Recherche de gènes associés à la tolérance à l'aluminium chez le blé Triticum aestivum et le riz Oryza sativa par de nouvelles approches moléculaires

L'aluminium est considéré comme le principal facteur limitant de la productivité des plantes dans les sols acides. Ces sols représentent 40% des terres cultivables du globe. En Amérique du Nord, ils constituent 40,9% des terres arables et sont essentiellement répartis sur les territoires canadiens. Face à l'augmentation de la population mondiale, il y a un enjeu économique important à augmenter la surface des terres cultivables et donc à pallier la phytotoxicité due à l'aluminium. Le but de cette thèse était d'isoler des gènes de plantes conférant une résistance au métal. En effet, certaines plantes présentent une résistance plus ou moins élevée face à l'aluminium en sols acides. Des études génétiques ont montré que ce phénotype était porté par le génome nucléaire et résultait de l'expression d'un à trois gènes selon le genre végétal étudié. Une fois ces gènes isolés, il serait donc possible de transférer le caractère de résistance vers des plantes à fort intérêt agronomique. Les travaux réalisés dans cette thèse ont consisté à appliquer deux approches de biologie moléculaire pour mettre en évidence des gènes impliqués dans la résistance : l'Hybridation Suppressive Soustractive (SSH) et la construction de banques d'expression de protéines via les systèmes GeneRacer/Gateway. Ces techniques ont été utilisées pour rechercher des gènes de tolérance chez deux types de plantes résistantes : le blé Atlas-66 et le riz (naturellement très résistant). Un nouveau système de banque d'expression de protéines a été mis au point pour permettre de réaliser des banques à partir de petites quantités d'ARNm et d'exprimer ces banques dans différents organismes. Aucun gène de résistance n'a pu être isolé à partir de ces banques. Face au faible rendement de la SSH, de nouveaux procédés de suppression PCR ont été élaborés. Ces procédés ont été nommés Suppression PCR par Remplissage des Extrémités et Suppression PCR par Ajout d'ARN. Ces améliorations techniques ont permis d'augmenter de manière significative la qualité des banques SSH obtenues. Un gène surexprimé chez la variété tolérante de blé Atlas-66 a été isolé : le gène 49A1. Ce gène confère une résistance modérée chez la levure Saccharomyces cerevisiae et présente de fortes homologies avec des transporteurs d'oligo-peptide proton dépendants. Son homologue chez le riz possède une dizaine d'hélices trans-membranaires et un promoteur avec de nombreux éléments de régulation DOFCOREZM impliqués dans le métabolisme du carbone. Le gène 49A1 pourrait s'avérer être un élément clef de la tolérance à l'aluminium chez les plantes par son implication hypothétique dans les mécanismes de sécrétion du malate. En effet, ces mécanismes sont considérés par certains chercheurs comme un élément pouvant mener à la résistance au métal. Son expression chez les plantes pourrait permettre d'obtenir des plantes génétiquement modifiées tolérantes au métal.\ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : Aluminium, Métaux lourds, Tolérance, Blé, Ri

Viral and host factors required for avian H5N1 influenza A virus replication in mammalian cells

Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions

Host Determinant Residue Lysine 627 Lies on the Surface of a Discrete, Folded Domain of Influenza Virus Polymerase PB2 Subunit

Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain) exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design experiments to elucidate the effects of mutations on polymerase–host factor interactions

GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries

There are many methods for introducing random mutations into nucleic acid sequences. Previously, we described a suite of programmes for estimating the completeness and diversity of randomized DNA libraries generated by a number of these protocols. Our programmes suggested some empirical guidelines for library design; however, no information was provided regarding library diversity at the protein (rather than DNA) level. We have now updated our web server, enabling analysis of translated libraries constructed by site-saturation mutagenesis and error-prone PCR (epPCR). We introduce GLUE-Including Translation (GLUE-IT), which finds the expected amino acid completeness of libraries in which up to six codons have been independently varied (according to any user-specified randomization scheme). We provide two tools for assisting with experimental design: CodonCalculator, for assessing amino acids corresponding to given randomized codons; and AA-Calculator, for finding degenerate codons that encode user-specified sets of amino acids. We also present PEDEL-AA, which calculates amino acid statistics for libraries generated by epPCR. Input includes the parent sequence, overall mutation rate, library size, indel rates and a nucleotide mutation matrix. Output includes amino acid completeness and diversity statistics, and the number and length distribution of sequences truncated by premature termination codons. The web interfaces are available at http://guinevere.otago.ac.nz/stats.html

Gene Composer in a structural genomics environment

For structural biology applications, protein-construct engineering is guided by comparative sequence analysis and structural information, which allow the researcher to better define domain boundaries for terminal deletions and nonconserved regions for surface mutants. A database software application called Gene Composer has been developed to facilitate construct design

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained

Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

Influenza A viruses may cross species barriers and transmit to humans with the potential to cause pandemics. Interplay of human- (PB2 627K) and avian-like (PB2 627E) influenza polymerase complexes with unknown host factors have been postulated to play a key role in interspecies transmission. Here, we have identified human importin-α isoforms (α1 and α7) as positive regulators of human- but not avian-like polymerase activity. Human-like polymerase activity correlated with efficient recruitment of α1 and α7 to viral ribonucleoprotein complexes (vRNPs) without affecting subcellular localization. We also observed that human-like influenza virus growth was impaired in α1 and α7 downregulated human lung cells. Mice lacking α7 were less susceptible to human- but not avian-like influenza virus infection. Thus, α1 and α7 are positive regulators of human-like polymerase activity and pathogenicity beyond their role in nuclear transport

The PB2-E627K Mutation Attenuates Viruses Containing the 2009 H1N1 Influenza Pandemic Polymerase

The swine-origin H1N1 influenza A virus emerged in early 2009 and caused the first influenza pandemic in 41 years. The virus has spread efficiently to both the Northern and the Southern Hemispheres and has been associated with over 16,000 deaths. Given the virus’s recent zoonotic origin, there is concern that the virus could acquire signature mutations associated with the enhanced pathogenicity of previous pandemic viruses or H5N1 viruses with pandemic potential. We tested the hypothesis that mutations in the polymerase PB2 gene at residues 627 and 701 would enhance virulence but found that influenza viruses containing these mutations in the context of the pandemic virus polymerase complex are attenuated in cell culture and mice

Charting the Host Adaptation of Influenza Viruses

Four influenza pandemics have struck the human population during the last 100 years causing substantial morbidity and mortality. The pandemics were caused by the introduction of a new virus into the human population from an avian or swine host or through the mixing of virus segments from an animal host with a human virus to create a new reassortant subtype virus. Understanding which changes have contributed to the adaptation of the virus to the human host is essential in assessing the pandemic potential of current and future animal viruses. Here, we develop a measure of the level of adaptation of a given virus strain to a particular host. We show that adaptation to the human host has been gradual with a timescale of decades and that none of the virus proteins have yet achieved full adaptation to the selective constraints. When the measure is applied to historical data, our results indicate that the 1918 influenza virus had undergone a period of preadaptation prior to the 1918 pandemic. Yet, ancestral reconstruction of the avian virus that founded the classical swine and 1918 human influenza lineages shows no evidence that this virus was exceptionally preadapted to humans. These results indicate that adaptation to humans occurred following the initial host shift from birds to mammals, including a significant amount prior to 1918. The 2009 pandemic virus seems to have undergone preadaptation to human-like selective constraints during its period of circulation in swine. Ancestral reconstruction along the human virus tree indicates that mutations that have increased the adaptation of the virus have occurred preferentially along the trunk of the tree. The method should be helpful in assessing the potential of current viruses to found future epidemics or pandemics