Identification in the bacterial symbiont Burkholderia insecticola of factors involved in antimicrobial peptide-resistance and colonization of the insect Riptortus pedestris

L’insecte phytophage Riptortus pedestris, appartenant au sous-ordre des Hétéroptères, est un ravageur notoire de cultures agricoles en Asie du sud-est qui se nourrit préférentiellement de plants de soja. Cette punaise est associée à une bactérie symbiotique du genre Burkholderia nommée Burkholderia insecticola, localisée dans une région spécifique de l’intestin de l’insecte appelée la région M4. Cette région M4, organisée en cryptes, constitue l’organe symbiotique dans lequel le symbiote prolifère de manière extracellulaire. Cette interaction favorise la croissance et le développement de la punaise. Récemment, il a été montré que Riptortus produit des peptides antimicrobiens au sein des cryptes, appelés “crypt-specific cysteine-rich peptides” ou peptides CCR pour lesquels le symbiote est particulièrement résistant. Il a été proposé que les peptides antimicrobiens de l’hôte,incluant les peptides CCR, participent à la colonisation spécifique de l’organe symbiotique par B. insecticola. Dans ce travail, une approche Tn-seq a été utilisée pour identifier les gènes bactériens impliqués dans la résistance aux peptides antimicrobiens et dans la symbiose. Dans un premier temps, la robustesse de la méthode Tn-seq a été évaluée en identifiant le génome essentiel de B. insecticola. Puis dans un second temps, les facteurs bactériens impliqués dans la résistance aux peptides antimicrobiens ont été caractérisés via une approche gènes-candidats et l’approche Tn-seq. Dans une dernière partie, une expérience de Tn-seq in vivo a permis d’évaluer l’ampleur du goulot d’étranglement sur la population symbiotique lors de l’infection de l’organe symbiotique et d’identifier les facteurs symbiotiques impliqués dans la colonisation de R. pedestris.The phytophagous insect Riptortus pedestris, belonging to the Heteroptera suborder, is a notorious crop pest in South-Eastern Asia which feeds preferentially on soybean plants. This bean bug is associated with a bacterial symbiont, a specific Burkholderia species named Burkholderia insecticola, located in the M4 region of the insect’s midgut. This M4 region is organized in crypts and constitutes the symbiotic organ where the symbiont proliferates extracellularly. This interaction promotes the growth and the development of the bean bug. Recently, it was demonstrated that Riptortus produces antimicrobial peptides in the midgut crypts called crypt-specific cysteine-rich peptides (CCR) for which the bacterial symbiont demonstrates a high resistance profile. It was proposed that host antimicrobial peptides, including the CCR peptides, contribute to the specific colonization of the symbiotic organ by B. insecticola. In this work, a Tn-seq approach was used to find bacterial fitness genes involved in antimicrobial peptide resistance and symbiosis. First, the robustness of the Tn-seq method was assessed by identifying the essential genome of B. insecticola. Second, the bacterial factors for antimicrobial peptide resistance were characterized, based on both a candidate-gene and the Tn-seq approach. Finally, a Tn-seq in vivo experiment was performed to reveal the infection bottleneck effect on the symbiotic population and to identify the bacterial symbiosis factors for the colonization of R. pedestris

Identification des facteurs de résistance aux peptides antimicrobiens et de colonisation de l’insecte Riptortus pedestris chez la bactérie symbiotique Burkholderia insecticola

The phytophagous insect Riptortus pedestris, belonging to the Heteroptera suborder, is a notorious crop pest in South-Eastern Asia which feeds preferentially on soybean plants. This bean bug is associated with a bacterial symbiont, a specific Burkholderia species named Burkholderia insecticola, located in the M4 region of the insect’s midgut. This M4 region is organized in crypts and constitutes the symbiotic organ where the symbiont proliferates extracellularly. This interaction promotes the growth and the development of the bean bug. Recently, it was demonstrated that Riptortus produces antimicrobial peptides in the midgut crypts called crypt-specific cysteine-rich peptides (CCR) for which the bacterial symbiont demonstrates a high resistance profile. It was proposed that host antimicrobial peptides, including the CCR peptides, contribute to the specific colonization of the symbiotic organ by B. insecticola. In this work, a Tn-seq approach was used to find bacterial fitness genes involved in antimicrobial peptide resistance and symbiosis. First, the robustness of the Tn-seq method was assessed by identifying the essential genome of B. insecticola. Second, the bacterial factors for antimicrobial peptide resistance were characterized, based on both a candidate-gene and the Tn-seq approach. Finally, a Tn-seq in vivo experiment was performed to reveal the infection bottleneck effect on the symbiotic population and to identify the bacterial symbiosis factors for the colonization of R. pedestris.L’insecte phytophage Riptortus pedestris, appartenant au sous-ordre des Hétéroptères, est un ravageur notoire de cultures agricoles en Asie du sud-est qui se nourrit préférentiellement de plants de soja. Cette punaise est associée à une bactérie symbiotique du genre Burkholderia nommée Burkholderia insecticola, localisée dans une région spécifique de l’intestin de l’insecte appelée la région M4. Cette région M4, organisée en cryptes, constitue l’organe symbiotique dans lequel le symbiote prolifère de manière extracellulaire. Cette interaction favorise la croissance et le développement de la punaise. Récemment, il a été montré que Riptortus produit des peptides antimicrobiens au sein des cryptes, appelés “crypt-specific cysteine-rich peptides” ou peptides CCR pour lesquels le symbiote est particulièrement résistant. Il a été proposé que les peptides antimicrobiens de l’hôte,incluant les peptides CCR, participent à la colonisation spécifique de l’organe symbiotique par B. insecticola. Dans ce travail, une approche Tn-seq a été utilisée pour identifier les gènes bactériens impliqués dans la résistance aux peptides antimicrobiens et dans la symbiose. Dans un premier temps, la robustesse de la méthode Tn-seq a été évaluée en identifiant le génome essentiel de B. insecticola. Puis dans un second temps, les facteurs bactériens impliqués dans la résistance aux peptides antimicrobiens ont été caractérisés via une approche gènes-candidats et l’approche Tn-seq. Dans une dernière partie, une expérience de Tn-seq in vivo a permis d’évaluer l’ampleur du goulot d’étranglement sur la population symbiotique lors de l’infection de l’organe symbiotique et d’identifier les facteurs symbiotiques impliqués dans la colonisation de R. pedestris

Burkholderia Gut Symbionts Associated with European and Japanese Populations of the Dock Bug Coreus marginatus (Coreoidea: Coreidae)

International audienceInsects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern

Mutational robustness of intrinsically disordered proteins: a comparative study between the potyvirus intrinsically disordered VPg and the plant susceptibility factor eIF4E

Mutational robustness of intrinsically disordered proteins: a comparative study between the potyvirus intrinsically disordered VPg and the plant susceptibility factor eIF4E. 16. Rencontres de Virologie Végétale (RVV 2017

Trans-cellular tunnels induced by the fungal pathogen Candida albicans facilitate invasion through successive epithelial cells without host damage

International audienceThe opportunistic fungal pathogen Candida albicans is normally commensal, residing in the mucosa of most healthy individuals. In susceptible hosts, its filamentous hyphal form can invade epithelial layers leading to superficial or severe systemic infection. Although invasion is mainly intracellular, it causes no apparent damage to host cells at early stages of infection. Here, we investigate C. albicans invasion in vitro using live-cell imaging and the damage-sensitive reporter galectin-3. Quantitative single cell analysis shows that invasion can result in host membrane breaching at different stages and host cell death, or in traversal of host cells without membrane breaching. Membrane labelling and three-dimensional ‘volume’ electron microscopy reveal that hyphae can traverse several host cells within trans-cellular tunnels that are progressively remodelled and may undergo ‘inflations’ linked to host glycogen stores. Thus, C. albicans early invasion of epithelial tissues can lead to either host membrane breaching or trans-cellular tunnelling

Mutational robustness of intrinsically disordered proteins: a comparative study between the potyvirus intrinsically disordered VPg and the plant susceptibility factor eIF4E

Mutational robustness of intrinsically disordered proteins: a comparative study between the potyvirus intrinsically disordered VPg and the plant susceptibility factor eIF4E. 16. Rencontres de Virologie Végétale (RVV 2017

The biotroph Agrobacterium tumefaciens thrives in tumors by exploiting a wide spectrum of plant host metabolites

International audienceAgrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g., sucrose, glutamate) and enriched (e.g., opines, GABA, GHB, pyruvate) metabolites that A. tumefaciens could use as nutrients. Transposon-sequencing and transcriptomics pinpointed the genes that are crucial and/or up-regulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, tricarboxylic acid cycle and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge) and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and transposon-sequencing to decipher the lifestyle of a plant pathogen. This article is protected by copyright. All rights reserved

Comparative analysis of mutational robustness of the intrinsically disordered viral protein VPg and of its interactor eIF4E.

Conformational intrinsic disorder is a feature present in many virus proteins. Intrinsically disordered regions (IDRs) have weaker structural requirement than ordered regions and mutations in IDRs could have a lower impact on the virus fitness. This could favor its exploration of adaptive solutions. The potyviral protein VPg contains IDRs with determinants for adaptation to its host plant. To experimentally assess whether IDRs are more resistant to mutations than ordered regions, the biologically relevant interaction between mutant libraries of both VPg and the eukaryotic translation initiation factor 4E (eIF4E) and their respective wild type partner was examined using yeast two hybrid assay. Our data shows that VPg is significantly more robust to mutations than eIF4E and as such belongs to a particular class of intrinsically disordered proteins. This result is discussed from the standpoint of IDRs involvement in the virus adaptive processes

Dissimilar gene repertoires of Dickeya solani involved in the colonization of lesions and roots of Solanum tuberosum

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum. They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda (=kdgA), gudD, garK, garL, and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients (dpp, ddp, dctA, and pst) including glucuronate (kdgK and yeiQ) and synthesis of metabolites: cellulose (celY and bcs), aryl polyene (ape), and oocydin (ooc). We constructed in-frame deletion mutants of bcsA, ddpA, apeH, and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the ΔpstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers

Differential Genetic Strategies of Burkholderia vietnamiensis and Paraburkholderia kururiensis for Root Colonization of Oryza sativa subsp. japonica and O. sativa subsp. indica , as Revealed by Transposon Mutagenesis Sequencing

International audienceBurkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth.Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCE Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction