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.

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.

SummaryWe have focused our interest on two cis-regulatory elements, named site II motif and telo box, identi®ed 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 cyclerelated 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 AtPura, 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-speci®c developmental pathways is discussed.

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 PromTCP20:CDS20∷GUS∷GFP 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 AtTCP20∷EAR 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.

LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogenfixing, 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.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

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In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 £ 10 10 colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species-and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.