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Summary • An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. • Agrobacterium rhizogenes transformed root explants were derived either from Myc +M. truncatula or from the infection-defective Myc − mutant TR26 ( dmi2–2 ), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. • Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc + roots, but not in epidermal cells in contact with appressoria in Myc − roots. • We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.

International audience; Nitrogen-fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little-researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection-associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing in vivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin-like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D. trinervis outer root tissues. This is accompanied by nucleo-cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen-fixing rhizobia.

Although it is now well-established that decorated lipo-chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre-infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear-localized cameleon Nup-YC2.1 into Casuarina glauca we show that cell-free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca(2+) spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca(2+) spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre-infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin-based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.

The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca(2+) spiking in the Medicago truncatula root epidermis. Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca(2+) spiking responses and LC-MS to characterize targeted molecules in GSEs. This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca(2+) spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca(2+) spiking in M. truncatula ROCs. We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization.

Contents 533 I. 533 II. 534 III. 536 IV. 536 537 References 537 SUMMARY: Root endosymbioses are beneficial associations formed between terrestrial plants and either bacterial or fungal micro-organisms. A common feature of these intracellular symbioses is the requirement for mutual recognition between the two partners before host-regulated microbial entry. As part of this molecular dialogue, symbiosis-specific microbial factors set in motion a highly conserved plant signal transduction pathway, of which a central component is the activation of sustained nuclear Ca2+ oscillations in target cells of the host epidermis. Here, we focus on recent findings concerning this crucial Ca2+ -dependent signalling step for endosymbiotic associations involving either arbuscular mycorrhizal fungi or nitrogen-fixing Frankia actinomycetes, and in particular how this knowledge is contributing to the identification of the respective microbial factors.

Taking a look at plant-microbe relationships Ever since plants colonized land, they have evolved a range of mutualistic associations with bacteria and fungi. Indeed, such associations were probably required for plants to grow on harsh, nutrient-poor surfaces. Martin et al. review the spectrum of plant-microbe symbioses and their evolution, including evidence from the Rhynie Chert of the Devonian period and modern associations. Surprisingly, diverse functional plant-microbial symbioses have several common conserved features, including signaling pathways, immune evasion, and root development. Science , this issue p. eaad4501

The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca(2+) ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca(2+) signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca(2+) spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

Contents 533 I. 533 II. 534 III. 536 IV. 536 537 References 537 SUMMARY: Root endosymbioses are beneficial associations formed between terrestrial plants and either bacterial or fungal micro-organisms. A common feature of these intracellular symbioses is the requirement for mutual recognition between the two partners before host-regulated microbial entry. As part of this molecular dialogue, symbiosis-specific microbial factors set in motion a highly conserved plant signal transduction pathway, of which a central component is the activation of sustained nuclear Ca

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

International audience; Legumes improve their mineral nutrition through nitrogen-fixing root nodule symbioses with soil rhizobia. Rhizobial infection of legumes is regulated by a number of transcription factors, including ERF Required for Nodulation1 (ERN1). Medicago truncatula plants defective in ERN1 are unable to nodulate, but still exhibit early symbiotic responses including rhizobial infection. ERN1 has a close homolog, ERN2, which shows partially overlapping expression patterns. Here we show that ern2 mutants exhibit a later nodulation phenotype than ern1, being able to form nodules but with signs of premature senescence. Molecular characterization of the ern2-1 mutation reveals a key role for a conserved threonine for both DNA binding and transcriptional activity. In contrast to either single mutant, the double ern1-1 ern2-1 line is completely unable to initiate infection or nodule development. The strong ern1-1 ern2-1 phenotype demonstrates functional redundancy between these two transcriptional regulators and reveals the essential role of ERN1/ERN2 to coordinately induce rhizobial infection and nodule organogenesis. While ERN1/ERN2 act in concert in the root epidermis, only ERN1 can efficiently allow the development of mature nodules in the cortex, probably through an independent pathway. Together, these findings reveal the key roles that ERN1/ERN2 play at the very earliest stages of root nodule development.

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International audience; Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont. In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene. First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-β-glucuronidase (GUS) expression in root hairs of transgenic M. truncatula. These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation. Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction. Finally, pMtENOD11-GUS was introduced into all three Nod−/Myc−dmi M. truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7. We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3. In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.

A blackbody optical fiber thermometer consists of an optical fiber whose sensing tip is given a metallic coating. The sensing tip of the fiber forms an isothermal cavity, and the emission from this cavity is approximately equal to the emission from a blackbody. Standard two-color optical fiber thermometry involves measuring the spectral intensity at the end of the fiber at two wavelengths. The temperature at the sensing tip of the fiber can then be inferred using Planck's law and the ratio of the spectral intensities. If, however, the length of the optical fiber is exposed to elevated temperatures, erroneous temperature measurements will occur due to emission by the fiber. The results presented in this article show it is possible to use additional spectral measurements to account for emission by the fiber and accurately measure the tip temperature. In addition, the proposed technique can be used to obtain an estimate of the temperature profile along the fiber.

International audience; The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4-5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T-1 descendents.

A blackbody optical fiber thermometer consists of an optical fiber whose sensing tip is given a metallic coating. The sensing tip of the fiber forms an isothermal cavity and the emission from this cavity is approximately equal to the emission from a blackbody. When a short length of the fiber is exposed to a high temperature environment, the temperature at the sensing tip can be inferred using the standard two-color approach. If, however, more than a short length of the fiber is exposed to elevated temperatures, emission by the fiber will result in erroneous temperature measurements. This paper presents experimental results that show it is possible to use additional spectral measurements to eliminate errors due to emission by the fiber and measure the tip temperature. In addition, the technique described in this paper can be used to obtain an estimate of the temperature profile along the fiber.

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.

International audience; In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.

Une correction a été ajoutée au début de l'article.; International audience; Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

A blackbody optical e ber thermometer consists of an optical e ber whose sensing tip is given a metallic coating. The sensing tip of the e ber forms an isothermal cavity, and the emission from this cavity is approximately equal to the emission from a blackbody. Temperature readings are obtained by measuring the spectral intensity at the end of the e ber at two wavelengths. The ratio of these measurements is used to infer the temperature at the sensing tip. However, readings from blackbody optical e ber thermometers are corrupted by self-emission when extended portions of the e ber are exposed to elevated temperatures. Two possible methods are described for correcting the problem caused by self-emission by the e ber. The e rst method is two-e ber optical e ber thermometry. In this method a second e ber is positioned parallel to the original e ber. The e bers are identical except that the second e ber has a ree ecting coating instead of a blackbody cavity at its tip. Because both the emitting and ree ecting e bers are exposed to the same thermal environment, measurements of the intensity at the end of the ree ecting e ber can be used to eliminate error caused by emission by the e ber. The second approach is spectral remote sensing. In this method the intensity exiting the e ber is measured in portions of the visible and infrared spectrum. The measured spectral intensities are used to reconstruct the temperature proe le along the e ber.

Depuis une vingtaine d’annees, les nombreuses recherches focalisees sur la plante modele Arabidopsis thaliana ont permis des progres considerables dans notre connaissance des bases moleculaires de la biologie des plantes. Avec l’achevement du sequencage du genome d’Arabidopsis [1], la palette complete des outils de genetique et de genomique maintenant disponibles devrait encore accelerer le rythme des decouvertes. De son cote, le riz fait l’objet de rapides developpements genomiques en tant que modele pour les monocotyledones. Cependant, il est clair que ces deux especes ne suffisent pas pour representer le monde vegetal dans toute sa diversite biologique [2]. Ce fait est particulierement bien illustre par le cas des Legumineuses (Fabacees) qui representent l’un des taxons vegetaux les plus importants, tant du point de vue de la biologie et de l’ecologie fondamentales que du point de vue agronomique et environnemental.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

The spatio-temporal expression pattern of the Medicago truncatula ENOD20 gene during early stages of nodulation has been analyzed with transgenic alfalfa (M. varia) expressing a pMtENOD20-GUS chimeric fusion. Our results show that transcriptional activation of this gene occurs initially in dividing inner cortical cells corresponding to sites of nodule primordium formation and subsequently in root hairs containing infection threads. Use of Sinorhizobium meliloti nod gene mutants that uncouple nodule organogenesis from infection has confirmed that early MtENOD20 transcription is tightly linked to cortical cell activation (CCA). Furthermore, these experiments have revealed that an S. meliloti nodH mutant, defective in Nod factor sulfation and epidermal cell activation, is nevertheless able to elicit low-level CCA. Purified S. meliloti Nod factors trigger MtENOD20 transcription very rapidly (within 12 to 24 h) in the root cortex, and studies with transgenic alfalfa show that Nod factors are able to elicit gene expression coupled to CCA at concentrations as low as 10-11 M. Finally, we have made use of a range of synthetic lipo-chitooligosaccharides to show that fatty acid chain length is an important structural parameter in the capacity of Nod factors to elicit CCA. Taken together, our results suggest that pMtENOD20-GUS transgenic lines should prove valuable tools in future studies of Rhizobium and Nod factor-elicited CCA.

We have identified two single-copy genes from the model legume Medicago truncatula (MtENOD16 and 20) whose expression can be correlated with early stages of root nodulation and whose predicted coding sequences are partially homologous to both pea/vetch ENOD5 and soybean N315/ENOD55. Database searching and sequence alignment have defined the encoded early nodulins as a distinct sub-family of phytocyanin-related proteins, although the absence of key ligands implies that they are unlikely to bind copper. Molecular modelling based on known phytocyanin structure has been used to predict the 3-dimensional conformation of the principle globular domain of MtENOD16/20. Additional structural features common to both early nodulin and phytocyanin precursors include an N-terminal transit peptide, a highly variable (hydroxy)proline-rich sequence which probably undergoes extensive post-translational modification, and a hydrophobic C-terminal tail.

In order to study the spatio-temporal patterns of lectin gene expression throughout the various developmental stages of the Rhizobium—legume interaction, promoter-gusA fusions for each of three lectin genes from Medicago truncatula (Mtlec1, 2 and 3) have been introduced into transgenic Medicago varia plants. Histochemical staining for β-glucuronidase activity has shown that Mtlec1 and Mtlec3 are expressed independently of symbiosis in inner cortical and vascular tissues of the root, respectively. Transcription of Mtlec3 in the root stele is markedly enhanced following inoculation with Rhizobium meliloti. Furthermore, both Mtlec1 and Mtlec3 are transcribed in dividing cells of the root nodule primordium, whether elicited by R. meliloti or by purified nodulation (Nod) factors. These findings suggest that these two lectins are involved in developmental processes early during nodule ontogeny. In contrast, Mtlec2 is not expressed in uninoculated roots or during early stages of the symbiotic interaction. At later stages, all three lectin promoters are functional in mature nodules. In this context, lectin gene expression is restricted to non-infected cells of both central and certain peripheral tissues and appears to be correlated with the nitrogen-fixing capacity of the nodule.

International audience; The arbuscular mycorrhizal symbiosis associates soil fungi with the roots of the majority of plants species and represents a major source of soil phosphorus acquisition. Mycorrhizal interactions begin with an exchange of molecular signals between the two partners. A root signaling pathway is recruited, for which the perception of fungal signals triggers oscillations of intracellular calcium concentration. High phosphate availability is known to inhibit the establishment and/or persistence of this symbiosis, thereby favoring the direct, non-symbiotic uptake of phosphorus by the root system. In this study, Medicago truncatula plants were used to investigate the effects of phosphate supply on the early stages of the interaction. When plants were supplied with high phosphate fungal attachment to the roots was drastically reduced. An experimental system was designed to individually study the effects of phosphate supply on the fungus, on the roots, and on root exudates. These experiments revealed that the most important effects of high phosphate supply were on the roots themselves, which became unable to host mycorrhizal fungi even when these had been appropriately stimulated. The ability of the roots to perceive their fungal partner was then investigated by monitoring nuclear calcium spiking in response to fungal signals. This response did not appear to be affected by high phosphate supply. In conclusion, high levels of phosphate predominantly impact the plant host, but apparently not in its ability to perceive the fungal partner.

Rhizobial nodulation factors (NFs) activate a specific signaling pathway in Medicago truncatula root hairs that involves the complex interplay of Nodulation Signaling Pathway1 (NSP1)/NSP2 GRAS and Ethylene Response Factor Required for Nodulation1 (ERN1) transcription factors (TFs) to achieve full ENOD11 transcription. ERN1 acts as a direct transcriptional regulator of ENOD11 through the activation of the NF-responsive “NF box.” Here, we show that NSP1, when combined with NSP2, can act as a strong positive regulator of ERN1 and ENOD11 transcription. Although ERN1 and NSP1/NSP2 both activate ENOD11, two separate promoter regions are involved that regulate expression during consecutive symbiotic stages. Our findings indicate that ERN1 is required to activate NF-elicited ENOD11 expression exclusively during early preinfection, while NSP1/NSP2 mediates ENOD11 expression during subsequent rhizobial infection. The relative contributions of ERN1 and the closely related ERN2 to the rhizobial symbiosis were then evaluated by comparing their regulation and in vivo dynamics. ERN1 and ERN2 exhibit expression profiles compatible with roles during NF signaling and subsequent infection. However, differences in expression levels and spatiotemporal profiles suggest specialized functions for these two TFs, ERN1 being involved in stages preceding and accompanying infection thread progression while ERN2 is only involved in certain stages of infection. By cross complementation, we show that ERN2, when expressed under the control of the ERN1 promoter, can restore both NF-elicited ENOD11 expression and nodule formation in an ern1 mutant background. This indicates that ERN1 and ERN2 possess similar biological activities and that functional diversification of these closely related TFs relies primarily on changes in tissue-specific expression patterns.

A well-established population of Burmese pythons resides in the Everglades of southern Florida. Prompted in part by a report that identified much of southern USA as suitable habitat for expansion or establishment of the Burmese python, we examined the plausibility of this snake to survive winters at sites north of the Everglades. We integrated daily low and high temperatures recorded from October to February from 2005–2011 at Homestead, Orlando and Gainesville, Florida; and Aiken, South Carolina, with minimum temperatures projected for python digestion (16 °C), activity (5 °C) and survival (0 °C). Mean low and high temperatures decreased northward from Homestead to Aiken and the number of days of freezing temperatures increased northward. Digestion was impaired or inhibited for 2 months in the Everglades and up to at least 5 months in Aiken, and activity was increasingly limited northward during these months. Reports of overwinter survivorship document that a single bout of low and freezing temperatures results in python death. The capacity for Burmese pythons to successfully overwinter in more temperate regions of the USA is seemingly prohibited because they lack the behaviors to seek refuge from, and the physiology to tolerate, cold temperatures. As tropical Southeast Asia is the source of the Everglades Burmese pythons, we predict it is unlikely that they will be able to successfully expand to or colonize more temperate areas of Florida and adjoining states due to their lack of behavioral and physiological traits to seek refuge from cold temperatures.

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

• The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. • Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.

International audience; The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes–mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.

International audience; The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::β-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca2+ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca2+ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca2+ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca2+ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca2+ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca2+ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca2+ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.