Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the advent of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at hight titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.

Development of a reverse genetics system enabling the rescue of recombinant avian influenza virus A/Turkey/England/50-92/91 (H5N1)

We previously described the use of an established reverse genetics system for the generation of recombinant human influenza A viruses from cloned cDNAs. Here, we have assembled a set of plasmids to allow recovery of the avian H5N1 influenza virus A/Turkey/England/50-92/91 entirely from cDNA. This system enables us to introduce mutations or truncations into the cDNAs to create mutant viruses altered specifically in a chosen gene. These mutant viruses can then be used in future pathogenesis studies in chickens and in studies to understand the host range restrictions of avian influenza viruses in humans.

Tackling feline infectious peritonitis via reverse genetics

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Controlling complexity: the clinical relevance of mouse complex genetics.

Experimental animal models are essential to obtain basic knowledge of the underlying biological mechanisms in human diseases. Here, we review major contributions to biomedical research and discoveries that were obtained in the mouse model by using forward genetics approaches and that provided key insights into the biology of human diseases and paved the way for the development of novel therapeutic approaches.

he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.

Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.

Peroxisomal beta-oxidation--a metabolic pathway with multiple functions.

Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.

A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants.

BACKGROUND: Genes involved in arbuscular mycorrhizal (AM) symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes (forward genetics). In addition, a number of AM-related genes has been identified by their AM-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches (reverse genetics). However, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. If such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. The goal of this study was to systematically search for AM-related genes with a bioinformatics strategy that is insensitive to these problems. The central element of our approach is based on the fact that many AM-related genes are conserved only among AM-competent species. RESULTS: Our approach involves genome-wide comparisons at the proteome level of AM-competent host species with non-mycorrhizal species. Using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the AM-competent species. Proteins of these clusters were then analyzed in an extended set of 16 plant species and ranked based on their relatedness among AM-competent monocot and dicot species, relative to non-mycorrhizal species. In addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. As a result we present a list of yet uncharacterized proteins that show a strongly AM-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in AM. Among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the S-locus receptor kinases involved in sporophytic self-incompatibility. CONCLUSIONS: We present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. This strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest.

Molecular genetic approaches to the biology of the moss "Physcomitrella patens"

La mousse haplobiontique Physcomitrella patens est utilisée comme système génétique modèle pour l'étude du développement des plantes. Cependant, l'absence d'un protocole efficace de transformation a constitué jusqu'à présent un gros désavantage méthodologique pour le développement futur de ce système expérimental. Les résultats présentés dans le premier chapitre relatent la mise au point d'un protocole de transformation basé sur la technique de transfert direct de gènes dans des protoplastes par précipitation au PEG. Un essai d'expression transitoire de gènes a été mis au point. Ce protocole a été adapté afin de permettre l'introduction in vivo d'anticorps dans des protoplastes. Le protocole modifié permet d'introduire simultanément du DNA et des IgG dans les cellules, et nous avons démontré que ces anticorps peuvent inactiver spécifiquement le produit d'un gène co-introduit (GUS), ainsi que certaines protéines impliquées dans des processus cellulaires (tubuline). Cet essai, baptisé "essai transitoire d'immuno-inactivation in vivo", devrait être directement applicable à d'autres protoplastes végétaux, et permettre l'élaboration de nouvelles stratégies dans l'étude de processus cellulaires. Le second chapitre est consacré aux expériences de transformation de la mousse avec des gènes conférant une résistance à des antibiotiques. Nos résultats démontrent que l'intégration de gènes de résistance dans le génome de P. patens est possible, mais que cet événement est rare. Il s'agit là néanmoins de la première démonstration d'une transformation génétique réussie de cet organisme. L'introduction de gènes de résistance aux antibiotiques dans les protoplastes de P. patens génère à haute fréquence des clones résistants instables. Deux classes de clones instables ont été identifiés. La caractérisation phénotypique, génétique et moléculaire de ces clones suggère fortement que les séquences transformantes sont concaténées pour former des structures de haut poids moléculaire, et que ces structures sont efficacement répliquées et maintenues dans les cellules résistantes en tant qu'éléments génétiques extrachromosomaux. Ce type de transformation nous permet d'envisager des expériences permettant l'identification des séquences génomiques impliquées dans la replication de l'ADN de mousse. Plusieurs lignées transgéniques ont été retransformées avec des plasmides portant des séquences homologues aux séquences intégrées dans le génome, mais conférant une résistance à un autre antibiotique. Les résultats présentés dans le troisième chapitre montrent que les fréquences de transformation intégrative dans les lignées transgéniques sont 10 fois plus élevées que dans la lignée sauvage, et que cette augmentation est associée à une coségrégation des gènes de résistance dans la plupart des clones testés. Ces résultats génétiques indiquent que l'intégration de séquences d'ADN étranger dans le génome de P. patens a lieu en moyenne 10 fois plus fréquemment par recombinaison homologue que par intégration aléatoire. Ce rapport homologue/aléatoire est 10000 fois supérieur aux rapports obtenus avec d'autres plantes, et fournit l'outil indispensable à la réalisation d'expériences de génétique inverse dans cet organisme à haplophase dominante. THESIS SUMMARY The moss Physcomitrella patens is used as a model genetic system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. But further development of this model system was hampered by the lack of a protocol allowing the genetic transformation of this plant. We have developed a transformation protocol based on PEG-mediated direct gene transfer to protoplasts. Our data demonstrate that this procedure leads to the establishment of an efficient transient gene expression assay. A slightly modified protocol has been developed allowing the in vivo introduction of antibodies in moss protoplasts. Both DNA and IgGs can be loaded simultaneously, and specific antibodies can immunodeplete the product of an expression cassette (GUS) as well as proteins involved in cellular processes (tubulins). This assay, named transient in vivo immunodepletion assay, should be applicable to other plant protoplasts, and offers new approaches to study cellular processes. Transformations have been performed with bacterial plasmids carrying antibiotic resistance expression cassette. Our data demonstrate that integrative transformation occurs, but at low frequencies. This is the first demonstration of a successful genetic transformation of mosses. Resistant unstable colonies are recovered at high frequencies following transformation, and two different classes of unstable clones have been identified. Phenotypical, genetic and molecular characterisation of these clones strongly suggests that bacterial plasmids are concatenated to form high molecular arrays which are efficiently replicated and maintained as extrachromosomal elements in the resistant cells. Replicative transformation in P. patens should allow the design of experiments aimed at the identification of genomic sequences involved in moss DNA replication. Transgenic strains have been retransformed with bacterial plasmids carrying sequences homologous to the integrated transloci, but conferring resistance to another antibiotic. Our results demonstrate an order of magnitude increase of integrative transformation frequencies in transgenic strains as compared to wild-type, associated with cosegregation of the resistance genes in most of these double resistant transgenic strains. These observations provide strong genetic evidence that gene targeting occurs about ten times more often than random integration in the genome of P. patens. Such ratio of targeted to random integration is about 10 000 times higher than previous reports of gene targeting in plants, and provides the essential requirement for the development of efficient reverse genetics in the haplodiplobiontic P. patens.