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Marquages et suivis phénotypiques de micro-organismes symbiotiques de plantes

Defense Maîtrise : Univ. Genève, 2017
Abstract In absence of sufficient quantities of reduced forms of nitrogen, most plants of the Leguminosae family can establish symbiotic interactions with nitrogen-fixing bacteria collectively called rhizobia. To reduce atmospheric nitrogen (N2) for the benefit of host plants, rhizobia use an enzymatic complex known as the nitrogenase. To maximize nitrogenase activity legume hosts shelter rhizobia within cells of root or more rarely stem nodules: organs that are devoted to symbiotic N2-fixation. This implies that rhizobia found on the surface of root systems must colonize the cytoplasm of nodule cells via a complex and extremely selective infection process. Generally, plants only tolerate a limited number of rhizobia species and reciprocally, rhizobia form proficient symbioses with a narrow spectrum of legumes. In this respect, Sinorhizobium (Ensifer) fredii NGR234 is clearly exceptional since it forms nodules on roots of more than 120 plant genera and fixes nitrogen with at least 135 species of legumes. Despite such a broad host range, NGR234 cannot form efficient associations with Glycine max, while taxonomically related S. fredii strains HH103 and USDA257 can. Initially, the aim of this master was to construct a set of fluorescently labeled derivatives of NGR234 and HH103 to facilitate comparison of the plant infection processes followed by both strains. Accordingly, a transcriptionally silent chromosomal region conserved in NGR234 and HH103 was identified. This locus was to be targeted with a modified antibiotic-resistant cassette expressing either the mCherry or BFP proteins. Unfortunately, we were unable to obtain clones carrying the complete chromosomal region of NGR234, apparently because this locus triggered transposition of insertion sequences in several recipient E. coli strains. As another research project had to be considered, we focused on genetic complementation of the NGR∆nifQ mutant. First described in Fumeaux et al. (2011), NGR∆nifQ was shown to have symbiotic proficiencies similar to NGR234 when inoculated on various host plants including cowpea, siratro and mungbean (Vigna radiata). This was interesting given that NifQ provides molybdenum during assembly of the essential iron-molybdenum (FeMo) cofactor of the nitrogenase complex. Yet, when compared to the parent NGR234 strain that formed normal N2-fixing pinkish nodules, the NGR∆nifQ mutant induced formation of much darker, almost purple nodules on roots of several hosts including V. radiata. To confirm that deletion of NifQ was responsible for this unusual phenotype, we cloned into the broad host-range vector pRK7813 a functional copy of nifQ downstream of its native NifA-sigma54 dependent promoter (pCRKnifQ). Unfortunately, because the vast majority of NGR∆nifQ::pCRKnifQ transconjugants lost the construct during infection of root tissues, our initial complementation attempts failed. However, when a similar construct was established in the more stable pRG960 vector (pCRGnifQ), NGR∆nifQ::pCRGnifQ transconjugants formed nodules of a normal color. This confirmed that, in NGR234, the absence of NifQ considerably modifies nodule homeostasis.
Keywords RhizobiaSinorhizobium frediiNGR234Nitrogen fixationTransposaseNifQ,
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Research group Unité de microbiologie
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DE MALEZIEUX DU HAMEL, Chloé. Marquages et suivis phénotypiques de micro-organismes symbiotiques de plantes. Université de Genève. Maîtrise, 2017. https://archive-ouverte.unige.ch/unige:94508

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Deposited on : 2017-05-29

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