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Short open reading frame (sORF)-encoded polypeptides (SEPs) have recently emerged as key regulators of major cellular processes. Computational methods for the annotation of sORFs combined with transcriptomics and ribosome profiling approaches predicted the existence of tens of thousands of SEPs across the kingdom of life. Although, we still lack unambiguous evidence for most of them. The method of choice to validate the expression of SEPs is mass spectrometry (MS)-based peptidomics. Peptides are less abundant than proteins, which tends to hinder their detection. Therefore, optimization and enrichment methods are necessary to validate the existence of SEPs. In this article, we discuss the challenges for the detection of SEPs by MS and recent developments of biochemical approaches applied to the study of these peptides. We detail the advances made in the different key steps of a typical peptidomics workflow and highlight possible alternatives that have not been explored yet.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

International audience; Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

SummaryBackgroundRecent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRNAs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster.ResultsWe perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes.ConclusionTaken together, these results reveal that several Drosophila pri-miRNAs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet un-described pri-microRNA encoded peptides in Drosophila and their regulatory potential on genome expression.

Additional file 1. Supplementary Figures S1 to S13.

International audience; The importance of microorganisms in plant development, nutrition, and stress resistance is unquestioned and has led to a more holistic approach of plant-microbe interactions, under the holobiont concept. The structure of the plant microbiota is often described as host driven, especially in the rhizosphere, where microbial communities are shaped by diverse rhizodeposits. Gradually, this anthropogenic vision is fading and being replaced by the idea that plants and microorganisms co-shape the plant microbiota. Through coevolution, plants and microbes have developed cross-kingdom communication channels. Here, we propose that miRNAs are crucial mediators of plant-microbe interactions and microbiota shaping in the rhizosphere. Moreover, we suggest, as an alternative to generally unsuccessful strategies based on microbial inoculants, miRNAs as a promising tool for novel holobiont engineering.

Additional file 6. Review history.

Additional file 5. Uncropped Western blots.

International audience; MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

Arbuscular mycorrhizal (AM) symbiosis associates most plants with fungi of the phylum Glomeromycota. The fungus penetrates into roots and forms within cortical cell branched structures called arbuscules for nutrient exchange. We discovered that miR171b has a mismatched cleavage site and is unable to downregulate the miR171 family target gene, LOM1 (LOST MERISTEMS 1). This mismatched cleavage site is conserved among plants that establish AM symbiosis, but not in non-mycotrophic plants. Unlike other members of the miR171 family, miR171b stimulates AM symbiosis and is expressed specifically in root cells that contain arbuscules. MiR171b protects LOM1 from negative regulation by other miR171 family members. These findings uncover a unique mechanism of positive post-transcriptional regulation of gene expression by miRNAs and demonstrate its relevance for the establishment of AM symbiosis.

International audience; - Root colonization by arbuscular mycorrhizal (AM) fungi is a complex and finely tuned process. Previous studies have shown that, among other plant hormones, auxin plays a role in this process but the specific involvement of Aux/IAAs, the key regulators of auxin responses, is still unknown. -In this study, we addressed the role of the tomato Sl-IAA27 during AM symbiosis by using Sl-IAA27-RNAi and pSL-IAA27::GUS stable tomato lines. - The data show that Sl-IAA27 expression is up-regulated by the AM fungus and that silencing of Sl-IAA27 has a negative impact on AM colonization. Sl-IAA27-silencing resulted in down-regulation of three genes involved in strigolactone synthesis, NSP1, D27 and MAX1, and treatment of Sl-IAA27-silenced plants with the strigolactone analog GR24 complemented their mycorrhizal defect phenotype. - Overall, the study identified an Aux/IAA gene as a new component of the signaling pathway controlling AM fungal colonization in tomato. This gene is proposed to control strigolactone biosynthesis via the regulation of NSP1.

MicroRNAs (miRNAs) are short RNA molecules negatively regulating the expression of many important genes in plants and animals. We have recently shown that plant primary transcripts of miRNAs encode peptides (miPEPs) able to increase specifically the transcription of their associated miRNA.(1) We discuss here the possibility of using miPEPs as a new tool for functional analysis of single members of miRNA families in plants, including in non-model plants, that could avoid transgenic transformation and minimize artifactual interpretation. We also raise several fundamental and crucial questions that need to be address for a deeper understanding of the cellular and molecular mechanisms underlining the regulatory activity of miPEPs.

Nodulation and arbuscular mycorrhization require the activation of plant host symbiotic programs by Nod factors, and Myc-LCOs and COs, respectively. The pathways involved in the perception and downstream signaling of these signals include common and distinct components. Among the distinct components, NSP1, a GRAS transcription factor, has been considered for years to be specifically involved in nodulation. Here, we analyzed the degree of conservation of the NSP1 sequence in arbuscular mycorrhizal (AM) host and non-AM host plants and carefully examined the ability of Medicago truncatula nsp1 mutants to respond to Myc-LCOs and to be colonized by an arbuscular mycorrhizal fungus. In AM-host plants, the selection pressure on NSP1 is stronger than in non-AM host ones. The response to Myc-LCOs and the frequency of mycorrhizal colonization are significantly reduced in the nsp1 mutants. Our results reveal that NSP1, previously described for its involvement in the Nod factor signaling pathway, is also involved in the Myc-LCO signaling pathway. They bring additional evidence on the evolutionary relatedness between nodulation and mycorrhization.

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter–GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants. Fil: Bazin, Jeremie. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia. Universite Paris Diderot - Paris 7; Francia Fil: Khan. Ghazanfar Abbas. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia Fil: Combier, Jean Philippe. Laboratoire de Recherche en Sciences Veg etales; Francia. Centre National de la Recherche Scientifique; Francia Fil: Bustos Sanmamed, Maria del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia Fil: Debernardi, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Rodriguez, Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Sorin, Celine. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia. Universite Paris Diderot - Paris 7; Francia Fil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Hartmann, Caroline. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia. Universite Paris Diderot - Paris 7; Francia Fil: Crespi, Martin. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia Fil: Lelandais Briere, Christine. Centre National de la Recherche Scientifique. Institut des Sciences du Veg etal; Francia. Universite Paris Diderot - Paris 7; Francia

Contents 22 I. 22 II. 24 III. 25 IV. 27 V. 29 VI. 10 31 References 32 SUMMARY: Plants have evolved a remarkable faculty of adaptation to deal with various and changing environmental conditions. In this context, the roots have taken over nutritional aspects and the root system architecture can be modulated in response to nutrient availability or biotic interactions with soil microorganisms. This adaptability requires a fine tuning of gene expression. Indeed, root specification and development are highly complex processes requiring gene regulatory networks involved in hormonal regulations and cell identity. Among the different molecular partners governing root development, microRNAs (miRNAs) are key players for the fast regulation of gene expression. miRNAs are small RNAs involved in most developmental processes and are required for the normal growth of organisms, by the negative regulation of key genes, such as transcription factors and hormone receptors. Here, we review the known roles of miRNAs in root specification and development, from the embryonic roots to the establishment of root symbioses, highlighting the major roles of miRNAs in these processes.

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.

International audience; In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.SdhR that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not aVected by 2 g l¡1 carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 g l¡1 of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR ampliWcation and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.SdhR cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less eYcient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%.

Mechanisms regulating legume root nodule development are still poorly understood, and very few regulatory genes have been cloned and characterized. Here, we describe EFD (for ethylene response factor required for nodule differentiation), a gene that is upregulated during nodulation in Medicago truncatula. The EFD transcription factor belongs to the ethylene response factor (ERF) group V, which contains ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in the regulation of nodulation was examined through the characterization of a null deletion mutant (efd-1), RNA interference, and overexpression studies. These studies revealed that EFD is a negative regulator of root nodulation and infection by Rhizobium and that EFD is required for the formation of functional nitrogen-fixing nodules. EFD appears to be involved in the plant and bacteroid differentiation processes taking place beneath the nodule meristem. We also showed that EFD activated Mt RR4, a cytokinin primary response gene that encodes a type-A response regulator. We propose that EFD induction of Mt RR4 leads to the inhibition of cytokinin signaling, with two consequences: the suppression of new nodule initiation and the activation of differentiation as cells leave the nodule meristem. Our work thus reveals a key regulator linking early and late stages of nodulation and suggests that the regulation of the cytokinin pathway is important both for nodule initiation and development.

We show here that MtMMPL1, a Medicago truncatula nodulin gene previously identified by transcriptomics, represents a novel and specific marker for root and nodule infection by Sinorhizobium meliloti. This was established by determining the spatial pattern of MtMMPL1 expression and evaluating gene activation in the context of various plant and bacterial symbiotic mutant interactions. The MtMMPL1 protein is the first nodulin shown to belong to the large matrix metalloendoproteinase (MMP) family. While plant MMPs are poorly documented, they are well characterized in animals as playing a key role in a number of normal and pathological processes involving the remodeling of the extracellular matrix. MtMMPL1 represents a novel MMP variant, with a substitution of a key amino acid residue within the predicted active site, found exclusively in expressed sequence tags corresponding to legume MMP homologs. An RNA interference approach revealed that decreasing MtMMPL1 expression leads to an accumulation of rhizobia within infection threads, whose diameter is often significantly enlarged. Conversely, MtMMPL1 ectopic overexpression under the control of a constitutive (35S) promoter led to numerous abortive infections and an overall decrease in the number of nodules. We discuss possible roles of MtMMPL1 during Rhizobium infection.

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells.

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.

International audience; Nod factors are key bacterial signaling molecules regulating the symbiotic interaction between bacteria known as rhizobia and leguminous plants. Studying plant host genes whose expression is affected by Nod factors has given insights into early symbiotic signaling and development. Here, we used a double supernodulating mutant line that shows increased sensitivity to Nod factors to study the Nod factor-regulated transcriptome. Using microarrays containing more than 16,000 70-mer oligonucleotide probes, we identified 643 Nod-factor-regulated genes, including 225 new Nod-factor-upregulated genes encoding many potential regulators. Among the genes found to be Nod factor upregulated, we identified and characterized MtRALFL1 and MtDVL1, which code for two small putative peptide regulators of 135 and 53 amino acids, respectively. Expression analysis confirmed that these genes are upregulated during initial phases of nodulation. Overexpression of MtRALFL1 and MtDVL1 in Medicago truncatula roots resulted in a marked reduction in the number of nodules formed and in a strong increase in the number of aborted infection threads. In addition, abnormal nodule development was observed when MtRALFL1 was overexpressed. This work provides evidence for the involvement of new putative small-peptide regulators during nodulation.

International audience; Legume root architecture is characterized by the development of two de novo meristems, leading to the formation of lateral roots or symbiotic nitrogen-fixing nodules. Organogenesis involves networks of transcription factors, the encoding mRNAs of which are frequently targets of microRNA (miRNA) regulation. Most plant miRNAs, in contrast with animal miRNAs, are encoded as single entities in an miRNA precursor. In the model legume Medicago truncatula, we have identified the MtMIR166a precursor containing tandem copies of MIR166 in a single transcriptional unit. These miRNAs post-transcriptionally regulate a new family of transcription factors associated with nodule development, the class-III homeodomain-leucine zipper (HD-ZIP III) genes. In situ expression analysis revealed that these target genes are spatially co-expressed with MIR166 in vascular bundles, and in apical regions of roots and nodules. Overexpression of the tandem miRNA precursor correlated with MIR166 accumulation and the downregulation of several class-III HD-ZIP genes, indicating its functionality. MIR166 overexpression reduced the number of symbiotic nodules and lateral roots, and induced ectopic development of vascular bundles in these transgenic roots. Hence, plant polycistronic miRNA precursors, although rare, can be processed, and MIR166-mediated post-transcriptional regulation is a new regulatory pathway involved in the regulation of legume root architecture.

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.

International audience; Aux/IAA genes play a pivotal role in auxin transcriptional regulation. Their functions were mainly studied in Arabidopsis through analysis of gain-of-function mutants. In the tomato, the Solanaceae reference species, different studies on Sl-IAA down-regulated lines showed specific role for Sl-IAA genes. Our recent work revealed that the Sl-IAA 27 gene displays a distinct behavior compared with most Aux/IAA genes, being down-regulated by auxin. Interestingly, the silencing of Sl-IAA27 leads to altered chlorophyll accumulation in leaves, reduced fertilization, altered fruit development and altered root formation. Here we report that IAA27 could be a key auxin signaling gene involved in AM in tomato and also in Medicago model plant. Indeed both Sl-IAA27 and its closest homolog in Medicago truncatula, Mt-IAA27, are overexpressed in mycorrhized roots. These data are in line with the putative role of auxin in arbuscular mycorrhization.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc¯), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc¯ mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc¯ mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc¯:myc+ segregation ratio was observed, suggesting that each myc¯ phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc¯ phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

The arbuscular mycorrhiza (AM), a symbiosis between fungi from the phylum Glomeromycota and nearly 80% of terrestrial plant species. It is characterized by a two-way exchange in which the fungus provides mineral nutrients to the plant in exchange for carbohydrates. However this "feeding" of the fungus during the symbiotic process represents a significant carbon cost for the plant. To maintain a mutualistic interaction the two symbiotic partners have to strictly control the extent of fungal development in the roots. This control is called autoregulation. Several proteins have been found to be important for the regulation of the different mycorrhizal steps: the stimulation of fungal growth in the rhizosphere by the strigolactones, the fungal entrance in the roots, the hyphal proliferation in the roots and the arbuscule formation. In this work we examine in more detail the role of two of these proteins known to be involved in the mycorrhization process, the transcriptional factors NSP1 and NSP2 (Nodulation Signaling Pathway). We first confirm in M. truncatula roots the direct implication of NSP1 in the regulation of two strigolactone biosynthesis genes, DWARF27 (D27) and MAX1, during the asymbiotic conditions. Then, we show that NSP1, unlike NSP2, is a factor that promotes the fungal entries in the root, presumably due to its activation of D27 and MAX1 resulting in a stimulation of strigolactone synthesis and presymbiotic fungal growth. Next, during the later stages of mycorrhization, we highlight that in the colonized tissues NSP1 is absent and the induction of both D27 and MAX1 is not anymore NSP1 dependent. NSP1 protein is then localized in cells which are not yet colonized but are close to a colonization zone. There, it controls negatively the hyphal propagation in the root and positively the formation of arbuscules. In contrast, NSP2 is present in the colonized tissue where it promotes hyphal propagation and arbuscule development, perhaps by interacting with other proteins. We also show that if NSP1 proteins are absent of the colonized tissues, NSP1 transcripts are present. Unexpectedly, we unveil that in those colonized cells, NSP1 mRNA can protect, by a micro RNA (miR171h) decoy action called target mimicry, NSP2 mRNA against miR171h-mediated degradation. This is the first demonstration that a coding RNA molecule can be a target mimic for a microRNA. In our context this finding reveals a positive regulation of NSP2 expression by NSP1 transcripts and brings to light an additional layer of complexity in the mycorrhizal dual role of these two transcription factors. Finally, in tomato, we highlight that SlNSP1 could be directly or indirectly regulated by the AUX/IAA protein, SlIAA27. As a link with auxin we presume that this AUX/IAA protein is a new component of the signaling pathway controlling AM fungal colonization in tomato, and we propose that it controls strigolactone biosynthesis via the regulation of SlNSP1. Overall our work provides new pieces of the mycorrhizal puzzle and shows how important it is to perform spatiotemporal investigations for a better understanding of highly integrated and complex biological processes.; La symbiose mycorhizienne à arbuscule est une interaction bénéfique entre les champignons du phylum Glomeromycota et près de 80% des espèces de plantes terrestres. Elle est caractérisée par un échange réciproque de nutriments dans lequel le champignon fournit des sels minéraux à la plante en échange de sucres issus de la photosynthèse. Cependant, cette "alimentation" du champignon au cours de la symbiose représente un coût carbone important pour la plante. Ainsi, les plantes doivent strictement maitriser le développement des champignons symbiotiques dans les racines. Ce contrôle est appelé autorégulation. Plusieurs protéines ont été démontrées comme étant importantes pour la régulation des différentes étapes de la colonisation : la stimulation de la croissance fongique dans la rhizosphère par les strigolactones, l'entrée dans les racines, la prolifération des hyphes au sein des racines et la formation des arbuscules. Dans ce travail, nous avons examiné plus en détail le rôle de deux de ces protéines connues pour être impliquées dans le processus de mycorhization, les facteurs de transcription NSP1 et NSP2 (Nodulation Signaling Pathway). Nous avons d'abord pu confirmer dans les racines de M. truncatula en conditions non-symbiotiques, l'implication directe de NSP1 dans la régulation de deux gènes de biosynthèse des strigolactones, DWARF27 (D27) et MORE AXILLARY GROWTH (MAX1). Ensuite, nous avons montré que NSP1, contrairement à NSP2, favorise l'entrée du champignon dans la racine, sans doute due à l'induction de la synthèse des strigolactones stimulant le champignon, via l'activation de D27 et de MAX1. Ensuite, au cours des étapes ultérieures de la mycorhization, nous avons montré que dans les tissus colonisés, NSP1 est absent et que l'induction de D27 et de MAX1 n'était plus NSP1 dépendante. À cette étape, l'expression de la protéine NSP1 est localisée dans les cellules justes en amont du front de colonisation fongique. Là, elle contrôle négativement la propagation des hyphes dans la racine et positivement la formation des arbuscules. En revanche, NSP2 est présente dans le tissu colonisé où elle favorise la propagation des hyphes et le développement des arbuscules, peut-être en interaction avec d'autres facteurs. Nous avons également montré chez M. truncatula que si les protéines NSP1 sont absentes des tissus colonisés, les transcrits de NSP1 sont présents. De façon inattendue, nous avons mis en évidence que l'ARN messager de NSP1 avait la capacité de protéger l'ARN messager de NSP2 contre sa dégradation par le microARN (miR171h), par une action de piégeage du miR171h, appelé effet mimicry. Ceci est la première démonstration qu'une molécule d'ARN codante peut être la cible mimétique d'un microARN. Dans notre contexte d'étude cette constatation révèle que les transcrits de NSP1 permettent une régulation positive de l'expression de NSP2, et met en lumière un niveau de complexité supplémentaire dans le rôle de ces deux facteurs de transcription dans la symbiose mycorhizienne. Enfin, dans la tomate, nous avons montré que Sl-NSP1 pourrait être directement ou indirectement régulée par une protéine AUX / IAA impliquée dans la réponse précoce à l'auxine, Sl-IAA27. Ce lien avec l'auxine nous fait présumer que cette AUX/AAI est un nouveau composant de la voie de signalisation du contrôle de la colonisation fongique dans la tomate, et nous proposons qu'il puisse avoir un rôle dans le contrôle de la biosynthèse des strigolactones via la régulation de Sl-NSP1. L'ensemble de ce travail fournit de nouvelles pièces du puzzle constituant la symbiose mycorhizienne et montre l'importance de l'analyse des régulations spatiotemporelles pour une meilleure compréhension de ces processus biologiques extrêmement complexes.