Jean Dénarié

Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza

Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.

Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses.

Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Molecular basis of symbiotic host specificity in rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

Medicago truncatula, a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula.

Using dual cultures of arbuscular mycorrhizal (AM) fungi andMedicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea,Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella andFusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all threeM. truncatulaNod−/Myc− mutants (dmi1,dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the Nod−/Myc− mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.

The Medicago truncatula LysM-receptor Kinase Gene Family Includes NFP and New Nodule-expressed Genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

Lipo-oligosaccharide nodulation factors: A new class of signaling molecules mediating recognition and morphogenesis

Jean Dbnari6 and Julie Cullimore Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes CNRS-INRA, 31326 Castanet-Tolosan Cedex France The legume-rhizobia symbiosis is estimated to fix per an- num as much nitrogen as the fertilizer industry and is of great agronomic and ecological importance. This efficient nitrogen-fixing association occurs as a result of the forma- tion of a new specialized organ, the root nodule, which is induced by the prokaryotic symbiont on its specific plant partner (see Nap and Bisseling, 1990). The rhizobial genes that determine the host recognition and nodulation have recently been found to specify the synthesis of excreted lipo-oligosaccharide signals (Figure 1) which are capable of eliciting, at extremely low concentrations, many of the plant responses characteristic of the bacteria themselves. These responses include the initiation of cell division, in- duction of specific changes in cell morphology, and trig- gering of a plant organogenic program leading to the for- mation of the nodule. This review focuses on the role of this new class of signaling molecules in host-partner rec- ognition and nodule development in the legume-rhizobia symbiosis. Rhizobia, Prokaryotes that Elicit a Specific Plant Organogenesis Rhizobia (now classified into three genera, Rhizobium, Sradyrhizobium and Azorhizobium) are soil bacteria that can elicit the formation of nitrogen-fixing nodules on the roots of selected species of the legume family. Some of these bacteria exhibit a narrow host range of nodulation; for example three of the species discussed in this review, R. meliloti, R. leguminosarum biovar viciae and B. japoni- cum nodulate respectively alfalfa, pea/vetch and soybean and not each other’s hosts. In contrast R. sp. NGR234 has a broad host range, nodulating over 70 legume genera and even a non-legume, Parasponia (see Denarie et al., ‘I 992). Symbiotic nodule formation is based on two processes, plant infection and induction of a novel organ (Nap and Bisseling, 1990). To penetrate into their hosts, rhizobia elicit stimulation and reorientations of plant cell wall growth, presumably due to a restructuring of the cytoskele- ton. These changes result in both the entrapment of the bacteria within root hair curls and also the initiation and development of infection threads, tubular structures ,through which bacteria pass on their way down the root hair to the underlying cortical cell layers (Figure 2A). Ahead of the advancing threads a major developmental switch is elicited in the root cortex. Cortical cells are induced to dedifferentiate and divide, and a meristem is then formed, whose functioning generates the nodule. Nodules have a particular ontogeny and anatomy; they are not mere tu- mors but fully differentiated organs divided into a number of cell-types and tissues. Alfalfa nodules for example (Fig- ure 28) contain a distal meristem, an infection zone, a zone where the cells are filled with nitrogen-fixing bacteria, and vascular bundles that ensure metabolic exchanges with the rest of plant. Genetic manipulation of the prokaryotic partner has led to the identification of a set of rhizobial genes (the nod genes) required for infection, nodulation and the control of host specificity (Figure 1). The expression these genes isdependent upon plant signals, usuallyflavonoids, excreted in root exudates. In the presence of appropriate plant inducers, rhizobial NodD regulatory proteins activate the transcription of thestructural nodgenes. Among these, and present in all rhizobia, are common nodA% genes, in which mutations lead to a complete abolition of infection and nodulation. In addition, there are nod genes that are species/strain specific, such as noV De- narie et al., 1992; Fisher and Long, 1992). Nodulation Genes Specify the Production of Lipo-Oligosaccharide Nod Factors What could be the biochemical function of the rhizobial nod gene products? A link between the genetic determi-

A GRAS-type transcription factor with a specific function in mycorrhizal signaling

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

Transcriptional Responses toward Diffusible Signals from Symbiotic Microbes Reveal MtNFP- and MtDMI3-Dependent Reprogramming of Host Gene Expression by Arbuscular Mycorrhizal Fungal Lipochitooligosaccharides

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn’t Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca2+ signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.