Ralstonia solanacearum lipopeptide induceschlamydospore development in fungi and facilitatesbacterial entry into fungal tissues
Joseph E Spraker1, Laura M Sanchez2, Tiffany M Lowe1, Pieter C Dorrestein2,3
and Nancy P Keller41Department of Plant Pathology, University of Wisconsin—Madison, Madison, WI, USA; 2Departments ofPharmacology, Chemistry and Biochemistry, Skaggs School of Pharmacy and Pharmaceutical Sciences,University of California—San Diego, La Jolla, CA, USA; 3Collaborative Mass Spectrometry Innovation Center,University of California—San Diego, La Jolla, CA, USA and 4Departments of Bacteriology, Medical Microbiology andImmunology, University of Wisconsin—Madison, Madison, WI, USA
Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which sharesa broad ecological range with many plant- and soil-associated fungi. We sought to determine if R.solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R.solanacearum produced a diffusible metabolite that induced conserved morphological differentiationin 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes).Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cellwalls. Some chlamydospores internally harbored R. solanacearum, indicating a newly describedendofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry andpeptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R.solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of thehybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycinsynthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydosporedevelopment in fungal coculture and invaded fungal hyphae less well than wild-type. We proposethat ralsolamycin contributes to the invasion of fungal hyphae and that the formation ofchlamydospores may provide not only a specific niche for bacterial colonization but also enhancedsurvival for the partnering fungus.The ISME Journal (2016) 10, 2317–2330; doi:10.1038/ismej.2016.32; published online 4 March 2016
Introduction
Ralstonia solanacearum is the causative agent oflethal bacterial wilt and is a globally distributed soil-borne plant pathogen known to infect well over 200different plant hosts (Hayward, 1991). The broaddistribution of this bacterium in a wide variety of soilenvironments suggests that R. solanacearum likelyencounters a diversity of other soil microbes,including fungi. Although the molecular interactionsbetween R. solanacearum and host plants have beenwell studied (Genin and Denny, 2012), there hasbeen little exploration of R. solanacearum’s interac-tions with soil cohabitants.
Soil is a heterogeneous, complex microcosmreplete with inter-organismal interactions, whichrestructure the physical and chemical compositionof the shared environment. Small molecule signalsare increasingly recognized as important commu-nication signals in microbial interactions (Tarkkaet al., 2009). Historically, many of these specializedmetabolites (Davies, 2013) have been harnessed forpharmaceutical purposes, and there has been aresurgence of interest in natural products based onmicrobial crosstalk (Cragg and Newman, 2013).Genome mining of R. solanacearum strainGMI1000 shows that it has the potential to producea variety of secondary metabolites. A few of thesemetabolites have been characterized includingralfuranone (Wackler et al., 2011) and theyersinabactin-like siderophore, micacocidin(Kreutzer et al., 2011). Micacocidin has moderateantimycoplasma and antifungal effects in vitrobecause of its metal chelating properties (Kobayashiet al., 2000). These and other compounds produced
Correspondence: NP Keller, Departments of Bacteriology, MedicalMicrobiology and Immunology, University of Wisconsin—Madison,1550 Linden Drive, Madison, WI 53706, USA.E-mail: [email protected] 22 October 2015; revised 21 January 2016; accepted23 January 2016; published online 4 March 2016
by R. solanacearum likely impact how it interactswith microbes in the soil.
Small molecule-mediated antibiosis has beendocumented in both bacterial and fungal systems(Haas and Défago, 2005; Burlinson et al., 2011), butantagonistic interactions are only one potentialinteraction outcome. Recent advances in the under-standing of bacterial–fungal interactions indicatethat bacterial natural products can stimulate mor-phological transitions in many fungi. In particular,bacterial metabolites commonly induce fungal spor-ulation. For example, Paenibacillus validus pro-duces trisaccharides that induce hyphal elongationand sporulation in the arbuscular mycorrhizalfungus Glomus intraradices (Hildebrandt et al.,2006). Similarly, the fungal plant pathogen Rhizopusmicrosporus sporulates only when it is infected withits endosymbiotic bacterial partner, Burkholderiarhizoxinica (Lackner et al., 2011a).
Other research has shown that bacterial volatileorganic compounds and diffusible small moleculescan induce developmental shifts in fungi aside fromsporulation. For example, reciprocal volatile signal-ing between R. solanacearum and the soil fungusAspergillus flavus represses A. flavus sporulationand inhibits production of R. solanacearum viru-lence factors (Spraker et al., 2014). Fengycin A, alipopeptide produced by Bacillus subtilis, causesdiverse fungal taxa to form chlamydospores, thick-walled spores associated with fungal survival inharsh environmental conditions (Li et al., 2012). Inaddition, phenazine redox metabolites produced byPseudomonas aeruginosa trigger Aspergillus spp. tosporulate along a diffusion gradient (Zheng et al.,2015). These developmental shifts may contribute tomicrobial niche securement in the soil by triggeringhard-wired defense or survival mechanisms, how-ever, these ecological mechanisms are largelyunexplored.
Here we characterize a small molecule-mediatedinteraction between the common soil-borne bacterialpathogen R. solanacearum and a taxonomic varietyof plant- and soil-associated fungi that results infungal chlamydospore development. Using matrix-assisted laser desorption/ionization (MALDI) ima-ging mass spectrometry (MALDI-IMS) and tandemmass spectrometry (MS/MS) technologies, we iden-tified a novel diffusible R. solanacearum lipopep-tide, here named ralsolamycin. We observed adevelopmental shift in all fungi ranging fromchlamydospore formation to growth inhibition alongan increasing gradient of ralsolamycin. InactivatingrmyA, a gene that encodes a hybrid non-ribosomalpeptide synthetase-polyketide synthase abolishedralsolamycin production. Further, the rmyA mutantdid not induce chlamydospore formation. Remark-ably, we also demonstrate that R. solanacearuminvades the chlamydospores, a previously unde-scribed niche that may contribute to the notedenvironmental persistence of this devastating plantpathogen.
Materials and methods
Media and growth conditions and cocultureexperimentsAll strains used in this study are listed inSupplementary Table S1. R. solanacearum strainswere routinely grown at 30 °C on casamino acids-peptone-glucose (CPG) agar (Hendrick and Sequeira,1984), and mucoid colonies were selected forsubsequent experiments. Liquid bacterial cultureswere grown overnight in CPG at 30 °C and 180 r.p.m.When appropriate, the antibiotic gentamycin(15mg l–1) was added to the media. Overnight liquidcultures of R. solanacearum were pelleted bycentrifugation, washed twice in equal volumes ofsterilized, double-distilled water, quantified usingOD600 values and adjusted to ~ 2×108 cells ml–1.Then 5 μl (1 × 106 cells) of bacterial suspension wasspotted at each point.
Aspergillus spp. were routinely grown at 30 °C onglucose minimal medium (Shimizu and Keller, 2001),and conidia were harvested by applying a 0.01%Tween 80 solution to the plate followed by agitationwith a plastic cell spreader. Conidial suspensionswere quantified using a hemocytometer and dilutedin sterile water to 2×105 sporesml–1. Then 5 μlcontaining 1×103 spores was applied to a spot 2 cmaway from the R. solanacearum spot on CPG plates.The droplets were allowed to dry in a NUAIREbiological safety cabinet (NU425-400), then plateswere wrapped with parafilm once and incubated atroom temperature. Owing to variable sporulationhabits, all other fungi were routinely grown on potatodextrose agar and allowed to grow at room tempera-ture. For assays, mycelial plugs were removed fromactively growing cultures using the wide end of a 1mlpipette tip and transferred to the assay plate.
For coculture experiments, R. solanacearum andfungi were plated as described above and incubatedfor 14 days at room temperature. Chlamydosporeformation for all fungi was assessed as the fungalcolonies made contact with the R. solanacearumcultures. As the colonies expanded and grew into oneanother, sections of the interaction zone were excisedfor microscopic evaluation. For IMS, experimentationcolonies were grown for 3 days at room temperaturebefore being excised and processed. For assessment ofendofungal bacteria, green fluorescent protein (GFP)strains of R. solanacearum were similarly coculturedwith A. flavus, but the colonies were inoculated indirect contact to allow for physical interactions.
Microscopy of chlamydosporesFungal mycelia were harvested from coculture platesby cutting 5mm×5mm sections from the interactionzone between the two cultures and examined forchlamydospore formation. Cell walls were stainedwith calcofluor white at a final concentration of1mgml–1 for 1min before microscopy. DAPI (4,6-diamidino-2-phenylindole) staining of nuclei was
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carried out by submerging these sections in 1mgml–1
DAPI solution for 30min followed by a wash in 0.1 M
phosphate-buffered saline pH 7.0. Lipid bodies werestained by submerging sections in 10 μgml–1 NileRed, followed by a wash with 0.1 M phosphate-buffered saline. Stained samples were wet mountedand imaged on a Zeiss Axio Imager A10 microscope(Carl Zeiss, Oberkochen, Germany) equipped with aZeiss EC Plan- NEOFLUAR 40× /1.3 Oil DIC/∞/0.17and Zeiss EC Plan- NEOFLUAR 100× /1.3 Oil DIC/∞/0.17 objective and a series 120 X-Cite light source(EXFO). When different filters could be used,samples were stained with multiple stains to providecomparable images for the different features. Imageswere collected with AxioVision Release 4.7 software(Carl Zeiss).
For counts of chlamydospores of all fungi, inter-action zones from coculture with GMI1000 and thermyA mutant were collected and stained withcalcofluor as described above. Three separate imagesof hyphae collected from these interaction zone werecaptured and chlamydospores per high-poweredfield were enumerated manually using the cell-counter plug-in of the Fiji software package(Schindelin et al., 2012).
Time-lapse microscopy experiments were carriedout on a Nikon Eclipse Ti inverted fluorescentmicroscope (Nikon Instruments, Melville, NY, USA)with a PlanFluor 10×objective attached to a Nikon DS-Qi1Mc CCD camera (Nikon Instruments). Four filtersfor four different excitation/emission wavelengthswere available: ex-350 nm/em-400 nm; ex-492 nm/em-517 nm; ex-572 nm/em-600 nm; ex-647 nm/em-700 nm. Images were collected with NIS-ElementsAR version 4.30.01 software (Nikon Instruments).
Confocal laser-scanning microscopy experimentswere prepared as above but performed on a Zeiss 510Meta microscope (Carl Zeiss). To verify endohyphallocalization of bacteria, 0.5 μm Z-sections were takenacross multiple planes of view. An argon ion laserwas used to excite GFP-labeled cells and linearunmixing calculations were carried out in the ZeissLSM software to reduce fungal autofluorescence.A helium laser was used to excite the FM4-64-labeled membranes. All confocal images were pro-cessed in the open source Fiji software package andall overlays of multiple fluorescence channels are ofsingle confocal Z-planes.
Plasmid construction and genetic manipulationThe primers and plasmids used in this work are listedin Supplementary Table S2. PCR amplification wascarried out on a C1000 Thermal Cycler from Bio-Rad(Hercules, CA, USA). For creation of the rmyA-targeted disruption cassette, flanking regions of thetargeted promoter and fatty acyl-AMP ligase (FAAL)domain were amplified from GMI1000 genomic DNAusing primers RSp0641_5′F and RSp0641_5′R for theupstream sequence and RSp0641_3′F andRSp0641_3′R for the downstream sequence, each
amplifying ~1-kb fragments. Both RSp0641_5′R andRSp0641_3′F were designed with overlappingsequences to the 0.8-kb gentamycin resistance cas-sette, Gmr, which was amplified from the plasmidpUCgm using the primer set, GentR_F and GentR_R.These PCR products were purified with a QIAquickgel extraction kit (Qiagen, Valencia, CA, USA) andquantified before being fused using standard doublejoint PCR protocols (Yu et al., 2004). The fusionproduct was blunt-end ligated into the Zero BluntPCR cloning kit (Invitrogen, Carlsbad, CA, USA) tocreate pJES12.3. R. solanacearum strain GMI1000 wastransformed with pJES12.3 by electroporation andplated on gentamycin media to select for appropriatetransformants. Colony PCR using RSp0641_5′F andRSp0641_3′R were used to screen for loss of thetargeted rmyA promoter and FAAL domain. All PCRsteps were carried out with Pfu Ultra II DNAPolymerases (Agilent, Santa Clara, CA, USA).
The GMI1000 strain constitutively expressing GFPwas made by using natural transformation to movethe Tn5::gfp (Liu et al., 2001) insertion from genomicDNA of strain K60GFP (Yao and Allen, 2006) into theGMI1000 chromosome and transformants wereselected on tetracycline plates at a concentration of15mgml–1 and confirmed with fluorescence micro-scopy. Subsequently, the ΔrmyA strain constitu-tively expressing GFP was made by using naturaltransformation to move the Tn5::gfp insertion fromthe GMI1000-GFP strain. These strains were selectedfor tetracycline and gentamycin resistance, con-firmed with fluorescence microscopy, and verifiedphenotypically to not induce chlamydospores.
Imaging mass spectrometryLiquid bacterial cultures were grown overnight inCPG at 30 °C and 180 r.p.m. Overnight liquidcultures of R. solanacearum were pelleted bycentrifugation, washed 2× in equal volumes ofsterilized, double-distilled water, quantified usingOD600 values and adjusted to 2× 108 cells ml–1. Then5 μl (~1× 106 cells) of this cell suspension wasspotted at each point. GMI1000 or K60 (5 μl) werespotted next to A. flavus (1 μl) on yeast extract/maltextract ISP2 agar (10ml) in 100×25mm Petri dishes(Yang et al., 2012). All organisms were inoculated onseparate Petri dishes as controls. Samples wereincubated at room temperature for 72 h. The regionof interest and controls were excised from the agarand transferred to the same MALDI MSP 96 anchorplate (Bruker Daltonics, Billerica, MA, USA).A photograph was taken and the aerial hyphae ofA. flavus were removed gently with a cotton swabdampened with acetonitrile (Moree et al., 2012).Following the removal of the aerial hyphae, anotherphotograph as taken and Universal MALDI matrix(Sigma-Aldrich, St Louis, MO, USA) was appliedusing a 53 μm stainless steel sieve (Hogentogler &Co., INC., Columbia, MD, USA). The plate was thentransferred to an oven and desiccated at 37 °Covernight.
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Following adherence to the MALDI plate, anotherphotographic image was taken and the sample wassubjected to MALDI-TOF mass spectrometry (Micro-flex from Bruker Daltonics) for IMS acquisition. Datawere acquired in positive reflectron mode, with a500 μm laser interval in the XY and a mass range of250–2500 Da. The resulting data were analyzedusing FlexImaging software v. 3.0 (Bruker DaltonikGmbH, Bremen, Germany).
Extraction and semi-purificationR. solanacearum 24-h liquid cultures were filtersterilized using a 0.2 μm pore-size filter and appliedto actively growing hyphae of A. flavus to determineif direct bacterial contact was necessary for theformation of the chlamydospore-like structures.Within o12 h, a large number of mature hyphalcells differentiated into chlamydospores, indicatingthat direct physical contact between the microbeswas unnecessary for chlamydospore developmentsuggesting diffusible metabolite was responsible forchlamydospore development.
R. solanacearum GMI1000, K60 and A. flavus andtheir interaction zones were excised from thin agar(1 ´ 1 cm) and transferred to separate glass scintilla-tion vials. Acetonitrile (1ml) was added to the samplesand sonicated for 10min. These samples were parti-tioned against both 50:50 ACN:H2O and butanol andthe resulting mixture was centrifuged at 10 000 r.p.m.for 1min. The soluble organic fraction was removedand dried in vacuo. Samples were then re-suspendedin 100 μl of MeOH for further analysis.
Bulk extraction of the metabolite was done fromliquid culture by growing GMI1000 for 4 days in 1 literof CPG with 100 g Amberlite XAD-16 resin (Sigma-Aldrich). The resin was separated from the culturebroth via vacuum filtration and rinsed with 500mldouble-distilled H2O to remove excess salts andcellular debris. The adsorbed compounds were elutedin 1:1 MeOH/DCM and organic fractions were dried invacuo. Subsequently, these fractions were subjected tosolid phase extraction using a Waters Sep-Pak C18
cartridge (5 g) eluting with a 20% step gradient ofMeOH/H2O, ranging from 20 to 100% MeOH.
MS/MS data acquisitionSamples were directly infused into the mass spectro-meter using a Triversa nanomate-electrospray ioni-zation source (Advion Biosystems, Ithaca, NY, USA)coupled to a 6.42 T Thermo LTQ-FT-ICR massspectrometer (Thermo-Electron Corporation, SanJose, CA, USA). For nanomate, samples were dilutedin 50:50 MeOH:H2O with 0.1% formic acid and thendirectly infused using a back pressure of 0.35–0.5 psiand a spray voltage of 1.3–1.45 kV. FT-MS and iontrap MS/MS spectra were acquired using Tune Plussoftware version 1.0 and Xcalibur software version1.4 SR1 (Thermo-Electron Corporation). The instru-ment was tuned on m/z 816, the 15+ charge state ofcytochrome C.
The instrument scan cycle consisted of one 10-minsegment, during which a profile FT scan with aresolution of 25 000 was cycled with four data-dependent scans in the ion trap. MS/MS data werecollected in a data-dependent manner, during whichprofile mode FT-MS scans (150–1600m/z) cycledwith four MS/MS scans in the ion trap. The fourmost abundant peaks were fragmented (2m/z isola-tion width, a normalized collision energy of 35%,and an activation time of 30ms) and then added toan exclusion list for 600 s.
Network analysesThe resulting spectra were converted to mzXMLformat using msconvert in the ProteoWizard soft-ware package (http://proteowizard.sourceforge.net/tools.shtml). The data were then subjected tomolecular networking (Watrous et al., 2012). Thedata were then clustered with MS-Cluster with aparent mass tolerance of 1.0 Da and a MS/MSfragment ion tolerance of 0.3 Da to create consensusspectra. Further, consensus spectra that containedo1 spectrum were discarded. A network was thencreated where edges were filtered to have a cosinescore above 0.65 and 46 matched peaks. Furtheredges between two nodes were kept in the network ifand only if each of the nodes appeared in eachother's respective top 10 most similar nodes.
Phylogenetic analysis of bacteriaBacterial 16S DNA sequences were obtained fromGenBank and aligned using Muscle in the MEGA6software package (Tamura et al., 2013) using defaultparameters. The evolutionary history was inferredusing the maximum parsimony method. Tree 1 out of2 most parsimonious trees (length = 1648) is shown.The consistency index is (0.480386), the retentionindex is (0.730577) and the composite index is0.372381 (0.350959) for all sites and parsimony-informative sites (in parentheses). The percentage ofreplicate trees in which the associated taxa clusteredtogether in the bootstrap test (1000 replicates) areshown next to the branches (Felsenstein, 1985). Themaximum parsimony tree was obtained using theSubtree–Pruning–Regrafting algorithm (Nei andKumar, 2000) with search level 1 in which the initialtrees were obtained by the random addition ofsequences (10 replicates). The analysis involved 29nucleotide sequences. All positions containing gapsand missing data were eliminated. There were a totalof 1211 positions in the final data set.
ResultsA diffusible compound produced by R. solanacearuminduces chlamydospore development across diversefungal taxaR. solanacearum inhabits a variety of soils andinfects over 200 plant hosts. Soils and plants are both
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associated with many different fungal species, so wehypothesized that R. solanacearum utilizes chemicalsignals to mediate interactions with a diversity offungi. Using the characterized and sequencedR. solanacearum strain GMI1000, we examined theinteraction of cocultures of the bacterium and 34species of fungi: 30 Ascomycota species, 1 Basidio-mycota species (Rhizoctonia solani) and 3 Zygomy-cota species (Phycomyces blakesleeanus, Mucorbacilliformis and M. hiemalis). We found that adistinct zone of fungal inhibition formed between 2and 14 days of coculture. In addition, all fungi
formed distinct hyphal swellings near (~1.5 cm) theR. solanacearum colony, referred to hereafter as the‘interaction zone’ (Figure 1a).
The hyphal swellings resembled chlamydospores,thick-walled spores associated with certain fungalgenera and thought to aid in fungal survival underharsh environments (Couteaudier and Alabouvette,1990b; Li et al., 2012). The rates of chlamydosporeformation varied considerably between species, butall of the fungi tested formed chlamydospores within14 days of coculture with R. solanacearum. Severalfungi only formed chlamydospores after being in
Figure 1 Examination of the interaction zone of the R. solanacearum/fungal coculture revealed fungal hypertrophy resemblingchlamydospores produced by other fungi (a) left—coculture set-up of bacteria (top) and fungi (bottom). Red box indicates the ‘interactionzone’ where many intercalated chlamydospore-like structures (right—black arrows) developed in response to coculture withR. solanacearum strain GMI1000. Control images of non-chlamydospore-inducing bacterial cocultures can bee seen in SupplementaryFigure S1. Histological analysis of chlamydospores shows that they are (b) polynucleate (small white arrows), have thickened cell walls(white arrowheads) and (c) accumulate lipid bodies. (d) Time-lapse microscopy of isolated chlamydospores placed in fresh media showsthese structures are independently viable, and they can germinate to form a new fungal colony. Complete time-lapse video can be viewedin Supplementary Video S1.
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direct contact with the R. solanacearum colony formultiple days: the necrotrophic plant pathogensBotrytis cinerea and Sclerotinia sclerotiorum, thecoprophilous fungus Sordaria fimicola, one Fusar-ium species (verticillioides) and the BasidiomyceteRhizoctonia solani.
To study the hypertrophic structures in detail, theinteraction zones of A. flavus were excised andcharacterized. We chose A. flavus because volatile-mediated interactions between A. flavus andR. solanacearum do not result in chlamydosporeformation (Spraker et al., 2014) and also because thegenus is generally not known to produce chlamy-dospores. Micromanipulation and fluorescent histo-logical microscopy confirmed that the hyphalswellings were thick walled, chlamydospores.Hoescht staining showed that most chlamydopsoresharbored multiple nuclei (Figure 1b) although somelacked nuclei. A fungal cell wall-specific stain,calcofluor white, demonstrated that chlamydosporecell walls were thicker than normal hyphae cellwalls (Figure 1b), and Nile Red staining showedchlamydospores accumulated lipid bodies(Figure 1c). Together, the histological evidencesuggested that A. flavus chlamydospores could beviable structures. To test this hypothesis, chlamy-dospores were isolated via micromanipulation,washed and placed in fresh media and monitoredfor germination. After 5 h, the chlamydosporesgerminated with multiple germ tubes (Figure 1d,Supplementary Video S1).
Endofungal lifestyle of R. solanacearumMany bacteria live as endofungal symbionts in avariety of fungal hosts. Ralstonia spp. are nestedwithin a larger clade of identified endofungalbacteria (Hoffman and Arnold, 2010; Figure 2a,Supplementary Table S3). In order to test thehypothesis that R. solanacearum was capable ofinvading fungal hyphae, a constitutive GFP-producing R. solanacearum strain was coculturedwith A. flavus for 72 h. After harvesting the resultingmycelial mat and thoroughly washing the hyphae toremove extracellular bacteria, samples were imagedwith a fluorescent microscope. Many bacterial cellsremained associated with the fungal hyphae, andbacterial aggregates were often associated withchlamydospores, many appearing to be within thechlamydospores. Confocal laser-scanning micro-scopy confirmed that a substantial number ofbacteria were localized to the intracellular space ofsome chlamydospores (Figure 2b, SupplementaryVideo S2). Bacteria were rarely found within thenon-differentiated hyphae. These data supported ourhypothesis that R. solanacearum can invade fungalhyphae with specificity to the chlamydospores.
Identification of the chlamydospore-inducingcompoundTo identify Ralstonia-specific metabolites thatinduced chlamydospore formation, we coculturedA. flavus with a phylogenetically diverse set of
Figure 2 R. solanacearum is nested among other known endofungal bacteria and can be found within fungal cells. (a) Phylogeneticanalysis of endofungal bacteria based on maximum parsimony analysis of 16S ribosomal RNA gene sequences. The percentage of replicatetrees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Branches witho70% support values were collapsed. GenBank accession numbers are listed in Supplementary Table S3. Well-known bacterial speciesnot characterized as endofungal symbionts were inserted for reference and are marked with a red asterisk (*). (b) Confocal laser-scanningmicroscopy image of GFP-labeled GMI1000 colonizing A. flavus chlamydospore. Scale bar (white) represents 10 um. Z-stack scan can beviewed in Supplementary Video S2.
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R. solanacearum strains to identify a non-chlamydospore-producing isolate. Under these con-ditions, strain GMI1000 was the only isolate toinduce substantial chlamydospore formation in A.flavus (Supplementary Figure S1). We selected thenon-chlamydospore-inducing R. solanacearumstrain, K60 for comparative metabolite studiesbecause like GMI1000, the K60 genome wassequenced and publicly available. GMI1000 andK60 interaction zones were excised at 72 h. At thistime point, chlamydospores were plentiful but thecultures were not in physical contact. The zoneswere coated with universal MALDI matrix andsubjected to MALDI-TOF IMS (Yang et al., 2012) toexplore the spatial distribution of secreted metabo-lites and to identify differential signals betweenthese isolates. In total, 22 ions were detected in thebacterial fungal interactions. We attributed produc-tion of most metabolites to the bacterium or thefungus based on their spatial distributions withrespect to the optical image and their presence inindividual cultures.
Isolate GMI1000 produced high amounts of fourions that K60 did not produce. Three of these ionsclearly diffused beyond the margins of the bacterialcolony and reached the margins of the fungal colony.These ions were different ionized forms (protonated,sodiated and potassiated species) of one compoundwith the exact mass of 1291.7142 (Figure 3a,Supplementary Figure S2). The diffusible nature ofthis compound suggested it was the chlamydospore-inducing metabolite. This compound was furthercharacterized by tandem mass spectrometry (MS/MS) coupled with molecular networking analysis.The molecular network analyses showed that thefragmentation pattern of this compound was uniqueto GMI1000 (Figure 3b, Supplementary Figure S3).
Mapping compound to gene cluster and assessingbacterial colonizationUsing tandem mass spectrometry, an automatedpeptidogenomics analysis (Kersten et al., 2011) andthe program Pep2Path (Medema et al., 2014), weidentified characteristic partial peptide fragmenta-tion patterns associated with the signal of interestand identified one fragment exhibiting the followingresidue sequence tag: Gly-Thr-Ser-Ser-Gly-Phe-Ala(Figure 3b). Although this sequence tag does notexplain the entire mass of the metabolite, it doesstrongly indicate that a non-ribosomal peptidesynthase (NRPS) was involved in the synthesis ofthis compound. Using the in silico analysis capabil-ities of Pep2Path, in combination with AntiSMASH(Medema et al., 2011), we examined the R. solana-cearum GMI1000 and K60 genomes to identifyputative secondary metabolic gene clusters where amajor criterion was to find NRPS clusters unique tothe GMI1000 genome that matched the partialsequence tag described above.
One such gene cluster contained two large genesencoding a non-ribosomal peptide synthetase-polyketide synthase hybrid (RSp0641) and a NRPS(RSp0642). Several lines of evidence suggested thisgene cluster produced the chlamydospore-inducingmetabolite: these genes were absent from the K60genome, and the domain architecture of bothRSp0641 and RSp0642 predicted a product bestmatched to the 1291.7m/z of the signal identified inthe IMS studies. In addition, the NRPS adenylationdomains were predictive of the amino-acid residuesdetected in our MS/MS studies (SupplementaryTable S4). We termed this new compound ralsola-mycin and named RSp0641 and RSp0642 ralsola-mycin synthase genes rmyA and rmyB, respectively(Figure 4a). The protein products of rmyA-rmyBappear to a large secondary metabolite megasynthasecontaining both PKS and NRPS domains (Figure 4b).
In silico analysis using a PKS-NRPS predictor(Bachmann and Ravel, 2009) and domain-specificBLAST searches, suggested that RmyA contains aninitiating FAAL domain at its N-terminus followedby a keto-synthase domain, and two acetyl ornithineaminotransferase domains coupled to luciferase-likemonooxygenases. The remainder of the rmyA geneproduct contains four canonical NRPS modulescontaining adenylation (A), peptide carrier protein(PCP) and condensation (C) domains. The predictedpeptide product of rmyA terminates with a PCPdomain. Conventional knowledge of PCP domainssuggests that the product would remain tethered toRmyA. Similar analysis of RmyB showed that itinitiates with a C domain, suggesting that RmyBfunctionally associates with the C-terminal PCPdomain of RmyA. Further, rmyB encodes five NRPSmodules, ending with a predicted thioesterase (TE)domain. We predict the TE domain cyclizes andreleases the final product. The FAAL and PKSdomains of RmyAB suggest the product is alipopeptide (a fatty acid-like moiety attached to anamino-acid chain). Supplementary Table S4 lists allpredicted domain substrates and their closest identi-fied homologs.
We disrupted the putative promoter and FAALdomain of rmyA (Liu et al., 2013). The rmyA mutanthad significantly reduced chlamydospore-inducingactivity (Po0.05) in 28 of the 34 assayed fungalcocultures (Table 1), including A. flavus (Figure 4c),and lost all ralsolamycin IMS signals (Figure 4d).Furthermore, addition of semi-purified ralsolamycinto A. flavus triggers chlamydospore formation(Supplementary Figure S4). These data show thatrmyA contributes to ralsolamycin biosynthesis andthat ralsolamycin induces fungal chlamydosporeformation.
Ralsolamycin contributes to bacterial invasionof fungal cellsAs we saw an abundance of R. solanacearum cellsinternalized in the chlamydospores but not in
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undifferentiated hyphae, we hypothesized that ral-solamycin is necessary for invasion of fungal tissues.To test this hypothesis, GFP-producing GMI1000wild-type and rmyA mutant were cocultured with A.flavus. Samples were examined with confocalmicroscopy using amphiphilic styryl dye FM4-64as a membrane counterstain. The ΔrmyA bacteriararely invaded the hyphae. In contrast, the wild-typecells were abundant in the chlamydospores(Figure 5a). These findings suggest that ralsolamycin
enhances R. solanacearum’s invasion of fungaltissues with specificity to chlamydospores.
Discussion
Fungal chlamydospore development in responseto ralsolamycinMany fungi produce chlamydospores under adverseconditions. However, because not all fungi are
Figure 3 Analysis of differential IMS data sets, MS-MS networks indicate a single lipopeptide produced by R. solanacearum strainGMI1000 is responsible for initiating chlamydospore formation. (a) IMS analysis of strains GMI1000 and K60 showing only differentialsignals from this data set. Complete data set is in Supplementary Figure S4. (b) Network analyses of microbial metabolites detected in theMS-MS studies. The network is composed of nodes representing ions associated with the microbial colonies. Nodes are connected byedges, which represent the relatedness of the fragmentation patterns of each spectrum. Nodes only associated with A. flavus colonies aregreen. Nodes only associated with only GMI1000 are dark blue. Nodes associated with the ‘interaction zone’ are labeled in pink, Nodesfound in the GMI1000 cultures and ‘interaction zone’ (A. flavus/GMI1000) are labeled purple. Nodes associated with any of the K60culture conditions or that are shared between k60 and GMI1000 are colored in light blue. Nodes common to two or more of the previousnode classes are labeled in gray. Nodes of interest from the IMS study (1–4) and those that are known products of the microbes (5–6) arehighlighted with red boxes and listed on the right of the figure. (c) MS/MS spectra of extracted metabolites shows a unique fragmentationpattern of G-T-S-S-G-F-A for the compound with a m/z of 1291(top right).
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known to produce chlamydospores, traditionaltaxonomic studies use these structures as a trait todelineate evolutionary relationships. Our histologi-cal data suggest that chlamydospores formedin response to ralsolamycin are similar to thosedescribed in the literature (van Eck, 1978).We provide the first evidence of chlamydosporesformation in many of these fungi including allof the Aspergillus species tested. Our results alsoindicate that these structures may be more commonacross the kingdom Fungi than is currentlyrecognized.
The finding that all tested Aspergillus species canproduce chlamydospores was unexpected as onlyA. parasiticus chlamydospores have been putativelydescribed (Abou-Gabal and Fagerland, 1981). Thesedata suggest a novel mode of persistence for thiswell-studied genus. Although most of the assayedfungi formed abundant chlamydospores in responseto ralsolamycin, a subset formed relatively fewchlamydospores (Table 1). We have not ruled outthat these taxa may form chlamydospores moreabundantly under other conditions. Perhaps differ-ences in plasma membrane composition or signalingcascades among these fungi temper the conservedchlamydospore formation response to ralsolamycin.Alternatively, these fungi may respond to specificcues shown to affect chlamydospore formation inother fungi including nutrients (Barran et al., 1977;Ohara and Tsuge, 2004), light (Kües et al., 1998),osmolarity (Eisman et al., 2006), pH (Regúlez et al.,1980), air (Barran et al., 1977), temperature (Oharaand Tsuge, 2004) and other interkingdom signals(Li et al., 2005; Staib and Morschhäuser, 2005;
Li et al., 2012). Regardless of quantity, the findingthat all of the fungi developed chlamydospores whenexposed to ralsolamycin supports a conserveddifferentiation process in the kingdom Fungi inresponse to this lipopeptide.
Endofungal lifestyle of R. solanacearumOur data support the hypothesis that R. solana-cearum colonizes fungal cells, and that formation ofthe chlamydospores facilitates bacterial entry.Recently, diverse fungal lineages have been shownto harbor endofungal bacteria, most of whichare Gram-negative taxa (Hoffman and Arnold,2010). Of these related genera, Burkholderia rhizox-inica and other closely related species havebeen particularly well studied for their endofungallifestyle and their role in the production of bioactivesecondary metabolites (Partida-Martinez et al., 2007;Lackner et al., 2009; Lackner et al., 2011a, b).Our data analyzing ΔrmyA and GMI1000suggest that although ralsolamycin productionis not essential for bacterial invasion of fungalhyphae, it does contribute significantly to thisprocess and/or that chlamydospores may provide amore suitable site for bacterial growth uponinvasion.
Many bacterial lipopeptides such as iturin (Maget-Dana and Peypoux, 2003), fengycin (Patel et al.,2011), lichenysin (Madslien et al., 2013) and syr-ingomycin (Hutchison et al., 1995) form pores andpermeabilize membranes. We presume thatralsolamycin-mediated membrane restructuring con-tributes to the initiation of chlamydospore formation
Figure 4 (a) Graphical representation of the predicted biosynthetic gene cluster as predicted by comparative genomics. Yellow boxindicates the region (putative promoter sequence and FAAL encoding sequence) targeted for creation of the rmyA mutant using agentamycin resistance cassette. Genes and their closest protein homologs are listed in Supplementary Table S6. (b) Predicted domainarchitecture of the hybrid PKS megasynthases RmyA and RmyB. FAAL, fatty acid acyl ligase domain; PKS, polyketides synthase domain;AT, aminotransferase domain; MO, monooxygenase domain; A, adenylation domain; TE, thioesterase domain; blue circles,phosphopantetheine attachment site; yellow circles, condensation domains. Adenylation domains are numbered for reference to theirpredicted substrates listed in Supplementary Table S4. (c) rmyA mutant does not induce fungal chlamydospores. (d) rmyA mutant doesnot produce the ralsolamycin signals (m/z of 1291 and 1313) but does produce the other GMI1000-specific compound (m/z 625).
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Table 1 Chlamydospore counts per high-power field (magnification: x200) from interactions with R. solanacearum strains GMI1000and ΔrmyA
aIndicates strains with significantly greater (Po0.05) chlamydospore counts in coculture with GMI1000 relative to strain ΔrmyA. Mean, s.d. andP-values are listed to show variations between strains.
Figure 5 Confocal laser-scanning microscopy shows that (a) the rmyA disruption does not completely inhibit bacterial invasion of fungalhyphae (arrowheads indicate GFP-expressing bacteria) but (b) much larger populations of wild-type bacteria can be found inchlamydospores. White bar indicates 10 μm.
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and may facilitate the passage of R. solanacearuminto fungal cells. R. solanacearum possesses otherfactors that could contribute to fungal endosym-bioses such as chitinolytic enzymes, known to beimportant in fungal cell wall reorganization (Adams,2004), and genes for all of the major Gram-negativeprotein secretion systems (Genin and Boucher,2004). The R. solanacearum type III secretionmachinery shows significant conservation of geneorder and primary sequence to that of B. rhizoxinica,a system that has been shown to be important formaintaining a stable endofungal symbiosis ofB. rhizoxinica with the fungus R. microsporus(Lackner et al., 2011a) and possibly a similarmechanism contributes to R. solanacearum invasionof fungal cells.
R. solanacearum is known to persist in soilsfor remarkably long periods of time relative to otherbacterial plant pathogens (Álvarez et al., 2010).The traits underlying environmental persistence arepoorly studied, but external factors such as non-hostreservoir plants, permissive soil temperaturesand high soil moisture content may contribute topersistence. Our findings suggest that R. solana-cearum colonization within chlamydospores is anovel persistence mechanism for bacterial survivalin the absence of host plants. The large numbersof bacteria within colonized chlamydospores indi-cates that they multiply within chlamydospores,which may contribute to increasing inoculum loadonce plants are introduced. The chlamydosporescontain substantial amounts of lipid droplets, whichcould contribute to bacterial replication in thesespecialized structures. Currently, little is knownregarding nutritional interactions between bacter-ial/fungal symbioses (Burlinson et al., 2011)and efforts toward elucidating the nutritional inter-actions and survival outcomes of the R. solana-cearum/chlamydospore complex are currentlyunderway in our lab.
As homologs of the ralsolamycin biosyntheticcluster are present in a subset of the R. solanacearumstrains sequenced to date, it is intriguing to considerthat the invasion of chlamydospores may be impor-tant to these strains. We did not find any phyloge-netic or geographic trends that suggest thatralsolamycin biosynthesis is restricted to a singlephylotype, geographic locale or host plant(Supplementary Table S5); however, as more isolatesare sequenced a pattern of occurrence may beuncovered. In addition, CMR15 homologs of RmyAand RmyB showed 94% and 93% identity althoughthis strain did not induce chlamydospore formation.These data suggest that either this cluster makes adifferent metabolite; that the genes do not producefunctional proteins; or that the cluster may bedifferentially regulated under the conditions testedhere. Continued research into these strain differ-ences as well as increasing genomic information willhelp elucidate the impact of ralsolamycin biosynth-esis on R. solanacearum biology.
Ralsolamycin identification, characterization andbiosynthetic genesThe combination of spatial data from our IMSexperiments and the MS/MS network analysisindicates a single compound, ralsolamycin, is largelyresponsible for the bacterial induction of fungalchlamydospore formation. Multiple in silico ana-lyses tools indicate that ralsolamycin is produced bythe megasynthase enzyme encoded by the genesrmyA-rmyB. These two backbone genes, rmyA(a PKS-NRPS hybrid) and rmyB (a NRPS), are thetwo largest ORFs in the GMI1000 genome, approxi-mately 20.7 and 17.9 kb, respectively. The bipartitegenomes of strains GMI1000 and K60 were analyzedvia AntiSMASH (Medema et al., 2011) to look forcanonical backbone secondary metabolite genes.This analysis identified 19 putative secondary meta-bolic clusters in GMI1000. Of these, only three werepredicted to contain putative NRPSs: themicacocidin-producing RSc1806-1811 (Kreutzeret al., 2011), the ralfuranone-producing RSp1419-1434 (Wackler et al., 2011) and the ralsolamycin-producing RSp0638-0642 cluster characterized here.Identification of the 1290 Da metabolite by IMS asthe key metabolite was supported by the Pep2Pathprogram, which can link NRPS-products to theirbiosynthetic genes based on predictions of adenyla-tion substrate recognition domains (Medema et al.,2014). This program mapped the partial amino-acidsequence tag G-T-S-S-G back to the hybrid non-ribosomal peptide synthetase-polyketide synthasebackbone gene RSp0641-0642, which encodes 10predicted adenylation domains in total.
The domain architecture of RmyA is very similarto that responsible for the production of theantifungal lipopeptide mycosubtilin, MycA, pro-duced by Bacillus subtilis (Duitman et al., 1999).The presence of the N-terminal FAAL domainfollowed by a keto-synthase and aminotransferasedomain is a known motif in bacterial lipopeptidebiosynthesis (Du et al., 2001). These domainscooperate to convert carboxylic acid moieties intoactivated β-amino acids, which are utilized bysubsequent NRPS modules. The remaining modulesin RmyA-B appear to have a canonical NRPSadenylation, peptide carrier, condensation domainstructure suggesting normal non-ribosomal peptidebiosynthesis beyond the initial FAAL domain. Weare currently working toward complete characteriza-tion of this compound to verify its amino-acidcomposition.
Although it appears that RmyA and RmyB con-stitute a single operon, examination of this regionsuggests that there may be up to five genes involvedin its biosynthesis and transport including rmyA,rmyB and three additional genes: RSp0638, RSp0639and RSp0640 (Figure 4a) encoding a transmembranecyclic peptide transporter, a metallo-beta-lactamaseand an MbtH-like protein, respectively (Figure 5b,Supplementary Table S6). Comparative analysis ofthe available R. solanacearum strain genomes
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showed that these five genes are located within acopper resistance cluster common to most R.solanacearum strains. Although we have not verifiedthe functions of RSp0638-40, we predict that theyparticipate in the production and transport ofralsolamycin as they are all common componentsof secondary metabolic clusters.
Conclusions
We have demonstrated that the bacterial plantpathogen R. solanacearum strain GMI1000 induceschlamydospore formation in a wide range of fungaltaxa, many of which were not known to producechlamydospores. We used IMS to rapidly identifypotential mediators of this diffusible interactionphenotype. Tandem mass spectrometry coupledwith in silico peptidogenomics tools and moleculargenetics identified a putative R. solanacearumlipopeptide, ralsolamycin and the PKS-NRPSresponsible for its production.
We provide the first evidence that R. solana-cearum can induce and invade specialized fungalcells, chlamydospores, which may have significantimplications for environmental persistence of R.solanacearum. Chlamydospores enhance persistenceof soil-borne fungi and related organisms(Couteaudier and Alabouvette, 1990a; Crone et al.,2013) and endosymbiotic mutualisms provide survi-val benefits (Nikoh et al., 2014; O’Connor et al.,2014). R. solanacearum has an unusually longsurvival time in soil, even when host plants areremoved (Álvarez et al., 2010). Our data suggestralsolamycin-induced chlamydospores contributesto bacterial invasion of fungal cells, but ralsolamycinis not the sole factor contributing to this process. Wespeculate that endofungal colonization benefits R.solanacearum survival in soil in the absence ofplant–host tissue, and R. solanacearum co-opts theaccumulated lipids in chlamydospores. Furtherresearch in our lab aims to determine if thissymbiotic phenotype impacts the survival ofR. solanacearum and if there is any impact on thefungi it interacts with.
Conflict of Interest
The authors declare no conflict of interest.
AcknowledgementsThis research was funded by an NSF Graduate ResearchFellowship under grant no. DGE-1256259 to JES and inpart by National Science Foundation Grant EmergingFrontiers in Research and Innovation 1136903 to NPK.This work was supported in part by GM097509 (PCD). PCDfurther acknowledges Bruker and NIH grantGMS10RR029121 for the support of the shared instrumen-tation infrastructure that enabled this work. LMS issupported by National Institutes of Health IRACDA K12GM068524 grant award. TML is funded by NIH National
Research Service Award T32 GM07215 and by Agricultureand Food Research Initiative Competitive grant # 2015-67011-22799 from the USDA National Institute of Foodand Agriculture. We appreciate GFP-labeled GMI1000from Tuan Tran and critical feedback from Dr CaitilynAllen, both of the UW-Madison Department of PlantPathology. The content is solely the responsibility of theauthors and does not necessarily represent the officialviews of the National Science Foundation. Confocal laser-scanning microscopy work was done at the UW-MadisonPlant Imaging Center with the help of Sara Swanson.
Author contributions
The majority of the experiments were designed andexecuted by JES under the guidance of NPK. The IMSand chemical characterization of ralsolamycin wasdone by LMS under the guidance of PCD. R.solanacearum transformations were done by JESand TML.
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