Thèse Intrasym Genetic Requirements In Caballeronia Insecticola For Intracellular Symbiosis Inside The Gut Epithelial Cells Of Phytophagous Insects H/F - 75

Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation
École doctorale : Sciences du Végétal : du gène à l'écosystème
Laboratoire de recherche : I2BC - Institut de Biologie Intégrative de la Cellule
Direction de la thèse : Peter MERGAERT ORCID 0000000259197317
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-06T23:59:59Intracellular symbiosis is important in the biology of extant insects and plants, as a major transition in the evolution of life and for biotechnological applications in plants, yeast and fungi. The PhD project is framed within an overall objective that is to disclose and understand the evolutionary innovations enabling intracellular symbiosis in insects and to apply this understanding to engineer, as a proof of concept, an extracellular association (extra-sym) into a new intracellular symbiosis (intra-sym). The motivation for the IntraSym project is our recent discovery that in the stinkbugs (insect infraorder Pentatomomorpha), closely related species have either extracellular bacterial symbionts in the lumen of the gut (extra-sym) or intracellular symbionts of the same bacterial species located inside the gut epithelial cells (intra-sym). Thus, the Pentatomomorpha provides an ideal and unique framework to identify and compare the genetic determinants for extra-sym and intra-sym in both, the insect host and the bacterial gut symbionts. The INTRASYM PhD project will identify the genetic determinants that are specifically or commonly crucial for extra-sym and intra-sym in the model gut symbiont Caballeronia insecticola. C. insecticola establishes an extra-sym in stinkbugs of the Alydidae and Coreidae families and an intra-sym in species of the Rhyparochromidae family. The PhD candidate will use functional genomics (Tn-seq and RNA-seq), comparative genomics and a candidate gene approach to identify the bacterial adaptations needed for symbiosis in different extra-sym and intra-sym hosts belonging to these stinkbug families. Candidate functions enabling C. insecticola to infect gut epithelial cells or chronically establish in them in intra-sym hosts will be further characterized in depth.

Symbiosis with specific bacterial species is widespread, and is particularly important in the insects where symbiotic bacteria are crucial for fitness in most species. Roles of symbioses include nutrition and food digestion, detoxification, immunity, stress tolerance, behavior and more (1,2). Symbionts are hosted in different body and cellular locations, which can be extracellular in cavities of the cuticle on the body surface, extracellular in the gut lumen or intracellular (aka endosymbiosis) in specialized cells, called bacteriocytes that are organized in specialized symbiotic organs known as bacteriomes or incorporated in organs with other functions like the fat body. Below, extra-sym designates extracellular symbioses and intra-sym intracellular ones. Different hypothetical scenarios for the evolution of intra-sym have been proposed. The most plausible one (Fig. 1A) consists in the appearance of an intermediary stage of intracellular infection of gut epithelial cells by a member of the gut microbiota and the multiplication and chronic maintenance of these intracellular bacteria (3,4). In subsequent evolutionary stages, clusters of colonized gut epithelial cells become dissociated from the gut epithelial layer to form a symbiotic organ located in the hemolymph, independent from the gut. The identification of insect cases with the intermediary state would lend strong support to this hypothesis, but thus far, such insects have not been reported in the literature.
The Pentatomomorpha infraorder (stinkbugs) is species-rich, composed mainly of phytophagous insects, many of which are pests of crops associated with yield losses. Species in the Pentatomomorpha infraorder critically depend on bacterial symbionts for their development and display a variety of symbiotic syndromes depending on the Pentatomomorpha family the species belong to (5,6). In some species, the bacterial symbionts are located inside bacteriocytes that cluster together in a bacteriome but the majority of stinkbug species carry their symbionts inside the gut, in a specific symbiotic region of the posterior midgut that is composed of crypts. For the in the literature reported species, the gut symbionts are extracellular and located in the lumen of the crypts (i.e. extra-sym). However, our collaborating team, directed by Yoshi Kikuchi (AIST, Sapporo, Japan), discovered recently that in several Pentatomomorpha families that were not surveyed in detail before, the environmentally-acquired crypt bacteria infect, from the crypt lumen, the epithelial cells and chronically establish intracellularly in large numbers (i.e. intra-sym). Excitingly, this newly discovered symbiotic syndrome represents thus the missing link in the evolutionary model for intracellular symbiosis in insects. Intra-sym has evolved repeatedly within the Pentatomomorpha clade in less than 100 My, a relatively short time from an evolutionary perspective, suggesting that a small number of adaptations are required to switch from extra-sym to intra-sym. Importantly, species with extracellular location of gut symbionts and with intracellular gut symbionts are colonized with closely related bacteria belonging to the Caballeronia genus. Moreover, our model symbiont, Caballeronia insecticola can colonize extracellularly or intracellularly, according to the host.

The motivation for the INTRASYM PhD project is the recent and unpublished discovery in the stinkbugs of this intermediary evolutionary state. The INTRASYM PhD project will focus on the identification and characterization of adaptations in the gut symbiont C. insecticola that are specifically required for intracellular infection and chronic establishment in the crypt epithelial cells of stinkbugs.

Rationale: Bacterial species like C. insecticola can establish functional extracellular symbiosis in Riptortus pedestris (Alydidae), Leptoglossus occidentalis, Coreus marginatus, Cletus punctiger and other tested species of the Coreidae family, as well as functional intracellular symbiosis in Togo hemipterus and Metochus uniguttatus, both belonging to the Rhyparochromidae family. This suggests that the extra-sym vs. intra-sym type of interactions are determined most strongly by host factors. Nevertheless, we expect that the required genetic repertoire in the bacterial symbiont is also different between extra-sym and intra-sym. The uptake by the host cells might require recognition of specific bacterial components, e.g. secreted or envelope proteins, and the intracellular environment, including nutritional and stress factors, might be distinct from the environment in the lumen of the gut. In addition, the mechanism of metabolic exchanges between symbiont and host might be different. In a preliminary candidate gene approach, we found that the bacA gene of C. insecticola (an orthologue of the bacA gene in rhizobium bacteria for which we have demonstrated in the past its crucial role in the rhizobium-legume symbiosis (7)) is required for intra-sym in T. hemipterus but not for extra-sym in R. pedestris (unpublished, in preparation), highlighting one of several remarkable convergent phenotypes of intra-sym in insects and plants. Since bacA is encoding a peptide transporter involved in the resistance to certain antimicrobial peptides, we can predict that the intracellular symbiosis involves specific families of AMPs, absent in insects with extracellular symbiosis. More generally, defining the functions of intra-sym-specific genes in Caballeronia is primordial because the nature of these genes might give strong indications about corresponding host functions that govern intra-sym.
Strategy: The PhD candidate will use comprehensive genetic screens that will potentially identify all genetic determinants that are specific or common for extra-sym and intra-sym. These screens will comprise functional genomics (Tn-seq and RNA-seq), comparative genomics and a candidate gene approach. To distinguish between genetic determinants that are host species-specific and that are specific for a symbiotic syndrome (extra-sym vs. intra-sym), the genetic screens will be performed in multiple host species for each symbiotic syndrome. Subsequently, intra-sym-specific functions enabling C. insecticola to infect gut epithelial cells or chronically establish in them in intra-sym hosts will be further characterized in depth with ad hoc methods to understand the underlying mechanism of their role in intra-sym.
Outline of the tasks: The PhD work is divided in two main tasks. Task 1 consists in four genetic approaches to identify genes of interest that control intracellular infection or chronic establishment. The host species for these screens are for extra-sym R. pedestris, L. occidentalis, C. marginatus, and/or C. punctiger and for intra-sym Togo hemipterus and Metochus uniguttatus. These species are all available in lab rearing systems, in I2BC or in the lab of the Kikuchi team in Japan. Additional intra-sym hosts will be collected from the local environment in France. The second task consists in the detailed functional characterization of genes of interest from the first task, using reverse genetics and detailed phenotypic characterization of the mutants in vitro and in interaction with intra-sym hosts as well as biochemical approaches. Details of the two tasks are outlined below.
Task 1
T1.1Candidate species with intra-sym that can be collected from the natural environment in France are Rhyparochromus vulgaris, Aphanus rolandri. Gastrodes grossipes, Peritrechus gracilicornis, Beosus maritimus. These species are frequent in France (https://www.gbif.org/). Lab rearing systems for the collected species will be set up and their gut symbiosis will be characterized by microscopy and isolation of the bacterial species present in the crypt midgut region and their taxonomic classification. The capacity of C. insecticola to colonize the crypt region in these species will be confirmed.
T1.2Tn-seq and RNA-seq will be performed in at least three different insect species for both extra-sym and intra-sym hosts, each colonized with C. insecticola. Tn-seq and RNA-seq of C. insecticola in the host R. pedestris have been performed before successfully by the laboratory (8,9). The data of these studies will serve as a benchmark to set up simplified Tn-seq and RNA-seq protocols that can be applied on larger scale in six different host species.
T1.3The candidate gene approach that revealed the bacA gene as an intra-sym-specific function (see above), will be extended to the large collection of C. insecticola mutants available in the Kikuchi and Mergaert labs. Available mutants are affected in surface polysaccharides (EPS, LPS), hopanoids, Outer Membrane Proteins, Type IV pili, TVISS, Tad Pili, Type 1 fimbriae, metabolism. Additional mutants, not yet available, in genes of interest (predicted secreted and membrane proteins) will be obtained from an ordered transposon mutant library that is under construction. The mutants will be tested for symbiosis in T. hemipterus and M. uniguttatus with a simple phenotypic screen that is based on the survival of symbiotic insects until adulthood in these two species, while apo-symbiotic insects, i.e. animals that were not able to obtain a symbiont, die in the instar stages.
T1.4A collection of Caballeronia, Paraburkholderia, Burkholderia and Pandorea strains are available in the Kikuchi and Mergaert labs. Among them are species that can or cannot infect the crypts of R. pedestris. This strain collection will be phenotyped for symbiosis in extra-sym and intra-sym insects. Strains will be grouped according to phenotype (positive in both symbioses, positive in extra-sym only, positive in intra-sym only). Comparative genomics will be used to identify genes specific for each group.
Task 2
Since the overall objective of the project is to understand how intra-sym is established and functions, Task 2 will focus on the characterization of a small number (1 to 3 are expected) intra-sym-specific functions identified in Task 1. The choice of genes of interest will depend on the nature of the gene list but criteria for selection will include prior knowledge of the gene functions in other bacteria-host interactions (symbiotic or pathogenic) or a potential match of the genes with intra-sym specific genes of the host, which will be determined in parallel by collaborators of this project (see below, positioning of the project in the international context) (Fig. 1B).
Mutants will be generated in the selected genes (or obtained from the ordered transposon mutant collection). Using ad hoc methods, depending on the nature of the selected genes, the phenotypes of the mutants will be characterized in vitro using methods in microbiology, molecular biology, genomics, cell biology, biochemistry, etc. The phenotype in symbiosis will be analyzed by microscopy on dissected guts of insects infected with the mutants.