Dominique Lauressergues

DRB4-Dependent TAS3 trans-Acting siRNAs Control Leaf Morphology through AGO7

trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.

An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs

Plants contain more DICER‐LIKE (DCL) enzymes and double‐stranded RNA binding (DRB) proteins than other eukaryotes, resulting in increased small RNA network complexities. Analyses of single, double, triple and quadruple dcl mutants exposed DCL1 as a sophisticated enzyme capable of producing both microRNAs (miRNAs) and siRNAs, unlike the three other DCLs, which only produce siRNAs. Depletion of siRNA‐specific DCLs results in unbalanced small RNA levels, indicating a redeployment of DCL/DRB complexes. In particular, DCL2 antagonizes the production of miRNAs and siRNAs by DCL1 in certain circumstances and affects development deleteriously in dcl1 dcl4 and dcl1 dcl3 dcl4 mutant plants, whereas dcl1 dcl2 dcl3 dcl4 quadruple mutant plants are viable. We also show that viral siRNAs are produced by DCL4, and that DCL2 can substitute for DCL4 when this latter activity is reduced or inhibited by viruses, pointing to the competitiveness of DCL2. Given the complexity of the small RNA repertoire in plants, the implication of each DCL, in particular DCL2, in the production of small RNAs that have no known function will constitute one of the next challenges.

Arabidopsis FIERY1, XRN2, and XRN3 Are Endogenous RNA Silencing Suppressors

The eukaryotic defense response posttranscriptional gene silencing (PTGS) is directed by short-interfering RNAs and thwarts invading nucleic acids via the RNA slicing activity of conserved ARGONAUTE (AGO) proteins. PTGS can be counteracted by exogenous or endogenous suppressors, including the cytoplasmic exoribonuclease XRN4, which also degrades microRNA (miRNA)-guided mRNA cleavage products but does not play an obvious role in development. Here, we show that the nuclear exoribonucleases XRN2 and XRN3 are endogenous PTGS suppressors. We also identify excised MIRNA loops as templates for XRN2 and XRN3 and show that XRN3 is critical for proper development. Independently, we identified the nucleotidase/phosphatase FIERY1 (FRY1) as an endogenous PTGS suppressor through a suppressor screen in a hypomorphic ago1 genetic background. FRY1 is one of six Arabidopsis thaliana orthologs of yeast Hal2. Yeast hal2 mutants overaccumulate 3′-phosphoadenosine 5′-phosphate, which suppresses the 5′→3′ exoribonucleases Xrn1 and Rat1. fry1 mutant plants recapitulate developmental and molecular characteristics of xrn mutants and likely restore PTGS in ago1 hypomorphic mutants by corepressing XRN2, XRN3, and XRN4, thus increasing RNA silencing triggers. We anticipate that screens incorporating partially compromised silencing components will uncover additional PTGS suppressors that may not be revealed using robust silencing systems.

Redundant and Specific Roles of the ARGONAUTE Proteins AGO1 and ZLL in Development and Small RNA-Directed Gene Silencing

The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5′-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2

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.

Auxin Perception Is Required for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis

The formation of arbuscules in the arbuscular mycorrhizal symbiosis is dependent on the plant hormone auxin. 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.

A neomorphic sgs3 allele stabilizing miRNA cleavage products reveals that SGS3 acts as a homodimer.

The putative RNA‐binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) protects RNA from degradation before transformation into dsRNA by the RNA‐dependent RNA polymerase RDR6 during plant post‐transcriptional gene silencing and trans‐acting small interfering (siRNA) pathways. In this study, we show that SGS3 acts as a homodimer, and that the point mutation sgs3‐3 impairs post‐transcriptional gene silencing in a dominant‐negative manner through the formation of SGS3/sgs3‐3 heterodimers. Unlike complete‐loss‐of‐function sgs3 mutants, which are impaired in the accumulation of both micro RNA‐directed TAS cleavage products and mature trans‐acting siRNAs, the sgs3‐3 mutant overaccumulates TAS cleavage products and exhibits slightly reduced trans‐acting siRNA accumulation. Together, these results suggest that sgs3‐3 is a neomorphic allele that shows increased RNA protective activity, resulting in decreased RNA processing by downstream post‐transcriptional gene silencing and trans‐acting siRNA pathway components.

Strigolactones contribute to shoot elongation and to the formation of leaf margin serrations in Medicago truncatula R108

Strigolactones were recently identified as a new class of plant hormones involved in the control of shoot branching. The characterization of strigolactone mutants in several species has progressively revealed their contribution to several other aspects of development in roots and shoots. In this article, we characterize strigolactone-deficient and strigolactone-insensitive mutants of the model legume Medicago truncatula for aerial developmental traits. The most striking mutant phenotype observed was compact shoot architecture. In contrast with what was reported in other species, this could not be attributed to enhanced shoot branching, but was instead due to reduced shoot elongation. Another notable feature was the modified leaf shape in strigolactone mutants: serrations at the leaf margin were smaller in the mutants than in wild-type plants. This phenotype could be rescued in a dose-dependent manner by exogenous strigolactone treatments of strigolactone-deficient mutants, but not of strigolactone-insensitive mutants. Treatment with the auxin transport inhibitor N-1-naphthylphtalamic acid resulted in smooth leaf margins, opposite to the effect of strigolactone treatment. The contribution of strigolactones to the formation of leaf serrations in M. truncatula R108 line represents a novel function of these hormones, which has not been revealed by the analysis of strigolactone mutants in other species.

Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis

Genomic traces of symbiosis loss A symbiosis between certain bacteria and their plant hosts delivers fixed nitrogen to the plants. Griesmann et al. sequenced several plant genomes to analyze why nitrogen-fixing symbiosis is irregularly scattered through the evolutionary tree (see the Perspective by Nagy). Various genomes carried traces of lost pathways that could have supported nitrogen-fixing symbiosis. It seems that this symbiosis, which relies on multiple pathways and complex interorganismal signaling, is susceptible to selection and prone to being lost over evolutionary time. Science, this issue p. eaat1743; see also p. 125 Genome-wide comparative analysis across species reveals the fragility of the plant-bacterial symbiosis needed for nitrogen fixation. INTRODUCTION Access to nutrients such as nitrogen is required for plant growth. Legumes and nine additional plant families benefit from the nitrogen-fixing root nodule (NFN) symbiosis, in which roots develop nodules that intracellularly host nitrogen-fixing bacteria. In this mutually beneficial symbiosis, the bacteria convert atmospheric nitrogen into ammonium and deliver it to the host plant. NFN symbiosis thus enables plant survival under nitrogen-limiting conditions in terrestrial ecosystems. In agriculture, this symbiosis reduces reliance on nitrogen fertilizer, thus reducing the costs, ecological impact, and fossil fuel consumption attendant on large-scale application of fertilizers. RATIONALE Molecular phylogenies show that NFN symbiosis is restricted to four angiosperm orders—Fabales, Fagales, Cucurbitales, and Rosales—that together form the monophyletic NFN clade. However, only 10 of the 28 plant families within this clade contain species engaged in the NFN symbiosis. Even within these 10 families, most genera do not form this symbiosis. The NFN symbiosis requires the coordinated function of more than 30 essential genes. Presence of this symbiosis in related families suggests that a genetic change in the ancestor of the NFN clade enabled evolution of NFN symbiosis in this clade. The scattered distribution of functional NFN symbiosis across the clade has led to the question of whether NFN symbiosis evolved multiple times independently in a convergent manner or was lost multiple times regardless of the number of times it arose. Fossil data have been unable to answer this question. Here we used molecular evidence to ask how the current pattern of plant species with NFN symbiosis evolved. RESULTS We sequenced the genomes of seven nodulating species belonging to the Fagales, Rosales, and Cucurbitales orders and the legume subfamily Caesalpinioideae. We complemented this dataset by sequencing three genomes of nonnodulating species from the Cucurbitales and from the legume subfamilies Cercidoideae and Papilionoideae. Using a genome-wide phylogenomic approach, we found that all legume genes with a characterized role in NFN symbiosis are conserved in nodulating species with one exception. We observed larger numbers of order-specific gene family expansions that, solely because of their phylogenetic distribution, may include genes contributing to multiple gains or subsequent refinements of the symbiosis. In parallel, we discovered signatures of multiple independent loss-of-function events for the gene encoding the indispensable NFN symbiosis regulator NODULE INCEPTION (NIN) in 10 of 13 genomes of nonnodulating species within the NFN clade. The pattern suggests at least eight independent losses of NFN symbiosis. CONCLUSION We found that multiple independent losses of NFN symbiosis occurred in the four orders of the NFN clade. These results suggest that NFN symbiosis has previously been more common than currently evident and that this symbiosis is subject to an underestimated adverse selection pressure. Phylogenomics and evolution of NFN symbiosis. Genome sequencing of nodulating and nonnodulating species combined with 27 previously available genomes resulted in a dataset spanning the NFN clade and species outside the NFN clade as an outgroup. Orthogroups were identified and filtered following three phylogenetic patterns. This genome-wide analysis identified two genes involved in NFN symbiosis, NIN and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG), that were lost in most nonnodulating species. The occurrence of multiple losses (red crosses) of NFN symbiosis suggests an adverse selection pressure. The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator NODULE INCEPTION in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.

Positive Gene Regulation by a Natural Protective miRNA Enables Arbuscular Mycorrhizal Symbiosis

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.