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Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola. Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

In addition to abiotic triggers, biotic factors such as microbial symbionts can alter development of multicellular organisms. Symbiont-mediated morphogenesis is well-investigated in plants and marine invertebrates but rarely in insects despite the enormous diversity of insect-microbe symbioses. The bean bug Riptortus pedestris is associated with Burkholderia insecticola which are acquired from the environmental soil and housed in midgut crypts. To sort symbionts from soil microbiota, the bean bug develops a specific organ called the “constricted region” (CR), a narrow and symbiont-selective channel, located in the midgut immediately upstream of the crypt-bearing region. In this study, inoculation of fluorescent protein-labeled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12–18 h, blocking any potential subsequent infection events. The “midgut closure” developmental response was irreversible, even after symbiont removal from the crypts by antibiotics. It never occurred in aposymbiotic insects, nor in insects infected with nonsymbiotic bacteria or B. insecticola mutants unable to cross the CR. However, species of the genus Burkholderia and its outgroup Pandoraea that can pass the CR and partially colonize the midgut crypts induce the morphological alteration, suggesting that the molecular trigger signaling the midgut closure is conserved in this bacterial lineage. We propose that this drastic and quick alteration of the midgut morphology in response to symbiont infection is a mechanism for stabilizing the insect-microbe gut symbiosis and contributes to host-symbiont specificity in a symbiosis without vertical transmission.

Ribosomally synthesized peptides have wide ranges of functions in plants being, for example, signal molecules, transporters, alkaloids, or antimicrobial agents. Legumes are an unprecedented rich source of peptides, which are used to control the symbiosis of these plants with the nitrogen-fixing bacteria. Here, we discuss the function and the evolution of these peptides playing an important role in the formation or functioning of the symbiotic organs, the root nodules. We distinguish peptides that can be either cell-autonomous or secreted short-range or long-range signals, carrying messages in or between plant cells or that can act as effectors interacting with the symbiotic bacteria. Peptides are further classified according to the stage of the symbiotic process where they act. Several peptide classes, including RALF, DLV, ENOD40, and others, control infection and the initiation of cell divisions and the formation of nodule primordia. CLE and CEP peptides are implicated in systemic and local control of nodule initiation during autoregulation of nodulation and in response to the nutritional demands of the plant. Still other peptides act at later stages of the symbiosis. The PSK peptide is thought to be involved in the suppression of immunity in nodules and the nodule-specific cysteine-rich, GRP, and SNARP (LEED..PEED) peptide families are essential in the functioning of the nitrogen fixing root nodules. The NCRs and possibly also the GRP and SNARPs are targeted to the endosymbionts and play essential roles in the terminal differentiation of these bacteria.

Symbiosis between rhizobia soil bacteria and legume plants results in the formation of root nodules where plant cells are fully packed with nitrogen fixing bacteria. In the host cells, the bacteria adapt to the intracellular environment and gain the ability for nitrogen fixation. Depending on the host plants, the symbiotic fate of bacteria can be either reversible or irreversible. In Medicago and related legume species, the bacteria undergo a host-directed multistep differentiation process culminating in the formation of elongated and branched polyploid bacteria with definitive loss of cell division ability. The plant factors are nodule-specific symbiotic peptides. Approximately 600 of them are nodule-specific cysteine-rich (NCR) peptides produced in the rhizobium-infected plant cells. NCRs are targeted to the endosymbionts, and concerted action of different sets of peptides governs different stages of endosymbiont maturation, whereas the symbiotic function of individual NCRs is unknown. This study focused on NCR247, a cationic peptide exhibiting in vitro antimicrobial activities. We show that NCR247 acts in those nodule cells where bacterial cell division is arrested and cell elongation begins. NCR247 penetrates the bacteria and forms complexes with many bacterial proteins. Interaction with FtsZ required for septum formation is one of the host interventions for inhibiting bacterial cell division. Complex formation with the ribosomal proteins affects translation and contributes to altered proteome and physiology of the endosymbiont. Binding to the chaperone GroEL amplifies the NCR247-modulated biological processes. We show that GroEL1 of Sinorhizobium meliloti is required for efficient infection, terminal differentiation, and nitrogen fixation.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) – an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs. The authors report the identification of phazolicin (PHZ) - a prokaryotic translation inhibitory peptide - and its structure in complex with the E. coli ribosome, delineating PHZ’s mode of action and suggesting a basis for its bacterial species-specific activity.

Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products. FR900359 (FR) is a Gq protein inhibitor depsipeptide isolated from an uncultivable plant endosymbiont and synthesized by non-ribosomal peptide synthetases. Here, the authors discover a cultivable bacterial FR producer and show that FrsA thioesterase domain catalyses intermolecular transesterification of the FR side chain to the depsipeptide core during biosynthesis, improving Gq inhibition properties.

Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides act either by membrane disruption or by inhibiting essential intracellular processes. Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer, Rhizobium sp. strain Pop5. Here, we show that the frequency of spontaneous PHZ-resistant mutants in Sinorhizobium meliloti is below the detection limit. We find that PHZ can enter S. meliloti cells through two distinct promiscuous peptide transporters, BacA and YejABEF, which belong to the SLiPT (SbmA-like peptide transporter) and ABC (ATP-binding cassette) transporter families, respectively. The dual-uptake mode explains the lack of observed resistance acquisition because the simultaneous inactivation of both transporters is necessary for resistance to PHZ. Since both BacA and YejABEF are essential for the development of functional symbiosis of S. meliloti with leguminous plants, the unlikely acquisition of PHZ resistance via the inactivation of these transporters is further disfavored. A whole-genome transposon sequencing screen did not reveal additional genes that can provide strong PHZ resistance when inactivated. However, it was found that the capsular polysaccharide KPS, the novel putative envelope polysaccharide PPP (PHZ-protecting polysaccharide), as well as the peptidoglycan layer jointly contribute to the sensitivity of S. meliloti to PHZ, most likely serving as barriers that reduce the amount of PHZ transported inside the cell. IMPORTANCE Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides act either by membrane disruption or by inhibiting essential intracellular processes. The Achilles’ heel of the latter type of antimicrobials is their dependence on transporters to enter susceptible cells. Transporter inactivation results in resistance. Here, we show that a rhizobial ribosome-targeting peptide, phazolicin (PHZ), uses two different transporters, BacA and YejABEF, to enter the cells of a symbiotic bacterium, Sinorhizobium meliloti . This dual-entry mode dramatically reduces the probability of the appearance of PHZ-resistant mutants. Since these transporters are also crucial for S. meliloti symbiotic associations with host plants, their inactivation in natural settings is strongly disfavored, making PHZ an attractive lead for the development of biocontrol agents for agriculture.