Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Transcription activator-like effectors from endosymbiotic bacteria control the reproduction of their fungal host.

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.

Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease.

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

Transcription activator-like effector protects bacterial endosymbionts from entrapment within fungal hyphae.

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.

Bacterial cell wall-degrading enzymes induce basidiomycete natural product biosynthesis.

Natural products play a vital role for intermicrobial interactions. In the basidiomycete arena an important representative is variegatic acid, a lactone natural product pigment whose ecological relevance stems from both inhibiting bacterial swarming and from indirect participation in breakdown of organic matter by brown-rotting fungi. Previous work showed that the presence of bacteria stimulates variegatic acid production. However, the actual external molecular trigger that prompts its biosynthesis in the mushroom hyphae remained unknown. Here, we report on the identification of Bacillus subtilis subtilisin E (AprE) and chitosanase (Csn) as primary inducers of pulvinic acid pigment formation. Using the established co-culture system of B. subtilis and Serpula lacrymans, we used activity-guided FPLC-based fractionation of B. subtilis culture supernatants and subsequent peptide fingerprinting to identify candidates, and their role was corroborated by means of a pigment production assay using heterologously produced chitosanase and subtilisin. B. subtilis mutants defective in either the aprE or the csn gene still triggered pigmentation, yet to a lower degree, which points to a multicausal scenario and suggests the combined activity of these cell wall polymer-attacking enzymes as true stimulus.