The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.DOI: http://dx.doi.org/10.7554/eLife.03007.001

Author contributions and conflict of interest Daniel Braga performed research, analyzed data (molecular biology, malachite green assay, mass spectrometry) and contributed to writing the manuscript, Daniel Last performed research and analyzed data (structure elucidation, Fno assay, biogas plant studies), Mahmudul Hasan performed research (CofC/D enzyme assays), Huijuan Guo performed research and analyzed data (structure elucidation), Daniel Leichnitz performed research (chemical synthesis), Zerrin Uzum performed research (microscopy), Ingrid Richter performed research (microscopy), Felix Schalk performed research (cofE constructs), Christine Beemelmanns designed research, acquired funding, analyzed data (structure elucidation, synthesis) and edited the manuscript, Christian Hertweck designed research, acquired funding and edited the manuscript, Gerald Lackner designed the study, acquired funding and wrote the original manuscript. The authors declare no conflict of interest.

The tight association of the pathogenic fungus Rhizopus microsporus and its toxin-producing, bacterial endosymbionts (Mycetohabitans spp.) is distributed worldwide and has significance for agriculture, food production, and human health. Intriguingly, the endofungal bacteria are essential for the propagation of the fungal host. Yet, little is known about chemical mediators fostering the symbiosis, and universal metabolites that support the mutualistic relationship have remained elusive. Here, we describe the discovery of a complex of specialized metabolites produced by endofungal bacteria under symbiotic conditions. Through full genome sequencing and comparative genomics of eight endofungal symbiont strains from geographically distant regions, we discovered a conserved gene locus (hab) for a non-ribosomal peptide synthetase as a unifying trait. Bioinformatics analyses, targeted gene deletions, and chemical profiling uncovered unprecedented depsipeptides (habitasporins) whose structures were fully elucidated. Computational network analysis and labeling experiments granted insight into the biosynthesis of their non-proteinogenic building blocks (pipecolic acid and β-phenylalanine). Deletion of the hab gene locus was shown to impair the ability of the bacteria to enter their fungal host. Our study unveils a common principle of the endosymbiotic lifestyle of Mycetohabitans species and expands the repertoire of characterized chemical mediators of a globally occurring mutualistic association.

23 Phone: +49 3631 532 1104 24 25 Author contributions and conflict of interest 26 Daniel Braga performed research, analyzed data (molecular biology, malachite green assay, mass spectrometry) 27 and contributed to writing the manuscript, Daniel Last performed research and analyzed data (structure 28 elucidation, Fno assay, biogas plant studies), Mahmudul Hasan performed research (CofC/D enzyme assays), 29 Huijuan Guo performed research and analyzed data (structure elucidation), Daniel Leichnitz performed research 30 (chemical synthesis), Zerrin Uzum performed research (microscopy), Ingrid Richter performed research 31 (microscopy), Felix Schalk performed research (cofE constructs), Christine Beemelmanns designed research, 32 acquired funding, analyzed data (structure elucidation, synthesis) and edited the manuscript, Christian Hertweck 33 designed research, acquired funding and edited the manuscript, Gerald Lackner designed the study, acquired 34 funding and wrote the original manuscript. The authors declare no conflict of interest. 35 Abstract 1

Specific Rhizopus microsporus pathovars harbor bacterial endosymbionts (Burkholderia rhizoxinica) for the production of a phytotoxin. Here, we present the draft genome sequences of two R. microsporus strains, one symbiotic (ATCC 62417), and one endosymbiont-free (CBS 344.29). The gene predictions were supported by RNA sequencing (RNA-seq) data. The functional annotation sets the basis for comparative analyses.

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

The association of the agriculturally significant phytopathogenic fungus Rhizopus microsporus with the bacterial endosymbiont Burkholderia rhizoxinica is a remarkable example of bacteria controlling host physiology and reproduction. Here, we show that a group of transcription activator-like effectors (TALEs) called Burkholderia TALE-like proteins (BATs) from B. rhizoxinica are essential for the establishment of the symbiosis. Mutants lacking BAT proteins are unable to induce host sporulation. Utilising novel microfluidic devices in combination with fluorescence microscopy we observed the accumulation of BAT-deficient mutants in specific fungal side-hyphae with accompanying increased fungal re-infection. High-resolution live imaging revealed septa biogenesis at the base of infected hyphae leading to compartmental trapping of BATdeficient endobacteria. Trapped endosymbionts showed reduced intracellular survival, suggesting a protective response from the fungal host against bacteria lacking specific effectors. These findings underscore the involvement of BAT proteins in maintaining a balance between mutualism and antagonism in bacterial-fungal interactions and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.Bacteria living in close association with eukaryotic hosts may control and exploit their host via pathogenic or mutualistic interactions 1 . For

The hard tick, Ixodes ricinus, a main Lyme disease vector, harbors an intracellular bacterial endosymbiont. Midichloria mitochondrii is maternally inherited and resides in the mitochondria of I. ricinus oocytes, but the consequences of this endosymbiosis are not well understood. Here, we provide 3D images of wild-type and aposymbiotic I. ricinus oocytes generated with focused ion beam-scanning electron microscopy. Quantitative image analyses of endosymbionts and oocyte mitochondria at different maturation stages show that the populations of both mitochondrion-associated bacteria and bacterium-hosting mitochondria increase upon vitellogenisation, and that mitochondria can host multiple bacteria in later stages. Three-dimensional reconstructions show symbiosis-dependent morphologies of mitochondria and demonstrate complete M. mitochondrii inclusion inside a mitochondrion. Cytoplasmic endosymbiont located close to mitochondria are not oriented towards the mitochondria, suggesting that bacterial recolonization is unlikely. We further demonstrate individual globular-shaped mitochondria in the wild type oocytes, while aposymbiotic oocytes only contain a mitochondrial network. In summary, our study suggests that M. mitochondrii modulates mitochondrial fragmentation in oogenesis possibly affecting organelle function and ensuring its presence over generations.