Sara M. Díaz-Moreno

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinklers, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Reprogramming of gene expression during compression wood formation in pine: Coordinated modulation of S-adenosylmethionine, lignin and lignan related genes

BackgroundTranscript profiling of differentiating secondary xylem has allowed us to draw a general picture of the genes involved in wood formation. However, our knowledge is still limited about the regulatory mechanisms that coordinate and modulate the different pathways providing substrates during xylogenesis. The development of compression wood in conifers constitutes an exceptional model for these studies. Although differential expression of a few genes in differentiating compression wood compared to normal or opposite wood has been reported, the broad range of features that distinguish this reaction wood suggest that the expression of a larger set of genes would be modified.ResultsBy combining the construction of different cDNA libraries with microarray analyses we have identified a total of 496 genes in maritime pine (Pinus pinaster, Ait.) that change in expression during differentiation of compression wood (331 up-regulated and 165 down-regulated compared to opposite wood). Samples from different provenances collected in different years and geographic locations were integrated into the analyses to mitigate the effects of multiple sources of variability. This strategy allowed us to define a group of genes that are consistently associated with compression wood formation. Correlating with the deposition of a thicker secondary cell wall that characterizes compression wood development, the expression of a number of genes involved in synthesis of cellulose, hemicellulose, lignin and lignans was up-regulated. Further analysis of a set of these genes involved in S-adenosylmethionine metabolism, ammonium recycling, and lignin and lignans biosynthesis showed changes in expression levels in parallel to the levels of lignin accumulation in cells undergoing xylogenesis in vivo and in vitro.ConclusionsThe comparative transcriptomic analysis reported here have revealed a broad spectrum of coordinated transcriptional modulation of genes involved in biosynthesis of different cell wall polymers associated with within-tree variations in pine wood structure and composition. In particular, we demonstrate the coordinated modulation at transcriptional level of a gene set involved in S-adenosylmethionine synthesis and ammonium assimilation with increased demand for coniferyl alcohol for lignin and lignan synthesis, enabling a better understanding of the metabolic requirements in cells undergoing lignification.

EuroPineDB: a high-coverage web database for maritime pine transcriptome

BackgroundPinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases.DescriptionEuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided.ConclusionsThe EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation

Fluorescent protein markers and a newly discovered specific inhibitor of callose synthesis identify discrete trafficking events during late cytokinesis. Although cytokinesis is vital for plant growth and development, our mechanistic understanding of the highly regulated membrane and cargo transport mechanisms in relation to polysaccharide deposition during this process is limited. Here, we present an in-depth characterization of the small molecule endosidin 7 (ES7) inhibiting callose synthase activity and arresting late cytokinesis both in vitro and in vivo in Arabidopsis (Arabidopsis thaliana). ES7 is a specific inhibitor for plant callose deposition during cytokinesis that does not affect endomembrane trafficking during interphase or cytoskeletal organization. The specificity of ES7 was demonstrated (1) by comparing its action with that of known inhibitors such as caffeine, flufenacet, and concanamycin A and (2) across kingdoms with a comparison in yeast. The interplay between cell plate-specific post-Golgi vesicle traffic and callose accumulation was analyzed using ES7, and it revealed unique and temporal contributions of secretory and endosomal vesicles in cell plate maturation. While RABA2A-labeled vesicles, which accumulate at the early stage of cell plate formation, were not affected by ES7, KNOLLE was differentially altered by the small molecule. In addition, the presence of clathrin-coated vesicles in cells containing elevated levels of callose and their reduction under ES7 treatment further support the role of endocytic membrane remodeling in the maturing cell plate while the plate is stabilized by callose. Taken together, these data show the essential role of callose during the late stages of cell plate maturation and establish the temporal relationship between vesicles and regulatory proteins at the cell plate assembly matrix during polysaccharide deposition.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium

Analysis of Brachypodium mutants with increased cellular steady state auxin content and root cell elongation sheds light on the importance of the Acid Growth Theory of plant cell elongation in roots. The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Toward a Pinus pinaster bacterial artificial chromosome library

Conifers are of great economic and ecological importance, but little is known concerning their genomic organization. This study is an attempt to obtain high-quality high-molecular-weight DNA from Pinus pinaster cotyledons and the construction of a pine BAC library. The preparation incorporates modifications like low centrifugation speeds, increase of EDTA concentration for plug maintenance, use of DNase inhibitors to reduce DNA degradation, use of polyvinylpyrrolidone and ascorbate to avoid secondary metabolites, and a brief electrophoresis of the plugs prior to their use. A total of 72 192 clones with an average insert size of 107 kb, which represents an equivalent of 11X pine haploid genomes, were obtained. The proportions of clones lacking inserts or containing chloroplast DNA are both approximately 1.6%. The library was screened with cDNA probes for seven genes, and two clones containing Fd-GOGAT sequences were found, one of them seemingly functional. Ongoing projects aimed at constructing a pine bacterial artificial chromosome library may benefit from the methods described here.RésuméLes conifères présentent un intérêt économique et écologique de premier plan mais restent très mal connus du point de vue de l’organisation de leur génome. Cette étude présente une tentative réussie de construction d’une banque BAC de séquences d’ADN de haute qualité et de poids moléculaire élevé à partir de cotylédons de Pinus pinaster. Le protocole de préparation se base sur des ajustements comme une baisse de la vitesse de centrifugation, une augmentation des concentrations d’EDTA dans les culots, l’utilisation d’inhibiteurs des ADNases pour limiter la dégradation de l’ADN, l’utilisation de polyvinylpyrrolidone et d’ascorbate pour éliminer les métabolites secondaires, et de brèves électrophorèses des culots. Un total de 72 192 clones a été obtenu, d’une dimension moyenne d’inserts de 107 kb et représentant l’équivalent de 11X du génome haploïde de pin. La proportion de clones dépourvus d’inserts ou contenant de l’ADN chloroplastique était de 1.6%. La banque a été testée avec des ADN complémentaires de 7 gènes, et deux clones contenant la séquence de la Fd-GOGAT ont été détectés. Des projets visant à construire une banque bactérienne artificielle (BAC) de chromosome de pin tireront bénéfice de l’utilisation de cette méthode.

A Web Tool to Discover Full-Length Sequences — Full-Lengther

Many Expressed Sequence Tags (EST) sequencing projects produce thousands of sequences that must be cleaned and annotated. This research presents the so-called Full-Lengther, an algorithm that can find out full-length cDNA sequences from EST data. To accomplish this task, Full-Lenther is based on a BLAST report using a protein database such as UniProt. Blast alignments will guide to locate protein coding regions, mainly the start codon. Full-Lengther contains an ORF prediction algorithm for those cases which is not homologous to any sequence. The algorithm is implemented as a web tool to simplify its use and portability. This can be worldwide accessible via http://castanea.ac.uma.es/genuma/full-lengther/

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

Significance Cellulose is an abundant natural polymer synthesized primarily by vascular plants in which it forms the load-bearing component of the cell wall. It is a linear polymer of glucose molecules synthesized by membrane-embedded cellulose synthases that couple polymer synthesis with its secretion across the plasma membrane. Plants express multiple cellulose synthase isoforms that are organized into large macromolecular assemblies of varying composition that are likely responsible for aligning the cellulose strands into microfibrils. Here we show that recombinantly expressed and purified Populus tremula x tremuloides (hybrid aspen) cellulose synthase-8 is sufficient for cellulose biosynthesis and produces cellulose microfibrils in vitro. Our results demonstrate that no other plant-derived factors are required for cellulose microfibril biosynthesis. Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Molecular and Functional Analyses Support a Role of Ornithine-δ-Aminotransferase in the Provision of Glutamate for Glutamine Biosynthesis during Pine Germination

We report the molecular characterization and functional analysis of a gene (PsδOAT) from Scots pine (Pinus sylvestris) encoding Orn-δ-aminotransferase (δ-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other δ-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsδOAT encodes a functional δ-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsδOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of δ-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsδOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsδOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that δ-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.

Gene expression profiling in the stem of young maritime pine trees : detection of ammonium stress-responsive genes in the apex

The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base of maritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along the stem, from the top to the bottom. After validation of the microarray data, additional gene expression analyses were also performed in the shoots of young maritime pine trees exposed to different levels of ammonium nutrition. Our results show that the apex of maritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsive genes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young trees in managed forests.