Defense and senescence interplay in legume nodules.

Immunity and senescence play a crucial role in the functioning of the legume symbiotic nodules. The miss-regulation of one of these processes compromises the symbiosis leading to death of the endosymbiont and the arrest of the nodule functioning. The relationship between immunity and senescence has been extensively studied in plant organs where a synergistic response can be observed. However, the interplay between immunity and senescence in the symbiotic organ is poorly discussed in the literature and these phenomena are often mixed up. Recent studies revealed that the cooperation between immunity and senescence is not always observed in the nodule, suggesting complex interactions between these two processes within the symbiotic organ. Here, we discuss recent results on the interplay between immunity and senescence in the nodule and the specificities of this relationship during legume-rhizobium symbiosis.

Impact of high atmospheric carbon dioxide on the biotic stress response of the model cereal species Brachypodium distachyon.

Losses due to disease and climate change are among the most important issues currently facing crop production. It is therefore important to establish the impact of climate change, and particularly of high carbon dioxide (hCO2), on plant immunity in cereals, which provide 60% of human calories. The aim of this study was to determine if hCO2 impacts Brachypodium distachyon immunity, a model plant for temperate cereals. Plants were grown in air (430 ppm CO2) and at two high CO2 conditions, one that is relevant to projections within the coming century (1000 ppm) and a concentration sufficient to saturate photosynthesis (3000 ppm). The following measurements were performed: phenotyping and growth, salicylic acid contents, pathogen resistance tests, and RNAseq analysis of the transcriptome. Improved shoot development was observed at both 1000 and 3000 ppm. A transcriptomic analysis pointed to an increase in primary metabolism capacity under hCO2. Alongside this effect, up-regulation of genes associated with secondary metabolism was also observed. This effect was especially evident for the terpenoid and phenylpropanoid pathways, and was accompanied by enhanced expression of immunity-related genes and accumulation of salicylic acid. Pathogen tests using the fungus Magnaporthe oryzae revealed that hCO2 had a complex effect, with enhanced susceptibility to infection but no increase in fungal development. The study reveals that immunity in B. distachyon is modulated by growth at hCO2 and allows identification of pathways that might play a role in this effect.

Insight into the control of nodule immunity and senescence during Medicago truncatula symbiosis.

Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix-). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix- nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix- nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules.

The transcriptional co-regulators NBCL1 and NBCL2 redundantly coordinate aerial organ development and root nodule identity in legumes.

Medicago truncatula NODULE ROOT1 (MtNOOT1) and Pisum sativum COCHLEATA1 (PsCOCH1) are orthologous genes belonging to the NOOT-BOP-COCH-LIKE (NBCL) gene family which encodes key transcriptional co-regulators of plant development. In Mtnoot1 and Pscoch1 mutants, the development of stipules, flowers, and symbiotic nodules is altered. MtNOOT2 and PsCOCH2 represent the single paralogues of MtNOOT1 and PsCOCH1, respectively. In M. truncatula, MtNOOT1 and MtNOOT2 are both required for the establishment and maintenance of symbiotic nodule identity. In legumes, the role of NBCL2 in above-ground development is not known. To better understand the roles of NBCL genes in legumes, we used M. truncatula and P. sativum nbcl mutants, isolated a knockout mutant for the PsCOCH2 locus and generated Pscoch1coch2 double mutants in P. sativum. Our work shows that single Mtnoot2 and Pscoch2 mutants develop wild-type stipules, flowers, and symbiotic nodules. However, the number of flowers was increased and the pods and seeds were smaller compared to the wild type. Furthermore, in comparison to the corresponding nbcl1 single mutants, both the M. truncatula and P. sativum nbcl double mutants show a drastic alteration in stipule, inflorescence, flower, and nodule development. Remarkably, in both M. truncatula and P. sativum nbcl double mutants, stipules are transformed into a range of aberrant leaf-like structures.

Identification of beneficial Lebanese Trichoderma spp. wheat endophytes.

Wheat is one of the most important crops in the world. Its production can be influenced by a diversity of beneficial and pathogenic rhizospheric microbes, including fungi. Amongst them, beneficial Trichoderma spp. can be used as alternatives to chemical fertilizers, as they are cheap and harmless to the environment. Our study aimed to isolate, identify, and characterize Trichoderma spp. from Lebanon associated with wheat. Two Trichoderma strains belonging to T. afroharzianum, and T. guizhouense species, were isolated and found to be endophytes, enhancing root growth and producing Indole-3-acetic acid. Inoculation also improved seedling development, and increased plant growth and yield. Furthermore, the two strains inhibit Fusarium growth in vitro. These Trichoderma spp. have thus the capacity to be used as organic fertilizers for wheat.

Arabidopsis thaliana SHOOT MERISTEMLESS Substitutes for Medicago truncatula SINGLE LEAFLET1 to Form Complex Leaves and Petals.

LEAFY plant-specific transcription factors, which are key regulators of flower meristem identity and floral patterning, also contribute to meristem activity. Notably, in some legumes, LFY orthologs such as Medicago truncatula SINGLE LEAFLET (SGL1) are essential in maintaining an undifferentiated and proliferating fate required for leaflet formation. This function contrasts with most other species, in which leaf dissection depends on the reactivation of KNOTTED-like class I homeobox genes (KNOXI). KNOXI and SGL1 genes appear to induce leaf complexity through conserved downstream genes such as the meristematic and boundary CUP-SHAPED COTYLEDON genes. Here, we compare in M. truncatula the function of SGL1 with that of the Arabidopsis thaliana KNOXI gene, SHOOT MERISTEMLESS (AtSTM). Our data show that AtSTM can substitute for SGL1 to form complex leaves when ectopically expressed in M. truncatula. The shared function between AtSTM and SGL1 extended to the major contribution of SGL1 during floral development as ectopic AtSTM expression could promote floral organ identity gene expression in sgl1 flowers and restore sepal shape and petal formation. Together, our work reveals a function for AtSTM in floral organ identity and a higher level of interchangeability between meristematic and floral identity functions for the AtSTM and SGL1 transcription factors than previously thought.

Effector-Dependent and -Independent Molecular Mechanisms of Soybean-Microbe Interaction.

Soybean is a pivotal staple crop worldwide, supplying the main food and feed plant proteins in some countries. In addition to interacting with mutualistic microbes, soybean also needs to protect itself against pathogens. However, to grow inside plant tissues, plant defense mechanisms ranging from passive barriers to induced defense reactions have to be overcome. Pathogenic but also symbiotic micro-organisms effectors can be delivered into the host cell by secretion systems and can interfere with the immunity system and disrupt cellular processes. This review summarizes the latest advances in our understanding of the interaction between secreted effectors and soybean feedback mechanism and uncovers the conserved and special signaling pathway induced by pathogenic soybean cyst nematode, Pseudomonas, Xanthomonas as well as by symbiotic rhizobium.

Brachypodium distachyon UNICULME4 and LAXATUM-A are redundantly required for development.

In cultivated grasses, tillering, leaf, and inflorescence architecture, as well as abscission ability, are major agronomical traits. In barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and brachypodium (Brachypodium distachyon), NOOT-BOP-COCH-LIKE (NBCL) genes are essential regulators of vegetative and reproductive development. Grass species usually possess 2-4 NBCL copies and until now a single study in O. sativa showed that the disruption of all NBCL genes strongly altered O. sativa leaf development. To improve our understanding of the role of NBCL genes in grasses, we extended the study of the two NBCL paralogs BdUNICULME4 (CUL4) and BdLAXATUM-A (LAXA) in the nondomesticated grass B. distachyon. For this, we applied reversed genetics and generated original B. distachyon single and double nbcl mutants by clustered regularly interspaced short palindromic repeats - CRISPR associated protein 9 (CRISPR-Cas9) approaches and genetic crossing between nbcl targeting induced local lesions in genomes (TILLING) mutants. Through the study of original single laxa CRISPR-Cas9 null alleles, we validated functions previously proposed for LAXA in tillering, leaf patterning, inflorescence, and flower development and also unveiled roles for these genes in seed yield. Furthermore, the characterization of cul4laxa double mutants revealed essential functions for nbcl genes in B. distachyon development, especially in the regulation of tillering, stem cell elongation and secondary cell wall composition as well as for the transition toward the reproductive phase. Our results also highlight recurrent antagonist interactions between NBCLs occurring in multiple aspects of B. distachyon development.

Avoidance of detrimental defense responses in beneficial plant-microbe interactions.

In the environment microbes interact with plants and provide them with benefits that include protection against biotic and abiotic stresses as well as improved nutrition. However, plants are also exposed to parasites and pathogens. To manage appropriate responses, evolution has resulted in improved tolerance of plants to beneficial microbes while keeping the ability to recognize detrimental ones and to develop defense responses. Here we review the mechanisms involved in these interactions. We also discuss how the interactions might be handled to improve crop resistance to pathogens without losing the ability to establish beneficial interactions.

WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn.

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous ArabidopsisWUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa.

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Roles of BdUNICULME4 and BdLAXATUM-A in the non-domesticated grass Brachypodium distachyon.

In cultivated grasses, tillering, spike architecture and seed shattering represent major agronomical traits. In barley, maize and rice, the NOOT-BOP-COCH-LIKE (NBCL) genes play important roles in development, especially in ligule development, tillering and flower identity. However, compared with dicots, the role of grass NBCL genes is underinvestigated. To better understand the role of grass NBCLs and to overcome any effects of domestication that might conceal their original functions, we studied TILLING nbcl mutants in the non-domesticated grass Brachypodium distachyon. In B. distachyon, the NBCL genes BdUNICULME4 (CUL4) and BdLAXATUM-A (LAXA) are orthologous, respectively, to the barley HvUniculme4 and HvLaxatum-a, to the maize Zmtassels replace upper ears1 and Zmtassels replace upper ears2 and to the rice OsBLADE-ON-PETIOLE1 and OsBLADE-ON-PETIOLE2/3. In B. distachyon, our reverse genetics study shows that CUL4 is not essential for the establishment of the blade-sheath boundary but is necessary for the development of the ligule and auricles. We report that CUL4 also exerts a positive role in tillering and a negative role in spikelet meristem activity. On the other hand, we demonstrate that LAXA plays a negative role in tillering, positively participates in spikelet development and contributes to the control of floral organ number and identity. In this work, we functionally characterized two new NBCL genes in a context of non-domesticated grass and highlighted original roles for grass NBCL genes that are related to important agronomical traits.

Medicago-Sinorhizobium-Ralstonia Co-infection Reveals Legume Nodules as Pathogen Confined Infection Sites Developing Weak Defenses.

Legumes have the capacity to develop root nodules hosting nitrogen-fixing bacteria, called rhizobia. For the plant, the benefit of the symbiosis is important in nitrogen-deprived conditions, but it requires hosting and feeding massive numbers of rhizobia. Recent studies suggest that innate immunity is reduced or suppressed within nodules [1-10]; this likely maintains viable rhizobial populations. To evaluate the potential consequences and risks associated with an altered immuni`ty in the symbiotic organ, we developed a tripartite system with the model legume Medicago truncatula [11, 12], its nodulating symbiont of the genus Sinorhizobium (syn. Ensifer) [13, 14], and the pathogenic soil-borne bacterium Ralstonia solanacearum [15-18]. We show that nodules are frequent infection sites where pathogen multiplication is comparable to that in the root tips and independent of nodule ability to fix nitrogen. Transcriptomic analyses indicate that, despite the presence of the hosted rhizobia, nodules are able to develop weak defense reactions against pathogenic R. solanacearum. Nodule defense response displays specificity compared to that activated in roots. In agreement with nodule innate immunity, optimal R. solanacearum growth requires pathogen virulence factors. Finally, our data indicate that the high susceptibility of nodules is counterbalanced by the existence of a diffusion barrier preventing pathogen spreading from nodules to the rest of the plant.

Sinorhizobium meliloti succinylated high-molecular-weight succinoglycan and the Medicago truncatula LysM receptor-like kinase MtLYK10 participate independently in symbiotic infection.

The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

Symbiosis Signaling: Solanaceae Symbiotic LCO Receptors Are Functional for Rhizobium Perception in Legumes.

A new study shows that plant receptor genes necessary for the ancient and widespread symbiosis with arbuscular mycorrhizal fungi were co-opted in legume plants, without modifications, to establish the evolutionarily more recent and more specific symbiosis with their bacterial rhizobium partners.

Author Correction: Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant.

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Despite the interest in these compounds, many steps of the biosynthetic pathway have not been elucidated. In this study, we identified the alkaloids produced in different organs of R. bifida under different growth conditions, set up the conditions for in vitro R. bifida regeneration and initiated the molecular characterization of two R. bifida genes involved in alkaloids biosynthesis: the Norbelladine 4'-O-Methyltransferase (RbN4OMT) and the Cytochrome P450 (RbCYP96T). We show that montanine is the main alkaloid produced in the different R. bifida organs and developed a direct organogenesis regeneration protocol, using twin-scale explants cultivated on media enriched with naphthalene acetic acid and benzyladenine. Finally, we analyzed the RbN4OMT and RbCYP96T gene expressions in different organs and culture conditions and compared them to alkaloid production. In different organs of R. bifida young, adult and regenerated plants, as well as under various growing conditions, the transcripts accumulation was correlated with the production of alkaloids. This work provides new tools to improve the production of this important pharmaceutical compound and for future biotechnological studies.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

OBJECTIVES: The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. RESULTS: We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days. CONCLUSION: This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Legume Nodules: Massive Infection in the Absence of Defense Induction.

Plants of the legume family host massive intracellular bacterial populations in the tissues of specialized organs, the nodules. In these organs, the bacteria, named rhizobia, can fix atmospheric nitrogen and transfer it to the plant. This special metabolic skill provides to the legumes an advantage when they grow on nitrogen-scarce substrates. While packed with rhizobia, the nodule cells remain alive, metabolically active, and do not develop defense reactions. Here, we review our knowledge on the control of plant immunity during the rhizobia-legume symbiosis. We present the results of an evolutionary process that selected both divergence of microbial-associated molecular motifs and active suppressors of immunity on the rhizobial side and, on the legume side, active mechanisms that contribute to suppression of immunity.

MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity.

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. Medicago truncatula NODULE ROOT1 (MtNOOT1), Pisum sativum COCHLEATA1 (PsCOCH1), and Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) are orthologs of Arabidopsis (Arabidopsis thaliana) AtBLADE-ON-PETIOLE1/2 and are members of the NBCL gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of MtNOOT1, PsCOCH1, and LjNBCL1 triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in M. truncatula, MtNOOT2MtNOOT2 is the paralog of MtNOOT1 and belongs to a second legume-specific NBCL subclade, the NBCL2 clade. MtNOOT2 expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. Mtnoot2 mutants did not present any particular symbiotic phenotype; however, the loss of function of both MtNOOT1 and MtNOOT2 resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. Mtnoot1 noot2 double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules.