Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction

Abstract 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.


Introduction
Bacterial-fungal interactions are critically important in agriculture and human health, and their relevance to ecology is now seen as the norm [1], with numerous studies focusing on identifying the individual microorganisms residing within bacterial-fungal communities (e.g.The Human Microbiome Project) [2].Among these interactions, fungal-bacterial endosymbiosis, in which bacteria reside within the cytosol of fungal hyphae, represents the most intimate relationship.Contemporary studies have revealed that endosymbiotic bacteria, commonly found in members of the Mucoromycota, are not unassuming background actors, but rather have considerable inf luence on fungal pathogenicity, physiology, and ecology [3].
The endosymbiosis between the Mucoromycota fungus Rhizopus microsporus and the bacterium Mycetohabitans rhizoxinica is a unique example of fungal pathogenicity being effectuated by an endosymbiont [4].In housing M. rhizoxinica, R. microsporus gains the ability to kill rice seedlings through the secretion of the bacterial secondary metabolite rhizoxin [5], which also protects the fungus from soil-dwelling micropredators [6].A fundamental process that is key for the persistence of the Rhizopus-Mycetohabitans symbiosis is that endosymbionts are translocated into the fungal spores during host reproduction [7].If endosymbionts are eliminated through antibiotic treatment, R. microsporus is unable to reproduce vegetatively (Fig. 1A) [7].Thus, the ability of R. microsporus to reproduce asexually via sporulation strictly depends on the presence of endobacteria.This endosymbiontdependent control of fungal sporulation is known to be mediated by a multitude of bacterially produced symbiosis factors such as a specialized lipopeptide O-antigen [8], transcription activator-like effectors [9], and the secondary metabolite habitasporin [10].Even though M. rhizoxinica is known for its high potential for secondary metabolite biosynthesis [10,11], it also produces unusual molecules linked to primary metabolism such as F O (7,8-didemethyl-8-hydroxy-5-deazaribof lavin) and 3PG-F 420 , a derivative of the deazaf lavin cofactor F 420 (Fig. 1B) [12].Although F O is the precursor of 3PG-F 420 , both metabolites have entirely distinct physiological roles.F O plays a key role as a lightharvesting chromophore in DNA photolyases across all three domains of life (Bacteria, Archaea, and Eukarya) [13][14][15].F 420 was first discovered as a cofactor of methanogenesis in anaerobic archaea [16].Beyond archaea, F 420 metabolism has only been extensively studied in the bacterial phylum Actinobacteria [17], where it mediates diverse catalytic reactions central to processes such as antibiotic biosynthesis in streptomycetes [18] and drug resistance in Mycobacterium tuberculosis [19].More recently, genomic studies uncovered a broad distribution of genes encoding enzymes responsible for the biosynthesis of F 420 in Gram-negative bacteria [20,21].Of these, three species were confirmed as F 420 producers [21].
F 420 production by Gram-negative bacteria that engage in a symbiotic lifestyle has been reported in two cases-namely, the sponge symbiont Candidatus Entotheonella factor [22] and M. rhizoxinica [12].So far, the physiological role of the deazaf lavin cofactor in these microorganisms remains unknown.The R. microsporus-M.rhizoxinica symbioses provide an ideal model system to investigate the function of F 420 because M. rhizoxinica can be cultured axenically and a readily discernible phenotype exists that is indicative of a stable symbiosis, i.e. sporulation of the fungal host.Other than the discernment of whether metabolites from M. rhizoxinica contribute to the ability of R. microsporus to reproduce, the Rhizopus-Mycetohabitans model allows earlier aspects of symbiosis establishment to be probed such as bacterial colonization of hyphae and intracellular distribution and survival [23][24][25][26].
Here, we show that homologous genes encoding the pathway to produce 3PG-F 420 are conserved in the genomes of several endofungal Mycetohabitans symbionts.Furthermore, we demonstrate that F O , the precursor of 3PG-F 420 , is a symbiosis factor that is instrumental in the maintenance of the phytopathogenic R. microsporus-M.rhizoxinica alliance.

Gene expression studies
RNA was isolated from M. rhizoxinica HKI-454 using the Quick-RNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA) following the manufacturers' recommendations.Because there is no standard method for RNA extraction from endofungal bacteria, we developed our own protocol for reliable RNA extraction from symbiotic M. rhizoxinica (see Supplementary File "Materials and Methods" for details).
Quantitative PCR (qPCR) was used to study expression levels of two genes (fbiC and cofC) in axenic M. rhizoxinica HKI-454 as well as in M. rhizoxinica HKI-454 living in symbiosis with R. microsporus (ATCC62417).The gene fbiC encodes F O synthase, which catalyzes the key step in F 420 biosynthesis leading to the formation of the metabolically active precursor F O (Fig. 2A) [30].A second important step, the side-chain biosynthesis of 3PG-F 420 -0 is catalyzed by CofC.The M. rhizoxinica rpoB gene was used as an internal control for calculation of expression levels and normalization.All qPCR primer pairs used are listed in Supplementary Table 2A.First, primer efficiencies were calculated from standard curves generated with serial dilutions (ranging from 1 to 10 −3 ) of axenic M. rhizoxinica cDNA.All primer pairs have an efficiency of 93% to 96% and were used in subsequent gene expression experiments (Supplementary Table 3) using MyTaq HS Mix (Bioline, London, UK) and EvaGreen ® Fluorescent DNA Stain (Jena Bioscience, Jena, Germany).Each sample was run in three technical replicates on a QuantStudio 5 Real-Time PCR machine (Applied Biosystems, Waltham, MA).A control reaction, in which sterile water replaced the cDNA, was carried out in parallel.Amplifications of each template were performed in three biological replicates (n = 3).The cycle threshold (C t ) values were calculated using the Design and Analysis Software v1.5.2 (Applied Biosystems).C t values were used for quantification of expression levels via the 2 − Ct method [31] in MS Excel.Statistical analysis was performed in GraphPad Prism 9.5.1 (GraphPad Software, La Jolla, CA, www.graphpad.com).An unpaired t-test with Welch's correction was used to study the gene expression level of fbiC and cofC in M. rhizoxinica wild type grown in axenic culture in relation to M. rhizoxinica living in symbiosis with R. microsporus.

Extraction of F O /3PG-F 420 -n from fungal mycelium and axenic Mycetohabitans spp.
Small pieces of fungal mycelium were inoculated in 100 ml nutrient broth medium (Merck Millipore, Darmstadt, Germany) in 500 ml baff led Erlenmeyer f lasks and incubated at 30 • C and 110 rpm.After 7 days of incubation, fungal mycelium was strained through a 40 μm cell strainer (Corning Inc.) and resuspended in 10 ml ice-cold HPLC-grade methanol (VWR Chemicals, Darmstadt, Germany).Axenic bacterial overnight cultures of Mycetohabitans spp.were inoculated in 50 ml MGY M9 minimal medium in 300 ml baff led Erlenmeyer f lasks and cultured at 30 • C and 110 rpm.After 7 days of incubation, bacterial cultures were snap-frozen in liquid nitrogen in 500 ml round-bottom f lasks and freeze-dried overnight (Christ Alpha loc-1 m, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).Dry Mycetohabitans spp.cultures were then resuspended in 10 ml ice-cold HPLC-grade methanol (VWR Chemicals).
All resuspended samples were sonicated for 20 min (Sonorex RK100 Ultrasonic bath, Bandelin, Berlin, Germany) and then shaken (250 rpm) for 1 h followed by centrifugation (10 000 × g) for 15 min (Centrifuge 5810R, Eppendorf, Hamburg, Germany).The supernatant was filtered through a paper filter into a 500 ml round-bottom f lask and the solvent was evaporated on a vacuum rotary evaporator (Laborota 4003, Heidolph, Schwabach, Germany).Dry extracts were redissolved in 2 ml LC-MS-grade water (Carl Roth, Karlsruhe, Germany).The aqueous extracts were loaded on to a 100 mg C18 Chromabond cartridge (Macherey-Nagel, Düren, Germany), which was washed with methanol and conditioned with LC-MS-grade water beforehand.The cartridge was then washed and desalted with 1 ml LC-MSgrade water, followed by elution of the sample in three fractions with methanol (0.5 ml, 1.0 ml, and 0.5 ml).F O was almost always concentrated in the second fraction.The samples were centrifuged at 10000 × g for 20 min and the supernatant was stored at −20 • C until further LC-MS analysis (see Supplementary File "Materials and Methods" for details).

Generation of M. rhizoxinica fbiC and M. rhizoxinica cofC using CRISPR/Cas cytidine base editing and subsequent genetic complementation
To investigate a possible role of F O and 3PG-F 420 -n in the symbiosis, two genes (fbiC and cofC) were deleted using cytidine base editing.To establish a working CRISPR/Cas cytidine base editing method for M. rhizoxinica HKI-454, we used our knowledge gained from the CRISPR/Cas modification of the Betaproteobacterium Burkholderia gladioli [32], where we utilized a temperature-sensitive, low-copy plasmid (pTsK-CasRed-Bt, Supplementary Table 4) containing among others a codon-optimized cas9 * gene.See Supplementary File "Materials and Methods" for details on the generation of the base editing plasmids pTsK-AnCU-fbiC (targeting fbiC) and pTsK-AnCU-cofC (targeting cofC) and subsequent transfer of the plasmids into M. rhizoxinica by electroporation.
Both M. rhizoxinica fbiC and M. rhizoxinica cofC were checked for deleterious mutations using whole genome sequencing (see Supplementary File "Materials and Methods" for details).Both mutants were subsequently genetically complemented.Brief ly, M. rhizoxinica fbiC and M. rhizoxinica cofC were transformed with the plasmids pRANGER-fbiC and pRANGER-cofC, respectively.These expression vectors carry the native promoter controlling expression of each gene, yielding the complemented strains M. rhizoxinica fbiC pRANGER-fbiC and M. rhizoxinica cofC pRANGER-cofC (see Supplementary File "Materials and Methods" for details).

Co-culture / sporulation bioassay
Liquid sporulation bioassays, containing apo-symbiotic R. microsporus and 100 μl of overnight cultures of M. rhizoxinica wild type (HKI-454), M. rhizoxinica fbiC, M. rhizoxinica cofC, M. rhizoxinica fbiC pRANGER-fbiC, or M. rhizoxinica cofC pRANGER-cofC were performed as previously described [9].Experiments were performed at least four times independently (n ≥ 4 biological replicates) with six technical replicates on each plate.GraphPad Prism 9.5.1 (GraphPad Software) was used for statistical analysis and graphing.Data from spore counts were compared between M. rhizoxinica strains using one-way analysis of variance (ANOVA) and Tukey Honestly Significant Difference (HSD) test function in GraphPad.P values (P) <.05 were considered statistically significant.The Brown-Forsythe test was used to test for equal variance and a P < .05 was considered significant.
To test whether F O alone could trigger fungal sporulation or whether the presence of bacteria is still necessary, we chemically synthesized F O (see Supplementary File "Materials and Methods" for details) and added varying concentrations of synthetic F O (312 ng/ml, 625 ng/ml, and 937 ng/ml) to co-cultures of R. microsporus and M. rhizoxinica fbiC.Apo-symbiotic R. microsporus was also grown in the absence of endobacteria in 48-well plates in either liquid or solid potato dextrose medium that was supplemented with a final concentration of 73 μg/ml synthetic F O .Spores were harvested and quantified as described previously [9], and the formation of sporangia was visualized using a Zeiss Axio Mycetohabitans spp.strains that were isolated from their corresponding R. microsporus host; (E) gene expression assay for two F 420 biosynthetic enzymes; the expression level of genes fbiC and cofC in M. rhizoxinica was measured in pure culture (axenic) and in symbiosis with R. microsporus (symbiotic) using qPCR; the gene rpoB was used to calculate expression levels using the 2 -Ct method; bars represent means of three independent biological replicates (n = 3) and error bars indicate standard deviation; unpaired t-test with Welch's correction ( * P<05, Supplementary Tables 5 and 6).

Imaging of endohyphal M. rhizoxinica strains
To visualize the localization of F 420 -producing and F 420 -deficient M. rhizoxinica strains within the fungal hyphae, strains were transformed with a plasmid encoding green f luorescent protein (GFP) (see Supplementary File "Materials and Methods" for details) and then used in the liquid sporulation assays as described previously [9].After 5 days of incubation, fungal mycelium was transferred to PDA Petri dishes.Sterile, high-performance cover glasses (D = 0.17 mm +/− 0.005 mm, refractive index = 1.5255 +/− 0.0015, Carl Zeiss Microscopy) were placed on the agar plate in close proximity to the mycelium.Plates were incubated for 1-2 days at 30 • C to allow fungal mycelium to grow on top of the cover glass.Once fungal mycelium had grown on top of the cover glass, the cover glasses were carefully removed from agar plates, and the mycelium was fixed for 5 min in fixing solution (3.7% formaldehyde, 25 mM KH 2 HPO 4 ).Samples were washed at least three times with 25 mM KH 2 HPO 4 before mounting the cover slide in embedding agent (ProLong Gold Antifade Mountant, Invitrogen) on a microscope slide (superfrost, ground 90 • , Thermo Scientific).Fluorescence microscopy was carried out using a f luorescence wide-field microscope (LaserWF mode in the ELYRA 7, Carl Zeiss Microscopy), and bacterial cells were visualized using a laser excitation at 488 nm and an emission bandpass from 495 nm to 590 nm.For superresolution images, the structured illumination mode was used with a grating of 27.5 μm period.Fluorescent wide-field images were used to quantify bacterial cells inside of fungal hyphae (see Supplementary File "Materials and Methods" for details).

Products of conserved F 420 biosynthetic gene clusters are produced under symbiotic conditions
The discovery that two Mycetohabitans strains encode a complete F 420 biosynthetic gene cluster [12,33], despite their highly reduced genomes [10,34], raised the question as to whether the cofactor might contribute to the integrity of the bacterialfungal symbiosis.Therefore, we searched the genomes of six additional Mycetohabitans strains that are endosymbionts of globally distributed R. microsporus strains for F 420 biosynthesis loci (Supplementary Table 1) [27].We found a complete, highly conserved F 420 biosynthesis locus, containing the requisite genes for F 420 biosynthesis, in all Mycetohabitans-symbionts of R. microsporus (Fig. 2A and Supplementary Fig. 1).The gene fbiC encodes F O synthase, which catalyzes the key step in F 420 biosynthesis leading to the formation of the metabolically active precursor F O (Fig. 2A) [30].A second important step, the side-chain biosynthesis of 3PG-F 420 -0 is catalyzed by CofC and CofD [12,33].Finally, a γ -glutamyl ligase (CofE) catalyzes the successive addition of l-glutamate residues (n) to 3PG-F 420 -0 yielding 3PG-F 420 -n [35].
To investigate under which conditions 3PG-F 420 and its precursor F O are produced, we extracted and analyzed the following cultures by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS): (i) eight R. microsporus strains containing their corresponding Mycetohabitans spp.endosymbionts, (ii) one R. microsporus strain (ATCC62417) cured of its endosymbionts (RMapo), (iii) R. microsporus strains that are naturally endosymbiont-free, and (iv) axenic Mycetohabitans spp.strains that were isolated from their corresponding R. microsporus host.The metabolic profiling showed that both F O and 3PG-F 420 -n species are predominantly produced in Mycetohabitans strains when living as endosymbionts of R. microsporus (Fig. 2B and Supplementary Fig. 2A).In contrast, apo-symbiotic and naturally endosymbiont-free R. microsporus strains do not produce any F O or 3PG-F 420 -n species (Fig. 2C and Supplementary Fig. 2B).We noted that endosymbiotic bacteria grown outside of their host sporadically produce F O and 3PG-F 420 -n under the conditions tested (Fig. 2D and Supplementary Fig. 2C).3PG-F 420 -2 and 3PG-F 420 -3 are the most abundant species in Mycetohabitans strains, although up to four glutamate residues were detected (3PG-F 420 -4) (Supplementary Fig. 2C).
In light of the finding that F O and 3PG-F 420 -n are predominantly produced under symbiotic conditions, we probed whether F O and 3PG-F 420 -n production is constitutive or dependent on the presence of the fungal host.Gene expression of fbiC and cofC was monitored in axenic M. rhizoxinica HKI-454 and in M. rhizoxinica HKI-454 living as endosymbiont in R. microsporus ATCC62417.These genes were chosen because they both encode enzymes that catalyze key steps in F 420 biosynthesis (Fig. 2A).A gene encoding the βsubunit of the bacterial RNA polymerase (rpoB) was used as an internal control for RNA integrity and cDNA synthesis efficiency.Although it is inherently difficult to isolate high-quality RNA from endofungal bacteria, we developed a reliable method for RNA extraction from symbiotic M. rhizoxinica (Supplementary Fig. 3).Using qPCR, we show that fbiC is 1.6-fold upregulated (unpaired ttest: t = 2.87, df = 4.0, P = .0455,Supplementary Table 5) and cofC is 4.6-fold upregulated (unpaired t-test: t = 4.221, df = 4.0, P = .0135,Supplementary Table 6) in symbiotic M. rhizoxinica compared to axenic bacterial cultures (Fig. 2E).
Based on the prevalence of F 420 -biosynthesis genes in the genomes of endofungal Mycetohabitans strains and the increased production of F O and 3PG-F 420 -n under symbiotic conditions, backed up by elevated transcription levels of F 420 biosynthetic genes, we deemed it important to further probe the role of these metabolites in the symbiosis.

Generation of F 420 -deficient M. rhizoxinica using CRISPR/Cas-assisted base editing
To facilitate subsequent investigations into the role of F O /3PG-F 420 in the Rhizopus-Mycetohabitans symbiosis, we performed targeted gene inactivation of fbiC and cofC.As both genes belong to one operon and are under the control of the same promotor (Fig. 2B), we intended to produce markerless gene inactivations to avoid possible polar effects.Specifically, our goal was to introduce a stop codon into the coding sequences of fbiC and cofC, creating two individual M. rhizoxinica mutant strains.We deemed cytidine base editing a promising method to introduce stop codons [36].
To generate base editing plasmids, a temperature-sensitive, low-copy plasmid containing a rhamnose-inducible and codonoptimized cas9 * gene was used [32].We added a gene that encodes a fusion protein consisting of APOBEC1 (a cytidine deaminase from rats), nCas9 (Cas9-nickase with a D10A mutation from Streptococcus pyogenes), and a uracil DNA glycosylase inhibitor (UGI) from a Bacillus phage (Fig. 3A).
The base editing plasmids pTsK-AnCU-fbiC and pTsK-AnCU-cofC (Supplementary Table 4) differed only in the synthetic guide RNA (sgRNA) sequences in which the N20 sequences determine the individual target site.Both plasmids were used for precisely targeting fbiC and cofC in the M. rhizoxinica genome to generate the mutants M. rhizoxinica fbiC::stop (M.rhizoxinica fbiC) and M. rhizoxinica cofC::stop (M.rhizoxinica cofC), respectively.Successful base editing in potential mutants was verified via Sanger sequencing (Fig. 3B).Whole genome sequencing of M. rhizoxinica fbiC and M. rhizoxinica cofC revealed no deleterious mutations in either strain, thus confirming the specificity of the chosen sgRNA.CRISPR/Cas-mediated base editing was previously developed for Gammaproteobacteria (Pseudomonas putida) [37] and Alphaproteobacteria (Brucella melitensis) [38].Here, we established this genome editing strategy for Betaproteobacteria (M.rhizoxinica).
As expected, metabolic profiling of the mutant strains using LC-MS/MS confirmed that F O and 3PG-F 420 biosynthesis is abolished in M. rhizoxinica fbiC, whereas M. rhizoxinica cofC is able to produce F O but no 3PG-F 420 -2 (Fig. 3C).In comparison, M. rhizoxinica wild type produces F O and 3PG-F 420 -2 in axenic culture under the same conditions.Axenically grown M. rhizoxinica fbiC displays growth behavior similar to the wild type when cultivated on a selection of carbon sources in liquid media (Supplementary Fig. 4).A broader screen for altered utilization of carbon sources using phenotypic microarrays did not reveal any metabolic deficit of F O -deficient M. rhizoxinica (Supplementary Table 7).

F O -deficient M. rhizoxinica abolish the sporulation ability of R. microsporus
The ability of R. microsporus to reproduce asexually via sporulation strictly depends on the presence of endobacteria as well as secondary metabolites produced by M. rhizoxinica [7,10,26].Thus, considering that F O and 3PG-F 420 are predominantly produced under symbiotic conditions, we aimed to explore the potential involvement of 3PG-F 420 and/or its precursor F O in fungal sporulation.The sporulation ability of R. microsporus containing either M. rhizoxinica fbiC or M. rhizoxinica cofC was investigated in a sporulation bioassay [24].Brief ly, apo-symbiotic R. microsporus mycelium is co-cultured with axenic M. rhizoxinica wild type or mutant strains in 48-well plates.After 5-7 days of incubation, the formation of spores can be observed indicating the successful establishment of the symbiosis [7].Apo-symbiotic R. microsporus does not sporulate (Fig. 4A).Subsequently, the absence/presence of F O and 3PG-F 420 in co-cultures was verified via LC-MS/MS analysis.
M. rhizoxinica cofC is able to produce F O , but not 3PG-F 420 , and readily triggers fungal sporulation to a similar degree as the wild type.(Fig. 4A and B and Supplementary Table 8).In contrast, in the case of co-cultivation with M. rhizoxinica fbiC, spores are absent and the sporulation efficiency is significantly reduced (P < .05)when compared to the M. rhizoxinica wild type (Fig. 4A and Supplementary Table 8).As expected, F O and 3PG-F 420 are absent in R. microsporus-M.rhizoxinica fbiC co-cultures (Fig. 4B).Thus, the inability to sporulate appears to be linked to a lack of F O but not 3PG-F 420 .In addition, growth of the fungal mycelium containing M. rhizoxinica fbiC is slower and fewer aerial hyphae are formed, which is comparable to apo-symbiotic fungal mycelium (Fig. 4A).The observation that F O -deficiency completely abolishes R. microsporus sporulation is unexpected, as a complete abrogation of fungal sporulation is rarely observed [24].
To confirm that the inability of M. rhizoxinica fbiC to induce fungal sporulation is solely due to disruption of the fbiC gene, we performed an in vivo trans-complementation experiment.To this end, we transformed M. rhizoxinica fbiC and M. rhizoxinica cofC with the plasmids pRANGER-fbiC and pRANGER-cofC, respectively.These expression vectors carry the native promoter controlling expression of each gene, yielding the complemented strains M. rhizoxinica fbiC pRANGER-fbiC and M. rhizoxinica cofC pRANGER-cofC (Supplementary Fig. 5).Recolonization of apo-symbiotic R. microsporus by cofC pRANGER-cofC leads to a level of fungal   sporulation similar to that imparted by the wild type (Fig. 4A).In addition, co-incubation of R. microsporus with fbiC pRANGER-fbiC readily triggers sporulation, thus restoring the wild-type phenotype (Fig. 4A).As expected, both complemented strains are able to produce F O and 3PG-F 420 -2 in co-culture with R. microsporus (Fig. 4B).These results show that a lack of 3PG-F 420 alone does not affect the ability of R. microsporus to sporulate.However, when 3PG-F 420 -deficiency is coupled with a lack of F O , fungal sporulation is abolished, signifying that F O is an essential mediator in the sporulation process of endosymbiont-dependent R. microsporus.

Chemical complementation of F O -deficient M. rhizoxinica restores sporulation in R. microsporus
The observation that F O -deficiency completely abolishes fungal sporulation raised the question as to whether F O alone can trigger R. microsporus sporulation or whether the presence of bacteria is necessary for this process.To answer this question, chemical complementation with synthetic F O was performed.Apo-symbiotic R. microsporus was co-incubated with M. rhizoxinica fbiC in a sporulation assay as described above.In addition, synthetic F O was added to individual wells containing aposymbiotic R. microsporus and M. rhizoxinica fbiC at varying concentrations (312 ng/ml, 625 ng/ml, or 937 ng/ml).When synthetic F O is added to non-sporulating R. microsporus-M.rhizoxinica fbiC co-cultures, spore formation is restored (Fig. 5A).Although the number of spores is lower than induced by the wild type, the sporulation in chemically complemented cocultures is significantly increased (P < .002)when compared to M. rhizoxinica fbiC (Fig. 5B and Supplementary Table 9).In fact, we observed a dose-dependent increase in the number of spores with increasing amounts of F O supplementation (Fig. 5B and Supplementary Table 9).
As these results clearly confirmed the importance of F O in fungal sporulation, we were curious as to whether F O itself could induce sporulation in R. microsporus in the absence of endobacteria.Apo-symbiotic R. microsporus was grown in 48well plates in either liquid or solid potato dextrose medium containing a final concentration of 73 μg/ml synthetic F O .Spore formation was not detected, even after 2 weeks of incubation (Supplementary Fig. 6).Together with the successful chemical complementation of M. rhizoxinica fbiC, these results indicate that F O alone is not sufficient to trigger sporulation of the host fungus and that other bacterial traits are involved in this process.

F O -deficient M. rhizoxinica are able to recolonize R. microsporus hyphae efficiently
A lack of sporulation in F O -deficient R. microsporus-M.rhizoxinica co-cultures could be caused by the inability of M. rhizoxinica fbiC to recolonize fungal hyphae as has been previously reported for M. rhizoxinica that lack a functional type 2 secretion system [25].To investigate whether M. rhizoxinica fbiC can enter fungal hyphae, we generated M. rhizoxinica strains that express  9) was applied; data points of RMapo + fbiC are the same as depicted in Fig. 4A.GFP constitutively.Apo-symbiotic R. microsporus was co-incubated with GFP-expressing M. rhizoxinica wild type, M. rhizoxinica fbiC, or M. rhizoxinica cofC.After 1 week of co-cultivation, superresolution f luorescence microscopy was performed to visualize individual bacterial cells and fungal hyphae.As expected, wildtype M. rhizoxinica is located inside fungal hyphae.M. rhizoxinica fbiC and M. rhizoxinica cofC are also clearly visible inside R. microsporus hyphae confirming successful fungal recolonization by both of these strains (Fig. 6A).Fungal mycelium recolonized by M. rhizoxinica fbiC displays a different morphology, both at the macroscopic and microscopic scale, when compared to that recolonized by M. rhizoxinica wild type and M. rhizoxinica cofC (Figs 4A and 6A).When grown on agar plates, the mycelium containing M. rhizoxinica fbiC grows f lat and patchy in contrast to the f luffy, aerial mycelium of the wild type and M. rhizoxinica cofC.A combination of widefield and f luorescence microscopy revealed hyphae with a larger diameter (Fig. 6A) containing a significantly higher number of bacterial cells (P < .0001)than hyphae containing M. rhizoxinica wild type or M. rhizoxinica cofC (Fig. 6B and Supplementary Table 10).
The high bacterial load of M. rhizoxinica fbiC in fungal hyphae is reversible through the addition of synthetic F O .By adding increasing concentrations of synthetic F O (312 ng/ml, 625 ng/ml, or 937 ng/ml) to apo-symbiotic R. microsporus-M.rhizoxinica fbiC co-cultures, the bacterial load of M. rhizoxinica fbiC inside the fungal hyphae is comparable to the wild type in all cases (Fig. 6A and Fig. 6B).Quantification of M. rhizoxinica fbiC pRANGER-fbiC (complemented M. rhizoxinica fbiC) reveals significantly fewer countable bacterial cells inside fungal hyphae compared to M. rhizoxinica fbiC (P <.0001, Fig. 6B and Supplementary Table 10).The ability of F O -deficient M. rhizoxinica to efficiently recolonize their fungal host combined with the abolishment of fungal sporulation shows that F O is not essential for host colonization but rather supports a process that triggers host sporulation in this bacterialfungal symbiosis.

Discussion
The specialized redox cofactor F 420 is instrumental for diverse biochemical processes in archaea and Actinobacteria, e.g.biosynthesis of antibiotics [39], methanogenesis [17], or the degradation of chemical pollutants [40].In recent years, F 420 has gained considerable interest due to its low redox potential, which allows for F 420 -dependent enzymes to be utilized in biotechnological applications, such as bioremediation and biosynthetic production of antibiotics [41][42][43][44].This has invigorated a search for new F 420 -dependent enzymes leading to the discovery of F 420 biosynthetic gene clusters in a wide range of Gram-negative bacteria.We have previously shown that the Gram-negative bacterium M. rhizoxinica HKI-454 (Class: Betaproteobacteria) is able to produce an unusual derivative of F 420 (3PG-F 420 ) [12].However, virtually nothing was known about the role of the products of the F 420 pathway in M. rhizoxinica and in Gram-negative bacteria in general.
Here, we show that the genetic inventory to produce cofactor 3PG-F 420 , although rather rare in related bacteria, is conserved in the genomes of endofungal Mycetohabitans strains that are known to form a stable symbiosis with their corresponding R. microsporus hosts [27].Gene expression analysis and metabolic profiling showed that biosynthesis of 3PG-F 420 and its precursor F O are particularly pronounced during symbiotic growth.By establishing a CRISPR-assisted cytidine base editing strategy for M. rhizoxinica, we were able to generate M. rhizoxinica strains deficient of F O and 3PG-F 420 (M.rhizoxinica fbiC) and deficient of only 3PG-F 420 (M.rhizoxinica cofC).Our CRISPR-based, markerless gene inactivation method represents an improvement over previous methods based on homologous recombination and counterselectable markers [24] as it avoids possible polar effects.Coculture experiments unequivocally demonstrate that sporulation of the host is only triggered by bacteria that produce F O or when supplemented with F O .M. rhizoxinica cofC, which produces F O but not 3PG-F 420 , does not show any obvious effect on sporulation    (Fig. 7).These results demonstrate that F O is a crucial symbiosis factor that controls fungal reproduction.
To probe whether F O is required by M. rhizoxinica itself, we tested if F O deficiency negatively impacts bacterial growth in vitro but did not observe any growth defects of M. rhizoxinica fbiC, at least in axenic culture.In vivo, we even observed an increased bacterial cell density of M. rhizoxinica fbiC in R. microsporus hyphae.Thus, it is unlikely that F O deficiency causes growth impairments or insufficient protective responses under symbiotic conditions, as such traits would result in poor host colonization and reduced intracellular survival of M. rhizoxinica [23].In fact, an increase in M. rhizoxinica fbiC cell density following fungal recolonization mirrors an unusual phenomenon recently reported in R. microsporus reinfected with M. rhizoxinica lacking Mycetohabitans transcription activator-like effector 1 (MTAL1) [23].Based on microf luidics and f luorescence microscopy, it was shown that MTAL1-deficient M. rhizoxinica are trapped in hyphae through septa formation leading to high cell numbers.Although we did not observe septa formation and subsequent trapping of M. rhizoxinica in this study, it is of course possible that F O deficiency causes a similar response by the fungus.Alternatively, F O may regulate bacterial cell numbers in vivo as a type of quorum sensing molecule.The process of how bacterial numbers are controlled inside hyphae is still unknown as M. rhizoxinica does not produce any of the "classical" quorum sensing molecules [11].Another possible explanation for the high bacterial load observed in this study is an accumulation of bacterial cells due to the lack of sporulation because wild-type bacteria would normally be packed into spores [7].In line with this hypothesis, R. microsporus recolonized by M. rhizoxinica cofC shows a normal bacterial load and produces a similar number of spores as the wild type.Because M. rhizoxinica supplies its fungal host with selected amino acids [11], it is conceivable that a bacterial nutrient that is abundant during vegetative growth is consumed by the fungus once the sporulation starts, thus becoming a growth limiting factor.The higher cell density might consequently not be directly related to altered metabolic capacities of the bacteria but rather to an altered metabolism of the fungus.
As symbionts often deliver essential metabolites like vitamins or amino acids to their host [45,46], F O might be secreted into the host cytosol and subsequently utilized by the fungus.Indeed, F O is present in the supernatant of M. rhizoxinica [12,47] and might, therefore, act as a secreted "vitamin" or "signal" that triggers host sporulation.In support of this hypothesis, M. rhizoxinica is not actively engulfed by R. microsporus during entry into hyphae and is thus lacking a fungal cell membrane around the bacteria [25].In addition, F O represents the deazaf lavin "head" moiety of 3PG-F 420 , which is redox active [48] and capable of harvesting light [13].The sporulation-triggering activity of F O could involve its photochemical properties [13].The light-harvesting properties of F O are exploited by some DNA photolyases, which are highly efficient DNA repair enzymes [49].Because DNA photolyases have a high binding-specificity to DNA, one could imagine a possible activation or repression of fungal genes in a F O -dependent manner.However, future studies will have to rely on biochemical methods such as chemical proteomics to reveal binding partners of F O within the endofungal proteome.
Intrigued by the complete abrogation of fungal sporulation in R. microsporus recolonized by M. rhizoxinica fbiC, we investigated whether F O alone can trigger R. microsporus sporulation and observed that the presence of M. rhizoxinica is a prerequisite for host sporulation.This observation is in line with previous studies suggesting that control of fungal sporulation involves a multitude of bacterially produced symbiosis factors such as transcription activator-like effectors [9], the secondary metabolite habitasporin [10], or a specialized lipopeptide O-antigen [8].It was shown that a lack of these molecules reduces the sporulation ability of R. microsporus when recolonized by M. rhizoxinica.Here, we report complete abrogation of sporulation after host colonization by M. rhizoxinica fbiC.Such a complete absence of sporulation was previously only reported for M. rhizoxinica strains unable to recolonize the apo-symbiotic host due to the absence of intact type 2 [25] or type 3 secretion systems [24].Our results demonstrate that F O , the precursor of 3PG-F 420 , is yet another symbiosis factor that is instrumental for the maintenance of the R. microsporus-M.rhizoxinica alliance.
The purpose of 3PG-F 420 production in M. rhizoxinica remains mysterious, as M. rhizoxinica cofC, which produces F O but not 3PG-F 420 , does not show any obvious effect on fungal sporulation or recolonization.It has been shown that 3PG-F 420 is typically concentrated in bacterial cell pellets [12,47], indicating that 3PG-F 420 may still play as-yet-unknown physiological roles in Mycetohabitans species.It is, however, questionable whether 3PG-F 420 actually acts as a cofactor in Mycetohabitans species, as bioinformatics-based evidence for the presence of genes encoding putative F 420 -dependent oxidoreductases in M. rhizoxinica could not be obtained [12].This finding speaks against a cofactor role, because F 420 -dependent enzymes typically belong to well-studied enzyme families and at least two such enzymes would be necessary to drive F 420 -dependent redox biochemistry [20,21].It is, however, still possible that uncharacterized enzymes catalyze the 3PG-F 420 -dependent reactions.Alternatively, the lack of F 420dependent oxidoreductases could potentially be explained by 3PG-F 420 fulfilling a non-cofactor role in M. rhizoxinica, as has been reported for the cofactor nicotinamide adenine dinucleotide [50,51] and for the F 420 -derivative factor F 390 in Methanothermobacter thermautotrophicus [52][53][54].
In this study, we combined targeted gene inactivation, metabolic profiling, colonization assays, and microscopy to functionally characterize deazaf lavins of the endofungal symbiont M. rhizoxinica.We found that F O , the redox-active moiety of F 420 , is necessary for sporulation of the fungal host, signifying that F O is an essential symbiosis factor for the Rhizopus-Mycetohabitans symbiosis (Fig. 7).This study represents an unprecedented case of a deazaf lavin metabolite controlling the reproduction of a eukaryotic host.It seems plausible that F O acts as a mediator of fungal sporulation, but the molecular details of how this non-cofactor role is implemented remain to be deciphered.It should be mentioned that addition of the tail moiety to F O , to form the ultimate product of the biosynthetic pathway, may direct 3PG-F 420 toward possibly acting as a classical cofactor within M. rhizoxinica.Although the role of atypical metabolites in symbioses and microbial communities in general has been hitherto overlooked, this study showcases the remarkable f lexibility of biochemical molecules derived from a relatively simple pathway.Our results should inspire future research avenues into the functional elucidation of F 420 pathway products involved in cellular communication and symbiosis.

Figure 1 .
Figure 1.(A) Schematic representation of M. rhizoxinica-dependent sporulation of R. microsporus; mature sporangia are absent in fungi cured of endosymbionts by antibiotic treatment (apo-symbiotic R. microsporus); co-cultivation of axenic wild-type M. rhizoxinica with the apo-symbiotic fungus leads to recolonization, and the host's ability to form sporangia is restored; (B) chemical structure of F O , F 420 , and 3PG-F 420 ; n indicates the number of γ -linked glutamate residues.

Figure 3 .
Figure 3. Generation of M. rhizoxinica fbiC and M. rhizoxinica cofC using CRISPR/Cas-assisted base editing; (A) schematic overview of the temperature-sensitive plasmid pTsK-AnCU-fbiC encoding a fusion protein consisting of a cytidine deaminase (APOBEC1), a Cas9-nickase (nCas9), and an UGI; the plasmid also carries the fbiC-specific sgRNA (left panel); APOBEC1 deaminates a cytosine (C) to uracil (U); the resulting mismatch leads to the replacement of guanosine (G) with adenosine (A) on the non-edited strand (middle panel); the artificially generated U-A base pair is converted to a T-A base pair during DNA replication; this leads to the conversion of glutamine to a stop codon (right panel); the plasmid pTsK-AnCU-cofC, carrying the cofC-specific sgRNA, was constructed in an analogous manner; Abbreviations: APOBEC1, rat cytidine deaminase; ncas9 * , codon-optimized gene for Cas9 nickase from S. pyogenes; ; kan R , kanamycin resistance cassette; P rhaB , rhamnose-inducible promoter; P J23119 , constitutive promoter; PAM, protospacer adjacent motif; N20, the 20-bp sequence at the 5 end upstream of the PAM; Gln, glutamine; * , stop codon; (B) verification of base editing in M. rhizoxinica fbiC (exchange of C to T) and in M. rhizoxinica cofC (exchange of C to T on the minus strand, resulting in an exchange of G to A on the complementary strand) via Sanger sequencing; (C) confirmation of metabolite production via LC-MS/MS analysis; EICs (5 ppm mass window) confirming the absence of both F O ([M+H] + m/z 364.11393) and 3PG-F 420 -2 ([M+H] + m/z 790.18149) production by M. rhizoxinica fbiC, the presence of F O and absence of 3PG-F 420 -2 production by M. rhizoxinica cofC, and the production of both metabolites by M. rhizoxinica wild type (HKI-454).

Figure 7 .
Figure 7. Schematic model of F O -dependent host control by endofungal bacteria; the recolonization of apo-symbiotic R. microsporus by the wild-type endosymbiont (M.rhizoxinica wild type) restores the host's ability to sporulate; M. rhizoxinica that is able to produce F O but lacks the cofactor 3PG-F 420 (M.rhizoxinica cofC) do not diminish the sporulation ability of R. microsporus; in contrast, M. rhizoxinica incapable of producing 3PG-F 420 and F O (M. rhizoxinica fbiC) does not trigger sporulation of R. microsporus.