RESUMO
IMPORTANCE: Interactions between fungi and bacteria are critically important in ecology, medicine, and biotechnology. In this study, we shed light on factors that promote the persistence of a toxin-producing, phytopathogenic Rhizopus-Mycetohabitans symbiosis that causes severe crop losses in Asia. We present an unprecedented case where bacterially produced transcription activator-like (TAL) effectors are key to maintaining a stable endosymbiosis. In their absence, fungal sporulation is abrogated, leading to collapse of the phytopathogenic alliance. The Mycetohabitans TAL (MTAL)-mediated mechanism of host control illustrates a unique role of bacterial effector molecules that has broader implications, potentially serving as a model to understand how prokaryotic symbionts interact with their eukaryotic hosts.
RESUMO
As an endosymbiont of the ecologically and medically relevant fungus Rhizopus microsporus, the toxin-producing bacterium Mycetohabitans rhizoxinica faces myriad challenges, such as evading the host's defense mechanisms. However, the bacterial effector(s) that facilitate the remarkable ability of M. rhizoxinica to freely migrate within fungal hyphae have thus far remained unknown. Here, we show that a transcription activator-like (TAL) effector released by endobacteria is an essential symbiosis factor. By combining microfluidics with fluorescence microscopy, we observed enrichment of TAL-deficient M. rhizoxinica in side hyphae. High-resolution live imaging showed the formation of septa at the base of infected hyphae, leading to the entrapment of endobacteria. Using a LIVE/DEAD stain, we demonstrate that the intracellular survival of trapped TAL-deficient bacteria is significantly reduced compared with wild-type M. rhizoxinica, indicative of a protective host response in the absence of TAL proteins. Subversion of host defense in TAL-competent endobacteria represents an unprecedented function of TAL effectors. Our data illustrate an unusual survival strategy of endosymbionts in the host and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.
Assuntos
Hifas , Efetores Semelhantes a Ativadores de Transcrição , Bactérias , SimbioseRESUMO
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.
Assuntos
Burkholderia/metabolismo , Macrolídeos/metabolismo , Rhizopus/metabolismo , Simbiose , Burkholderia/genética , Burkholderia/ultraestrutura , Quitinases/metabolismo , Microscopia Crioeletrônica , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Hifas/genética , Hifas/metabolismo , Hifas/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteoma/metabolismo , Proteômica/métodos , Rhizopus/genética , Rhizopus/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like ß-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.
Assuntos
Ácido Bongcréquico/biossíntese , Burkholderia gladioli/metabolismo , Ácido Bongcréquico/química , Conformação MolecularRESUMO
BACKGROUND: Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of B. rhizoxinica HKI 0454 - a type strain of endofungal Burkholderia species. RESULTS: The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living Burkholderia species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, B. rhizoxinica lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors. CONCLUSIONS: B. rhizoxinica is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that B. rhizoxinica is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. In silico analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction.
Assuntos
Burkholderia/genética , Burkholderia/fisiologia , Evolução Molecular , Genômica/métodos , Rhizopus , Simbiose/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Burkholderia/efeitos dos fármacos , Burkholderia/metabolismo , Farmacorresistência Bacteriana/genética , Resistência a Múltiplos Medicamentos/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Genoma Bacteriano/genética , Lipopolissacarídeos/metabolismo , Pseudogenes/genética , Rhizopus/metabolismoRESUMO
Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic fungus Rhizopus microsporus. The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of B. rhizoxinica HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.
Assuntos
Burkholderia/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Cromossomos Bacterianos , Dados de Sequência Molecular , Plasmídeos , Análise de Sequência de DNARESUMO
Burkholderia rhizoxinica and Rhizopus microsporus form a unique symbiosis in which intracellular bacteria produce the virulence factor of the phytopathogenic fungus. Notably, the host strictly requires endobacteria to sporulate. In this study, we show that the endofungal bacteria possess a type III secretion system (T3SS), which has a crucial role in the maintenance of the alliance. Mutants defective in type III secretion show reduced intracellular survival and fail to elicit sporulation of the host. Furthermore, genes coding for T3SS components are upregulated during cocultivation of the bacterial symbiont with their host. This is the first report on a T3SS involved in bacterial-fungal symbiosis. Phylogenetic analysis revealed that the T3SS represents a prototype of a clade of yet uncharacterized T3SSs within the hrp superfamily of T3SSs from plant pathogenic microorganisms. In a control experiment, we demonstrate that under laboratory conditions, rhizoxin production was not required for establishment of the symbiotic interaction.