RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

Nitric oxide (NO) is a signaling molecule involved in plant responses to abiotic and biotic stresses. While there is evidence for NO accumulation during legume nodulation, almost no information exists for arbuscular mycorrhizas (AM). Here, we investigated the occurrence of NO in the early stages of Medicago truncatula–Gigaspora margarita interaction, focusing on the plant response to fungal diffusible molecules. NO was visualized in root organ cultures and seedlings by confocal microscopy using the specific probe 4,5-diaminofluorescein diacetate. Five-minute treatment with the fungal exudate was sufficient to induce significant NO accumulation. The specificity of this response to AM fungi was confirmed by the lack of response in the AM nonhost Arabidopsis thaliana and by analyzing mutants impaired in mycorrhizal capacities. NO buildup resulted to be partially dependent on DMI1, DMI2, and DMI3 functions within the so-called common symbiotic signaling pathway which is shared between AM and nodulation. Significantly, NO accumulation was not induced by the application of purified Nod factor, while lipopolysaccharides from Escherichia coli, known to elicit defense-related NO production in plants, induced a significantly different response pattern. A slight upregulation of a nitrate reductase (NR) gene and the reduction of NO accumulation when the enzyme is inhibited by tungstate suggest NR as a possible source of NO. Genetic and cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to AM symbiosis.     

Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism

GC-MS-based metabolite profiling was used to analyse the response of Medicago truncatula cell cultures to elicitation with methyl jasmonate (MeJa), yeast elicitor (YE), or ultraviolet light (UV). Marked changes in the levels of primary metabolites, including several amino acids, organic acids, and carbohydrates, were observed following elicitation with MeJa. A similar, but attenuated response was observed following YE elicitation, whereas little response was observed following UV elicitation. MeJa induced the accumulation of the triterpene beta-amyrin, a precursor to the triterpene saponins, and LC-MS analysis confirmed the accumulation of triterpene saponins in MeJa-elicited samples. In addition, YE induced a slight, but significant accumulation of shikimic acid, an early precursor to the phenylpropanoid pathway, which was also demonstrated to be YE-inducible by LC-MS analyses. Correlation analyses of metabolite relationships revealed perturbation of the glycine, serine, and threonine biosynthetic pathway, and suggested the induction of threonine aldolase activity, an enzyme as yet uncharacterized from plants. Members of the branched chain amino acid pathway accumulated in a concerted fashion, with the strongest correlation being that between leucine and isoleucine (r2=0.941). While UV exposure itself had little effect on primary metabolites, the experimental procedure, as revealed by control treatments, induced changes in several metabolites which were similar to those following MeJa elicitation. Sucrose levels were lower in MJ- and YE-elicited samples compared with control samples, suggesting that a portion of the effects observed on the primary metabolic pool are a consequence of fundamental metabolic repartitioning of carbon resources rather than elicitor-specific induction. In addition, beta-alanine levels were elevated in all elicited samples, which, when viewed in the context of other elicitation responses, suggests the altered metabolism of coenzyme A and its esters, which are essential in secondary metabolism.     

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 and Medicago 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 and Fusarium 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 three M. truncatula Nod(-)/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.     

Repeated leaf wounding alters the colonization of Medicago truncatula roots by beneficial and pathogenic microorganisms

In nature, plants are subject to various stresses that are often accompanied by wounding of the aboveground tissues. As wounding affects plants locally and systemically, we investigated the impact of leaf wounding on interactions of Medicago truncatula with root-colonizing microorganisms, such as the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the pathogenic oomycete Aphanomyces euteiches and the nitrogen-fixing bacterium Sinorhizobium meliloti. To obtain a long-lasting wound response, repeated wounding was performed and resulted in locally and systemically increased jasmonic acid (JA) levels accompanied by the expression of jasmonate-induced genes, among them the genes encoding allene oxide cyclase 1 (MtAOC1) and a putative cell wall-bound invertase (cwINV). After repeated wounding, colonization with the AM fungus was increased, suggesting a role of jasmonates as positive regulators of mycorrhization, whereas the interaction with the rhizobacterium was not affected. In contrast, wounded plants appeared to be less susceptible to pathogens which might be caused by JA-induced defence mechanisms. The effects of wounding on mycorrhization and pathogen infection could be partially mimicked by foliar application of JA. In addition to JA itself, the positive effect on mycorrhization might be mediated by systemically induced cwINV, which was previously shown to exhibit a regulatory function on interaction with AM fungi.     

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.     

Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress

The effects of nitric oxide (NO) on cadmium toxicity in Medicago truncatula seedlings were studied by investigating root growth and uptake of antioxidants, IAA and ions. Exposure to cadmium reduced root growth and NO accumulation, and increased the production of reactive oxygen species (ROS) in roots. Supplementation with NO improved root growth and reduced ROS accumulation in roots. The NO-scavenger cPTIO, the nitrate reductase (NR) inhibitor tungstate, and the NO synthase (NOS) inhibitor L-NAME all inhibited the accumulation of NO in roots and reversed the effects of NO in promoting the root growth and accumulation of proline and glutathione. Application of NO reduced auxin degradation by inhibiting the activity of IAA oxidase. Exogenous NO also enhanced the uptake of K⁺ and Ca²⁺. These results suggest that NO improves cadmium tolerance in plants by reducing oxidative damage, maintaining the auxin equilibrium and enhancing ion absorption.     

LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H]⁺. Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS³ experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula.     

The mitochondrial proteome of the model legume Medicago truncatula

Legumes carry out special biochemical functions, e.g. the fixation of molecular nitrogen based on a symbiosis with proteobacteria. At the cellular level, this symbiosis has to be implemented into the energy metabolism of the host cell. To provide a basis for future analyses, we have characterized the protein complement of mitochondria of the model legume Medicago truncatula using two-dimensional isoelectric focussing (IEF) and blue-native (BN)-SDS-PAGE. While the IEF reference map resulted mainly in resolution of those proteins associated with the mitochondrial matrix, the BN proteomic map allowed separation of protein subunits from the respiratory chain protein complexes, which are located in the organelle's inner membrane. The M. truncatula mitochondrial BN reference map revealed some striking similarities to the one from Arabidopsis thaliana but at the same time exhibited also some special features: complex II is of increased abundance and additionally represented by a low molecular mass form not reported for Arabidopsis. Furthermore three highly abundant forms of prohibitin complexes are present in the mitochondrial proteome of M. truncatula. Special features with respect to mitochondrial protein complexes might reflect adaptations of legumes to elevated cellular energy requirements enabling them to develop symbiotic interactions with rhizobial bacteria.     

Virus-host interactions: new insights from the small RNA world

RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions.     

FtsZ characterization and immunolocalization in the two phases of plastid reorganization in arbuscular mycorrhizal roots of Medicago truncatula

We have analyzed plastid proliferation in root cortical cells of Medicago truncatula colonized by arbuscular mycorrhizal (AM) fungi by concomitantly labeling fungal structures, root plastids, a protein involved in plastid division (FtsZ1) and a protein involved in the biosynthesis of AM-specific apocarotenoids. Antibodies directed against FtsZ1 have been generated after heterologous expression of the respective gene from M. truncatula and characterization of the gene product. Analysis of enzymatic activity and assembly experiments showed similar properties of this protein when compared with the bacterial proteins. Immunocytological experiments allowed two phases of fungal and plastid development to be clearly differentiated and plastid division to be monitored during these phases. In the early phase of arbuscule development, lens-shaped plastids, intermingled with the arbuscular branches, divide frequently. Arbuscule degradation, in contrast, is characterized by large, tubular plastids, decorated by a considerable number of FtsZ division rings.     

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T₀ generation, but these mutations failed to transmit to the T₁ generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.