et al., 2005). This common pathway has been referred as the SYM (symbiotic pathway) and contains a receptor like kinase, nuclear pore proteins, nuclear ion channels, a calcium/calmodulin-dependent protein kinase and an associated protein (Cyclops) of unknown function (for recent reviews see Parniske, 2008; Oldroyd et al., 2009). In particular, three SYM proteins have been extensively studied and shown to be early transmitters in both symbioses. In M. truncatula they were named DMI proteins (does not make infections). Molecular and genetic analyses of these genes showed that DMI1 (POLLUX in L. japonicus), encoding a cation-permeable channel, and DMI2 (SYMRK in L. japonicus), encoding a receptor like kinase, are hierarchically situated above DMI3, the calcium and calmodulin-dependent protein kinase (Wais et al., 2000). Both proteins are required to induce a calcium oscillation signal that is decoded by DMI3 (Kalóet al., 2005; Smit et al., 2005). While it was long known that NF induce calcium spiking as one of the earliest responses in root hairs, only recently it was shown that AM fungi elicit both, a transient cytosolic calcium elevation (Navazio et al., 2007) and calcium oscillation (Kosuta et al., 2008). In the first case, this happens in response to a fungal diffusible signal constitutively produced (Navazio et al., 2007), while calcium oscillation appears to be dependent on the presence of branched strigolactone-induced hyphae (Kosuta et al., 2008). Although the AM fungal diffusible constitutive signal was able to transcriptionally activate all DMI genes (Navazio et al., 2007), it is not known whether the transmission of this signal travels through this cascade. By contrast, calcium oscillation in response to a diffusible signal released by branched AM hyphae was shown to be dependent on DMI2 and DMI1 (Kosuta et al., 2008).
There are several examples of the divergence in the perception of NF and AM fungal signals, indicating the existence of more than one pathway for the establishment of root symbioses. These pathways are likely to share some components and to be triggered by more than one signal. Thus, while the symbiotic activation of the early nodulin MtENOD11 by NF is DMI dependent, its activation in AM symbiosis is DMI-independent when triggered by a diffusible fungal signal (Kosuta et al., 2003) or DMI-dependent when triggered by appressorium formation (Chabaud et al., 2002). Similarly, the induction of lateral root formation (LRF) induced by both NF and diffusible AM fungal signals requires different elements of the DMI cascade (Oláh et al., 2005). Hence, while NF-induced LRF requires all three components, DMI3 is dispensable for LRF triggered by a diffusible AM fungal signal. This indicates that if calcium oscillations are produced and needed for LRF during mycorrhiza symbiosis, they are not decoded by DMI3. Interestingly, induction of LRF in rice has been recently shown to be independent of the DMI1 and DMI3 rice orthologues (Gutjahr et al., 2009). This could suggest that calcium oscillation might not be necessary for perception of the diffusible AM fungal signal that induces LRF.
In this work we describe a novel M. truncatula gene (MtMSBP1) that is early induced by a diffusible AM fungal signal produced by branched hyphae. The inactivation of this gene leads to changes in the development of the fungus inside the cortex with the appearance of abnormal appressoria and aberrant or collapsing arbuscules. The transcriptional analysis of MtMSBP1 and of the other identified early marker genes clearly shows that at least two different signaling cascades exist for perception of an AM fungal diffusible signal. Even more, it may suggest the possibility that more than one fungal diffusible signal might exist. Thus, while one of these signals would travel independently of the receptor-like kinase DMI2, the perception of the second signal would be DMI2 dependent.
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- Materials and Methods
- Supporting Information
The establishment and maintenance of a mutualistic symbiosis between plants and microbes is a complex process that requires constant signal exchange between both partners to avoid defense reactions that would jeopardize the association. In the arbuscular mycorrhizal symbiosis this signal exchange occurs before physical contact in both directions. Strigolactones released from host roots have been shown to induce hyphal branching in AM fungi (Akiyama et al., 2005) and one or more diffusible signal molecules from AM fungi elicit transient cytosolic calcium elevation in host plant cells, induction of symbiosis related genes and calcium oscillation (Kosuta et al., 2003, 2008; Weidmann et al., 2004; Navazio et al., 2007). However, the nature of the fungal signal(s) is still unknown. The perception of AM fungal signals travels at least partly through the SYM pathway, but evidence of a second signal cascade has been recently shown (reviewed in Oldroyd et al., 2009). Here we present further evidence of the existence of at least two different signal cascades in M. truncatula for perception of AM fungal diffusible signals.
In this work we identified MtMSBP1 in a screening for early mycorrhiza induced genes together with other early markers (TC107197, TC106351 and TC112474). MtMSBP1 encodes a protein of the recently identified membrane-bound steroid-binding protein family that in plants is associated with the regulation of cell elongation (Yang et al., 2005). Although this gene is highly expressed in roots during asymbiosis in the central cylinder and in the meristem of the root apex, its expression is transiently induced two- to three-fold at very early stages of the AM symbiosis by a diffusible signal. The induced expression was observed in epidermal and subepidermal cells in the vicinity of approaching hyphae and in appressorium-enriched root areas. The induction in appressorium-enriched samples coincides with an induction of the early marker gene MtENOD11 (Journet et al., 2001). MtENOD11, a repetitive hydroxyproline-rich protein, has been widely used as space and time marker of mycorrhization (Chabaud et al., 2002; Kosuta et al., 2003). Similarly to the mycorrhizal-induced expression of MtMSBP1 described in this work, MtENOD11 is induced in root areas underneath branched hyphae (Kosuta et al., 2003). This might indicate that not all hyphae from AM fungi are able to produce (enough) signal(s) inducing expression of these two genes. Therefore it is likely, that the prestimulation of branching by plant strigolactones not only facilitates docking of the fungus to the root but also makes AM fungal hyphae competent for symbiosis.
In our work here, time-lapse video microscopy and cellophane experiments clearly showed that MtMSBP1 expression was induced before fungal contact. This shows that the branching hypha produces a secreted fungal compound, able to pass through a cellophane membrane, that triggers expression within 3 h. This rapid transcriptional induction suggests that the signal perception is transmitted through a pre-established signal cascade, perhaps involving phosphorylation. To date, only the SYM signal cascade mentioned above has been genetically defined for the perception of AM fungal signals. However, evidence that a second cascade might exist is accumulating. Thus, while MtENOD11 is activated upon perception of a diffusible signal in mutant plants defective in three genes of the SYM pathway (DMI2, DMI1 and DMI3) (Kosuta et al., 2003), calcium oscillation induced by a diffusible signal from AM fungi requires DMI2 and DMI1 (Kosuta et al., 2008). Additional indication of an alternative signaling cascade to the SYM route has been recently observed in rice (Gutjahr et al., 2009). The work presented here provides further indication of two different signal cascades operating in this symbiosis. The evidence is based on the differential activation of these target genes among wild-type and mutant plants defective in the symbiotic receptor kinase DMI2. In contrast to wild-type plants, that are able to perceive the AM-diffusible fungal signal/s and activate the four marker genes studied here, in DMI2 defective plants only one of the marker genes (TC106351) is clearly activated, indicating the existence of a DMI2-independent pathway. In addition to two signaling cascades, it is possible that more than one fungal signal might exist. Thus, one of these signals would travel through the SYM pathway and the second through a yet unidentified route. Biochemical analyses will help to discern about the existence of more than one AM fungal signal.
In order to ascertain the role that MtMSBP1 plays in the mycorrhiza symbiosis we localized the protein subcellularly and inactivated it using RNA interference. Surprisingly, although the A. thaliana protein was described to locate to the plasma membrane, we clearly showed here that the M. truncatula protein localizes to the ER as it has been shown for yeast and animal homologues (Rohe et al., 2009). Furthermore, heterologous localization of MtMSBP1 in A. thaliana protoplasts and in N. benthamiana leaves showed ER localization. This is consistent with steroids being molecules able to travel through biological membranes and to reach receptors within the cell lumen. Inactivation of MtMSBP1 by RNA interference showed a surprising but interesting phenotype. Thus, while we did not observe any morphological alterations in root development in the absence of mycorrhiza, the phenotype of the symbiotic structures within the root was affected. Hyphal penetration from appressoria was often aborted by septation of penetrating hyphae. Septation also occurred in intercellular hyphae but, most significantly, the morphology of arbuscules was distorted. Arbuscules developed much less frequently and with a crippled morphology in the RNAi hairy root lines, resembling the phenotype observed in the pea late mutant (RisNod24) or in the M. truncatula phosphate transporter mutant, mtpt4-1, when mycorrhized (Gianinazzi-Pearson, 1996; Lapopin et al., 1999; Javot et al., 2007). These results provide genetic evidence of the relevance of MtMSBP1 for the mycorrhiza symbiosis.
Membrane-bound steroid-binding proteins are a family of proteins first described in animals and later found in plants and fungi (Falkenstein et al., 1996; Hand et al., 2003; Iino et al., 2007). In contrast to plasma steroid-binding proteins that locate to the cytoplasm and shuttle to the nucleus after steroid binding where they act as transcription factors, membrane-bound steroid-binding proteins contain a single transmembrane domain and are presumed to act through second messengers (Lösel et al., 2003). In plants their role has been linked to the control of cell expansion/elongation as it was observed that hypocotyl length changes correlated to induction or repression of AtMSBP1 (Yang et al., 2005). However, recent work with the mammalian and yeast/fission yeast homologues have shown that this family of proteins might have a conserved role in the control of the sterol biosynthesis by binding and regulating ER-located cytochrome P450 enzymes (Mallory et al., 2005; Hughes et al., 2007). In this respect, Yang et al. (2005) also noted that in A. thaliana, from the 115 induced genes in transgenic lines overexpressing AtMSBP1, 13% were related to the steroid/sterol metabolism and signaling. The homeostasis of sterols appears to be of major importance for the establishment of root symbioses. Thus, experiments using sterol biosynthesis inhibitor fungicides have shown that the pattern of root sterols changes dramatically with application of fenpropimorph and this, in turn, impedes arbuscular mycorrhizal symbiosis (Campagnac et al., 2008). Fenhexamide, another sterol biosynthesis inhibitor fungicide does not induce major changes in the sterol profile of the root, or modify the total colonization by G. intraradices, but arbuscule frequency is significantly reduced, phenocopying the effect of inactivation of MtMSBP1. During symbiosis with rhizobia, the DMI2 receptor kinase has been shown to interact with 3-hydroxy-3-methylglutaryl CoA reductase (MtHGMR1), an enzyme of the mevalonate biosynthetic pathway required for the synthesis of isoprenoids (Kevei et al., 2007). Accordingly, application of lovastatin,which inhibits HGMR activity, or inactivation of HGMR by RNAi leads to a decrease in nodulation. We have evidence that DMI2 also interacts with HGMR during mycorrhiza formation (N. Rieger & N. Requena, unpublished results). MtHMGR1 has also been described to be induced at early stages of mycorrhiza symbiosis (Liu et al., 2003). Therefore, it appears that the control of sterol homeostasis is essential to accommodate mutualistic symbionts in the root. We propose that MtMSBP1 could be involved in this regulation by interaction with P450 ER enzymes during mycorrhiza formation.
In summary, we show here that AM fungi are able to produce diffusible signal(s) that are transmitted through two signaling pathways to induce an array of downstream effectors. We propose that one of these signals travels through the well-established SYM pathway or at least enters through this pathway requiring a functional DMI2. This diffusible signal activates TC107197, TC112474 and possibly MtMSBP1, novel markers for early mycorrhization. The second signal travels through a yet unknown signaling pathway to activate the specific mycorrhiza marker TC106351. At present, we cannot rule out whether these are all constitutive or induced signals. However, induction of MtMSBP1 correlated with the presence of branched hyphae. Our results for the inactivation of MtMSBP1 show that AM fungal induction of MtMSBP1 might be related to the need of altering the sterol metabolism to allow plasma membrane invagination and intracellular accommodation of the symbiont in the cortex.