PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula.

PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses.

Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula.

Myc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control. Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated. Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3- and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3- and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors. These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP.

Role of N-glycosylation sites and CXC motifs in trafficking of medicago truncatula Nod factor perception protein to plasma membrane.

The lysin motif receptor-like kinase, NFP (Nod factor perception), is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulfide bridges, and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulfide bridges involving CXC motifs in the extracellular region that are required for its biological function.

How many E3 ubiquitin ligase are involved in the regulation of nodulation?

In plants, as in animals, recent work has established that many developmental and defence response pathways are regulated by E3 ubiquitin ligases which control the level or the activity of key proteins through ubiquitination. Nodule formation is a tightly regulated process that integrates specific signal exchange and the coordinated activation of developmental mechanisms to synchronize bacterial infection and organ development. In the last decade, the characterization of several E3 ubiquitin ligase with roles during nodulation has been reported. These are mainly RING-finger and U-Box proteins involved either in nodule organogenesis or in the infection process. In this review, we summarize the knowledge in this field and conclude that the major challenge will be the identification of the regulation and targets of these E3 ubiquitin ligases.

The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with the LYK3 symbiotic receptor and negatively regulates infection and nodulation.

LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box-dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant-microbe interactions and development through interacting with receptor-like kinases is discussed.

A remorin protein interacts with symbiotic receptors and regulates bacterial infection.

Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.

In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development.

AtTCP20 is a transcription factor belonging to the Arabidopsis (Arabidopsis thaliana) TCP-P subfamily, characterized by its capacity to bind to site II motifs (TGGGCY). Our aim was to understand the role of AtTCP20 in plant development. The expression pattern of a translational fusion of Prom(TCP20):CDS20GUSGFP suggested a function for AtTCP20 in several plant organs and stages of development. The role of AtTCP20 was challenged in planta by inducing expression of AtTCP20 proteins fused with either a transcriptional activator domain (VP16) or a repressor domain (EAR). Expression of both modified proteins led to severe developmental phenotypes. In-depth analysis suggested that AtTCP20 may participate in the regulation of cell expansion, cell division, and cell differentiation. Gene expression profiling in roots and hypocotyls revealed that 252 genes were down-regulated in both organs after induction of the AtTCP20EAR repressor gene. Site II motifs (TGGGCY) were underrepresented in their promoters. Conversely, GG(A/T)CCC sequences related to binding sites identified for TCP proteins in rice (Oryza sativa) were overrepresented, and a TCP20 fusion protein was shown to bind to these sequences in vitro. Gene ontology indicated that many targeted genes were involved in cell wall biogenesis and modification during expansion and also encoded numerous transcription factors controlling plant development. Our results are consistent with the previous proposal that AtTCP20 is involved in cell division and growth coordination. Moreover, they further suggest that AtTCP20 also contributes to cell expansion control and indicate a different involvement of this protein in plant morphogenesis depending on the organ and the developmental stage.

TCP transcription factors predate the emergence of land plants.

TCP proteins are plant-specific transcription factors identified so far only in angiosperms and shown to be involved in specifying plant morphologies. However, the functions of these proteins remain largely unknown. Our study is the first phylogenetic analysis comparing the TCP genes from higher and lower plants, and it dates the emergence of the TCP family to before the split of the Zygnemophyta. EST database analysis and CODEHOP PCR amplification revealed TCP genes in basal land plant genomes and also in their close freshwater algal relatives. Based on an extensive survey of TCP genes, families of TCP proteins were characterized in the Arabidopsis thaliana, poplar, rice, club-moss, and moss genomes. The phylogenetic trees indicate a continuous expansion of the TCP family during the diversification of the Phragmoplastophyta and a similar degree of expansion in several angiosperm lineages. TCP paralogues were identified in all genomes studied, and Ks values indicate that TCP genes expanded during genome duplication events. MEME and SIMPLE analyses detected conserved motifs and low-complexity regions, respectively, outside of the TCP domain, which reinforced the previous description of a "mosaic" structure of TCP proteins.

Intrarectal immunization with rotavirus 2/6 virus-like particles induces an antirotavirus immune response localized in the intestinal mucosa and protects against rotavirus infection in mice.

Rotavirus (RV) is the main etiological agent of severe gastroenteritis in infants, and vaccination seems the most effective way to control the disease. Recombinant rotavirus-like particles composed of the viral protein 6 (VP6) and VP2 (2/6-VLPs) have been reported to induce protective immunity in mice when administered by the intranasal (i.n.) route. In this study, we show that administration of 2/6-VLPs by the intrarectal (i.r.) route together with either cholera toxin (CT) or a CpG-containing oligodeoxynucleotide as the adjuvant protects adult mice against RV infection. Moreover, when CT is used, RV shedding in animals immunized by the i.r. route is even reduced in comparison with that in animals immunized by the i.n. route. Humoral and cellular immune responses induced by these immunization protocols were analyzed. We found that although i.r. immunization with 2/6-VLPs induces lower RV-specific immunoglobulin G (IgG) and IgA levels in serum, intestinal anti-RV IgA production is higher in mice immunized by the i.r. route. Cellular immune response has been evaluated by measuring cytokine production by spleen and Peyer's patch cells (PPs) after ex vivo restimulation with RV. Mice immunized by the i.n. and i.r. routes display higher gamma interferon production in spleen and PPs, respectively. In conclusion, we demonstrate that i.r. immunization with 2/6-VLPs protects against RV infection in mice and is more efficient than i.n. immunization in inducing an anti-RV immune response in intestinal mucosa.

Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti.

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.

Internal telomeric repeats and 'TCP domain' protein-binding sites co-operate to regulate gene expression in Arabidopsis thaliana cycling cells.

We have focused our interest on two cis-regulatory elements, named site II motif and telo box, identified within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene. A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins. Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter. This expression is strongly enhanced by addition of a telo box within this chimaeric promoter. We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family. In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPuralpha, which was previously shown to bind telo boxes. An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells. The implication of these observations in plant-specific developmental pathways is discussed.