Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures

Structure and function of two GABA-binding proteins of the plant pathogen Agrobacterium tumefaciens

L’acide γ-aminobutyrique (GABA) est synthétisé par la plante en réponse à des stress abiotiques et biotiques dont l’infection par le pathogène bactérien A. tumefaciens. Des travaux antérieurs ont montré que le GABA induit, chez A. tumefaciens C58, l’expression d’une lactonase BlcC (=AttM) qui inactive ses propres signaux quorum-sensing (QS), donc module le transfert horizontal du plasmide Ti. La protéine périplasmique de liaison (Periplasmic Binding Protein, PBP) Atu2422 et le transporteur ABC Bra sont respectivement impliqués dans la perception et le transport du GABA. L’importation du GABA est nécessaire à l’induction de l’expression de BlcC, donc à la dégradation des signaux QS. Les caractéristiques structurales des récepteurs/senseurs du GABA ne sont connus ni chez les bactéries, ni chez les eucaryotes.Ce travail de doctorat a permis de définir la structure et la fonction de deux senseurs du GABA, les PBPs Atu2422 et Atu4243 d’A. tumefaciens C58.La structure cristalline d’Atu2422 a été résolue en présence de GABA ou d’acides aminés antagonistes de la liaison au GABA comme la proline et l’alanine. L'analyse structurale du site de fixation du ligand d’Atu2422 a permis d’identifier deux résidus clés, Phe77 et Tyr275, respectivement impliqués dans la sélectivité du ligand et la liaison du GABA. L’analyse phénotypique de mutants ponctuels a révélé le rôle crucial de ces deux résidus aminés dans l’interaction entre A. tumefaciens C58 et deux plantes hôtes, tomate et tabac. De plus, ces travaux ont défini les caractéristiques moléculaires d’une sous-famille de PBPs présentes chez différentes protéobacteries interagissant avec des hôtes eucaryotes et capables de fixer le GABA et des acides aminés compétiteurs comme la proline ou l’alanine.Ce travail a également révélé une deuxième PBP (appelée GABA2) impliquée dans la perception et l’importation du GABA chez A. tumefaciens. Cette PBP a été identifiée grâce au séquençage du génome et l’analyse du transcriptome de mutants spontanés, issus d’un mutant-KO atu2422 d’A. tumefaciens C58, mais devenus capables de transporter le GABA. La construction d’un mutant défectif pour la PBP GABA2 a permis d’évaluer son rôle dans la signalisation GABA et l’interaction A. tumefaciens-plante hôte. La structure cristalline de cette PBP en présence de GABA a permis d’identifier les résidus clés impliqués dans la fixation du GABA dont le rôle a été validé par l’analyse de mutations ponctuelles. Enfin, une analyse phylogénétique des orthologues de GABA2 a révélé leur présence au sein de nombreuses protéobactéries pathogènes et symbiotiques interagissant avec les plantes. L’ensemble de ces travaux aboutit à la proposition de deux modèles de référence quant aux mécanismes moléculaires associés à la perception du GABA, médiateur de communications inter-cellulaires et inter-organismes. Ce travail illustre l’association des approches de biologie structurale et de génétique pour la compréhension des interactions plantes-microorganismes.Γ-aminobutyric acid (GABA) is synthesized by plants in response to abiotic and biotic stresses, including infection with A. tumefaciens. Previous works have revealed that GABA induces the expression of the A. tumefaciens BlcC (=AttM) lactonase, which cleaves quorum-sensing (QS) signals, thus modulates QS-regulated functions such as horizontal transfer of the plasmid Ti. Periplasmic binding protein (PBP) Atu2422 and ABC transporter Bra of A. tumefaciens are involved in GABA transport from plant to A. tumefaciens. The structural characteristics of the receptors/sensors of GABA are still unknown in bacteria or eukaryotes.I have studied two GABA-binding PBPs of A. tumefaciens C58, Atu2422 and Atu4243 by a combination of structural, genetic and functional approaches.The crystal structure of Atu2422 was solved in the presence of GABA and competitive amino acids, such as proline and alanine. Structural analysis of the ligand binding site revealed two key residues, Phe77 and Tyr275, which are involved in the ligand selectivity and GABA binding, respectively. Analysis of two constructed point-mutants confirmed the critical role of these two residues in the interaction between A. tumefaciens C58 and two host plants, tomato and tobacco plants. Using characteristics of the GABA-binding site, a subfamily of GABA-PBPs was identified in Proteobacteria of which most of them interact with eukaryotic hosts.This work also revealed a second PBP (GABA2) involved in the GABA uptake in A. tumefaciens. This PBP was identified by whole-genome sequencing and transcriptomic analysis of two spontaneous mutants, which derived from the atu2422 mutant. A mutant GABA2 was constructed to validate GABA2 involvement in the transport of GABA, degradation of QS signal, conjugal transfer of the plasmid Ti, and aggressiveness of A. tumefaciens. X-ray structure of GABA-liganded PBP GABA2 revealed key-residues required for GABA-binding. Their role in the GABA uptake has been confirmed by analysis of point mutations. A phylogenetic approach showed that all GABA2-related proteins exhibiting these key-residues were clustered in the same PBPs subfamily.This study has contributed to a better understanding of the A. tumefaciens-plant host interaction, and has permitted to determine two GABA binding modes for PBPs

TssA forms a gp6-like ring attached to the type VI secretion sheath

The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS

Glutathione-dependent redox status of frataxin-deficient cells in a yeast model of Friedreich's ataxia.

International audienceFriedreich's ataxia is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulphur cluster defects and high sensitivity to oxidative stress. Glutathione is a major protective agent against oxidative damage and glutathione-related systems participate in maintaining the cellular thiol / disulfide status and the reduced environment of the cell. Here, we present the first detailed biochemical study of the glutathione-dependent redox status of wild-type and frataxin-deficient cells in a yeast model of the disease. There was five times less total glutathione (GSH+GSSG) in frataxin-deficient cells, imbalanced GSH/GSSG pools and higher glutathione peroxidase activity. The pentose phosphate pathway was stimulated in frataxin-deficient cells, glucose-6-phosphate dehydrogenase activity was three times higher than in wild-type cells, and this was coupled to a defect in the NADPH/NADP(+) pool. Moreover, analysis of gene expression confirms the adaptative response of mutant cells to stress conditions and we bring evidence for a strong relation between the glutathione-dependent redox status of the cells and iron homeostasis. Dynamic studies show that intracellular glutathione levels reflect an adaptation of cells to iron stress conditions, and allow to distinguish constitutive stress observed in frataxin-deficient cells from the acute response of wild-type cells. In conclusion, our findings provide evidence for an impairment of glutathione homeostasis in a yeast model of Friedreich's ataxia and identify glutathione as a valuable indicator of the redox status of frataxin-deficient cells

Concerted transfer of the virulence Ti plasmid and companion at plasmid in the Agrobacterium tumefaciens-induced plant tumour

International audienceThe plant pathogen Agrobacterium tumefaciensC58 harbours three independent type IV secretion (T4SS) machineries. T4SS(T-DNA) promotes the transfer of the T-DNA to host plant cells, provoking tumour development and accumulation of opines such as nopaline and agrocinopines. T4SS(pTi) and T4SS(pAt) control the bacterial conjugation of the Ti and At plasmids respectively. Expression of T4SS(pTi) is controlled by the agrocinopine-responsive transcriptional repressor AccR. In this work, we compared the genome-wide transcriptional profile of the wild-type A.tumefaciens strain C58 with that of its accR KO-mutant to delineate the AccR regulon. In addition to the genes that encode agrocinopine catabolism and T4SS(pTi), we found that AccR also regulated genes coding for nopaline catabolism and T4SS(pAt). Further opine detection and conjugation assays confirmed the enhancement of nopaline consumption and At plasmid conjugation frequency in accR. Moreover, co-regulation of the T4SS(pTi) and T4SS(pAt) correlated with the co-transfer of the At and Ti plasmids both in vitro and in plant tumours. Finally, unlike T4SS(pTi), T4SS(pAt) activation does not require quorum-sensing. Overall this study highlights the regulatory interplays between opines, At and Ti plasmids that contribute to a concerted dissemination of the two replicons in bacterial populations colonizing the plant tumour

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and alpha-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K-D of 0.6 mu M) greater than that for nopaline (KD of 3.7 mu M). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline/assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco

Involvement of NocT in the fitness of <i>A. tumefaciens</i> in the plant tumour.

<p>A, nopaline level in plant tumours induced by <i>A. tumefaciens</i> C58-control and <i>nocT</i> derivative; B, cell number of <i>A. tumefaciens</i> C58-control and its <i>nos</i>, <i>nocT</i> and <i>ocd</i> derivatives when they individually colonize plant tumour; C, competitive infection between the <i>A. tumefaciens</i> C58-control strain and <i>nocT</i> or <i>ocd</i> derivatives, all acting as Ti-plasmid donors in the presence of the recipient <i>A. tumefaciens</i> C58.00. Proportion (%) of the Ti-plasmid genotypes among donor strains in the inoculum (0 dpi) and the mature tumours (32 dpi) and among transconjugants (32 dpi) is indicated. Exact Fischer test was used for statistical analysis (p-value thresholds are indicated on the graph).</p