ORIGINAL PAPER

Abstract A 7.4-kilobase (kb) DNA plasmid was isolatedfrom Glomerella musae isolate 927 and designatedpGML1. Exonuclease treatments indicated that pGML1was a linear plasmid with blocked 5′ termini. Cell-frac-tionation experiments combined with sequence-specificPCR amplification revealed that pGML1 resided in mito-chondria. The pGML1 plasmid hybridized to cesium chlo-ride-fractionated nuclear DNA but not to A + T-rich mito-chondrial DNA. An internal 7.0-kb section of pGML1 wascloned and did not hybridize with either nuclear or mito-chondrial DNA from G. musae. Sequence analysis re-vealed identical terminal inverted repeats (TIR) of 520 bpat the ends of the cloned 7.0-kb section of pGML1. Theoccurrence of pGML1 did not correspond with the pathog-enicity of G. musae on banana fruit. Four additional iso-lates of G. musae possessed extrachromosomal DNA frag-ments similar in size and sequence to pGML1.

Key words Mitochondria · TIR · PCR · Banana pathogen

Introduction

Linear DNA plasmids have been isolated from several fil-amentous fungal plant pathogens including Claviceps pur-purea (Tudzynski et al. 1983), Fusarium oxysporum f. sp.conglutinans (Kistler and Leong 1986), F. solani f. sp. cu-curbitae (Samac and Leong 1988), Gaeumannomycesgraminis var. tritici (Honeyman and Currier 1986), Rhi-zoctonia solani (Hashiba et al. 1984), Cochliobolus heter-ostrophus (Garber and Yoder 1984) and Tilletia spp. (Kimet al. 1990). However, no DNA plasmids have been re-ported in any species of Colletotrichum.

Most of the fungal linear plasmids have several com-mon features. They are of mitochondrial origin, have ter-minal inverted repeats (TIR), and have proteins covalentlybound to the 5′-end (Meinhardt et al. 1990). In addition,open reading frames (ORFs) have been observed in sev-eral linear plasmids which have sequences similar to viralDNA and RNA polymerases, suggesting an evolutionarylink to viral genomes (Jung et al. 1987; Oeser and Tudzyn-ski 1989; Meinhardt et al. 1990; Rohe et al. 1991; Li andNargang 1993).

Although the precise functions of most fungal linearplasmids are unknown, a small number of fungal plasmidsare involved in growth senescence and toxin production(Bertrand et al. 1986; Meinhardt et al. 1990). Conflictingreports exist regarding the involvement of fungal linearplasmids with virulence and pathogenicity. In F. oxy-sporum, the presence of certain linear plasmids is asso-ciated with host specificity (Kistler and Leong 1986; Kistler et al. 1987). Hypovirulent isolates of R. solani havebeen shown to harbor linear plasmids which were lackingin highly virulent isolates (Hashiba et al. 1984). In con-trast, hypovirulent isolates of G. graminis appeared to havelost their plasmids (Honeyman and Currier 1986).

In the present paper we report the isolation and charac-terization of the first DNA plasmid from the filamentousfungal genus Colletotrichum. This plasmid, designatedpGML1, was isolated from Glomerella musae, the causa-tive agent of anthracnose on banana.

Curr Genet (1997) 32: 152–156 © Springer-Verlag 1997

Received: 27 June 1996 / 2 April 1997

Stanley Freeman · Regina S. RedmanGeorge Grantham · Russell J. Rodriguez

Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae[Anamorph: Colletotrichum musae (Berk. & Curt.) Arx.]

ORIGINAL PAPER

S. FreemanDepartment of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

R. S. Redman · R. J. Rodriguez (½)Northwest Biological Science Center, Biological Resources Division, US Geological Survey, 6505 NE 65th, Seattle, WA 98115, USA

G. GranthamDepartment of Plant Pathology, University of California Riverside, CA 92521, USA

Communicated by H. Bertrand

Materials and methods

Fungal isolates and growth conditions. G. musae isolates were ob-tained from Dr. A. Johanson, ODNRI, Kent, England. The fungi werecultured on either liquid or solid modified Mathur’s medium (MS)(Tu 1985) as previously described (Rodriguez and Owen 1992).

Mitotic inheritance of pGML1. Serial transfer experiments were in-itiated by inoculating the center of a Petri plate containing MS me-dium with conidia and allowing mycelia to grow and conidiate. Twoweeks after inoculation, conidia were transferred from the edge ofthe colony to the center of a fresh plate. This process was repeatedevery 2 week for 2 months, followed by liquid culture and DNA ex-traction (see below).

DNA isolation, purification and manipulation. DNA was extractedfrom liquid mycelial cultures and purified as previously described(Freeman et al. 1993; Rodriguez 1993). The pGML1 plasmid wasseparated from genomic DNA on agarose gels and purified by thegene-clean method (Bio101). In addition, the plasmids were frac-tionated from genomic DNA by cesium chloride/bis-benzimide cen-trifugation (Garber and Yoder 1983). DNA-blot analysis, generationof radioactive DNA probes, and restriction-enzyme analyses wereperformed using standard protocols (Sambrook et al. 1989). ThepGML1 plasmid was restriction enzyme-digested with StuI and PstI,cloned as two fragments into pUC19, and the DNA sequenced usingstandard protocols (Sanger et al. 1977; Sambrook et al. 1989).

Cell fractionation. Mitochondria were isolated by modification ofan existing protocol (Graham 1992). Mycelia were harvested by vac-uum filtration, frozen at –80°C, ground in liquid nitrogen, and re-suspended in 20 ml of buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, and 0.5 M sucrose). Cellular debris was removed bysuccessive centrifugations at 1500 g and 3500 g for 10 min at 4°C.The supernatant was then centrifuged at 20 000 g for 10 min at 4°Cto pellet mitochondria. The pellet was re-suspended in 0.5 ml of mit-ochondrial buffer (50 mM Tris-HCl pH 7.5 and 0.5 M sucrose) andtreated with DNase 1 (2000 u/ml) and RNase (5 u/ml) for 2 h at 22°C.Prior to enzyme treatment, 500 ng of the plasmid pHA1.3 (Redmanand Rodriguez 1994) was added as an internal control to assess theextent of nuclease digestion. Following enzyme treatment, sampleswere phenol- and chloroform-extracted, the debris pelleted at20 000 g for 10 min at 4°C, and mtDNA precipitated from the super-natant (Sambrook et al. 1989).

Nuclease assays. RNase and DNase treatment of pGML1 was per-formed in buffer containing 10 mM Tris (pH 8.0), 2.5 mM MgCl2,and 2 units of either RNase or DNase. The reaction mixture for Ex-onuclease III (Exo III) contained 50 mM Tris-HCl (pH 8.0), 5 mMMgCl2, 10 mM β-mercaptoethanol, and 5 units of Exo III. The lamb-da exonuclease reaction mixture contained 10 mM Tris-HCl(pH 9.0), 2.5 mM MgCl2, and 5 units of lambda exonuclease. All nu-clease reactions contained 500 ng of genomic DNA containingpGML1, were incubated at 37°C for 60 min, and analyzed by aga-rose-gel electrophoresis (Sambrook et al. 1989).

Polymerase chain reaction (PCR) analysis. PCR (Saiki et al. 1985;Mullis and Faloona 1987) were performed in 20-µl volumes contain-ing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.2%Triton X-100, 200 µM each of dATP, dCTP, dGTP, dTTP, 0.2 unitsTaq DNA Polymerase, 500 ng of each oligonucleotide primer, and20–200 ng of fungal genomic DNA. PCR reactions consisted of in-cubation at 93°C for 2 min followed by 35 cycles of the followingtemperature regime: 93°C for 15 s, 64°C for 1.5 min, and 72°C for1.5 min with no ramp times between temperatures. PCR productswere separated in 2% agarose gels, stained with ethidium bromide,and visualized with UV light. Oligonucleotide primers were basedon sequences from pGM3 and pGM4 (p705 5′-cctttaatccgcacctt-ccgt/p706 5′-tagcttcttctgatttaatac), the hygromycin B gene (GenBank# UO9715; p420 5′-cttgagtggcgctgcagacag/p421 5′-gtctcaactccgga-gctgaca), a 16s-like rDNA gene (GenBank # M55640; p689 5′-tccc-

ttaacgaggaacaattgg/p691 5′-cattcaatcggtagtagcgac), and the pectinlyase gene (Templeton et al. 1994; p658 5′-ggaattcgtcgacagcggtgt-catcaaggg/p659 5′-ggaattcggatccagtaacgtggtcgatccagac).

Pathogenicity evaluation. Banana fruit were surface-sterilized witha 1.5% NaOCl solution for 20 min and spot-inoculated with 10 µldrops of conidia (1 × 106 conidia/ml) from the different isolates ofG. musae. The fruit were incubated in a moist chamber at 25°C inthe dark. Disease was indicated by the occurrence of brown lesionswithin 3–4 days.

Results

Identification of a DNA plasmid in G. musae

Electrophoretic analysis of intact genomic DNA from sev-eral Colletotrichum species resulted in the identificationof a number of small extrachromosomal nucleic-acid frag-ments ranging in size from 0.5 to 8 kb (data not shown).To determine the chemical basis of the extrachromosomalnucleic acids, the fragments were assessed for sensitivityto RNase and DNase. Most of the extrachromosomal nu-cleic acids were sensitive to RNase but not DNase. How-ever, the extrachromosomal nucleic acid observed in iso-late 927 of G. musae was sensitive to DNase and not RNaseindicating that the fragment was DNA. In order to deter-mine the mitotic stability of the G. musae extrachromoso-mal fragment (designated pGML1), ten separate culturesof isolate 927 were serially transferred on solid growth me-dium and then liquid cultures were grown for DNA extrac-tion. In all of the liquid cultures, pGML1 was maintainedand was therefore considered to be a mitotically stable, au-tonomously replicating plasmid.

Restriction enzyme mapping, cloning, and sequence analysis of pGML1

An initial restriction endonuclease map of pGML1 wasconstructed which revealed that a StuI site was symmetri-cally located approximately 200 base pairs from each ter-minus (Fig. 1). In addition, a PstI site was asymmetricallylocated near the center of the plasmid. The internal regionof pGML1 (approximately 7.0 kb between the StuI sites)was then cloned as two fragments into the PstI-SmaI sitesof pUC19 for further restriction enzyme and sequence anal-ysis. The cloned fragments were designated pGM3 andpGM4 and were 3.0 and 4.0 kb in size, respectively. Ad-ditional restriction-enzyme analysis indicated that the ter-minal regions of pGML1 were identical for 500 to 1000 bpand that there was no additional symmetry in the majorityof the plasmid (Fig. 1).

The termini of pGM4 and pGM3 were sequenced andfound to be terminal inverted repeats (TIRs) (gen bank ac-cession AFO13292). The inverted repeat sequence ex-tended from the StuI sites for a distance of 520 bp. Since200 bp were lost from each end of pGML1 during the clon-ing process, the TIRs of pGML1 could be as large as 720 bp.

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Cellular location, chemical composition, and physical structure of pGML1

Cell-fractionation experiments combined with sequence-specific PCR amplification were used to determine the cel-lular location of pGML1. Mitochondria were isolated bydifferential centrifugation followed by DNase treatmentand DNA extraction. In order to assess the efficiency ofDNase digestion, 500 ng of a plasmid (pHA1.3) contain-ing the hygromycin B gene was added to purified mito-chondria prior to nuclease treatment. The purity of mtDNAwas then assessed by comparing the amplification of ge-nomic DNA, purified mtDNA, and pHA1.3 with PCRprimers specific to the hygromycin B gene, the pGML1plasmid, the mitochondrial 16s ribosomal gene, and the nuclear gene pectate lyase. The expected product sizes

from these primers were 1400 bp, 520 bp, 1100 bp, and600 bp respectively. These analyses indicated that pHA1.3was completely digested by nuclease treatment (Fig. 2,lanes 2–4), both pGML1 and 16s ribosomal sequences were present in purified mtDNA and genomic DNA (Fig. 2, lanes 5–10), and the nuclear pectate lyase gene wasamplified from genomic DNA but not mtDNA (Fig. 2,lanes 11–13). These data indicated that the mtDNA wasfree of nuclear-DNA contamination and that the pGML1plasmid was located in mitochondria.

To determine if pGML1 was linear or circular, genomicDNA containing the plasmid was exposed to Exo III andlambda exonuclease which digest dsDNA from open 3′- and 5′-ends, respectively. Exo III-treatment resulted incomplete digestion of both genomic DNA and pGML1while lambda exonuclease digested genomic DNA but hadno effect on the native plasmid (Fig. 3). These data indi-cated that pGML1 was a linear plasmid with nuclease-pro-tected 5′ termini.

Species distribution of pGML1

DNA was extracted from ten different isolates of G. mu-sae, size-fractionated by agarose-gel electrophoresis, andhybridized to radiolabelled pGML1. Hybridization analy-sis indicated that five of the ten G. musae isolates con-tained extrachromosomal DNAs which were similar in sizeto pGML1 (Fig. 4). In addition, there was hybridizationbetween pGML1 and genomic DNA regardless of the pres-ence or absence of autonomous plasmid sequences.

To determine which regions of pGML1 hybridized togenomic DNA, the cloned internal region was digestedwith PstI and HindIII, the digestion fragments isolated andused as hybridization probes. None of the probes hybri-dized to genomic DNA indicating that the regions of ge-

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Fig. 1 A partial restriction-endonuclease map of pGML1 from G. musae isolate 927 with sizes indicated in kb. The terminal boxesindicate terminal inverted repeats and the letters indicate restrictionenzyme sites: h = HindIII, m = MboI, p = PstI, s = StuI, x = XbaI,and w = HpaII. The orientation and relative sizes of the clonedpGML1 fragments (pGM3 and pGM4) are shown below the restric-tion map

Fig. 2 PCR analysis of DNA obtained from cell fractions of G. mu-sae isolate 927. Lanes 2–4 represent pHA1.3 plasmid, mtDNA, andtotal genomic DNA amplified with primers specific to the hygromy-cin B gene. Lanes 5–7 represent pHA1.3 plasmid, mtDNA, and to-tal genomic DNA amplified with primers specific to pGML1. Lanes8–10 represent pHA1.3 plasmid, mtDNA, and total genomic DNAamplified with primers specific to 16s RNA. Lanes 11–13 representpHA1.3 plasmid, mtDNA, and total genomic DNA amplified withprimers specific to the nuclear gene pectate lyase. Lanes 1 and 14contain 1-kb DNA size-markers

Fig. 3 Genomic DNA from G. musae isolate 927 in the absence andpresence of lambda exonuclease and Exo III. Lane 2 genomic DNAincubated in lambda exonuclease buffer devoid of enzyme; lane 3genomic DNA incubated in lambda exonuclease buffer containingenzyme; lane 4 genomic DNA incubated in Exo III buffer contain-ing enzyme; lane 5 genomic DNA incubated in Exo III buffer de-void of enzyme. Lanes 1 and 6 contain HindIII digested lambda DNAsize-markers

nomic similarity were outside the cloned TIRs of pGM4and pGM3 (data not shown).

To determine if pGML1 was hybridizing to sequencesin mtDNA or nuclear DNA (nDNA), DNA from thepGML1-deficient isolate 3–3 (Fig. 4) was fractionated onCsCl/bis-benzimide gradients, and the fractions analyzedon agarose gels. Although no hybridization was observedbetween pGML1 and mtDNA (A + T-rich fraction), theplasmid strongly hybridized to nDNA (data not shown) ina pattern identical to that observed with genomic DNAshown in Fig. 4.

Pathogenicity

All G. musae isolates produced infection and caused equiv-alent disease on banana fruit. No significant differenceswere observed in lesion size or rate of decay of banana fruit inoculated with isolates of G. musae that contained,or were devoid of, autonomous plasmid sequences (datanot shown).

Discussion

The 7.4-kb linear DNA plasmid pGML1 of G. musae iso-late 927 hybridized to plasmids of similar size in four otherG. musae isolates (Fig. 4). In addition, pGML1 was simi-lar to a plasmid identified in isolate 1116 with respect tophysical structure, restriction-endonuclease map, andDNA-hybridization analyses (data not shown). Five of theC. musae isolates analyzed here were devoid of plasmidDNA.

All ten isolates of G. musae produced equivalent lesionson banana fruit upon inoculation. Therefore, no apparentphytopathological effects were ascribed to the presence orabsence of pGML1, or similar plasmids, in G. musae. Therole of fungal plasmids in pathogenicity has varied in dif-

ferent fungal systems. G. musae appears to be similar toC. purpurea (Gessner-Ulrich and Tudzynski 1994) and R. solani (Jabaji-Hare et al. 1994) where the presence ofplasmids has no effect on virulence.

Certain features of pGML1 were common to linearDNA plasmids from other filamentous phytopathogenicfungi. Cell-fractionation experiments, PCR analysis, andCsCl/bisbenzimide centrifugation indicated that pGML1was located in mitochondria and the plasmid sequence wasA + T rich. Terminal inverted repeats were also present inpGML1; however, the 200-bp terminal sequences were notcharacterized because the termini were not cloned. ThepGML1 TIR sequences were between 520 and 720-bp long,similar to the TIRs of F. solani f. sp. cucurbitae (Samacand Leong 1989).

It is interesting that the pGML1 plasmid hybridized tonDNA but not to mtDNA, and that the cloned internal por-tion of pGML1 did not hybridize to either nDNA ormtDNA. This is contrary to other mitochondrial linear plas-mids which often have some homology to mitochondrialDNA (Meinhardt et al. 1990; Robinson and Horgen 1996).Further studies are required to define the sequence simi-larity between pGML1 and nDNA in G. musae, and the ev-olutionary significance of that similarity.

Acknowledgements We thank Dr. Gael Kurath and Dr. Harry Mirken for a critical review of the manuscript. This research wassupported in part by grant No. 6350-1-94 from The Israeli Ministryof Science and the Arts (SF as PI), a joint NSF, DOE, USDA grant92-37310-7821 (R.J.R. as co-principal investigator), a USDA grant9401082 (R.J.R. as PI), and the Biological Resources Division of theUS Geological Survey (R.J.R. as PI).

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Fig. 4 Undigested genomic DNA from ten different isolates (lanes1–10) of G. musae hybridized to 32P-radiolabelled pGML1 (the na-tive plasmid) of isolate 927. Lane 1 isolate 1–11; 2 isolate 2–27a; 3 isolate 3–15; 4 isolate 3–3; 5 isolate 3–38; 6 isolate 3–4; 7 isolate818; 8 isolate 927; 9 isolate 1116; 10 isolate 1410. Lanes 11 and 12represent purified plasmid DNA from isolates 927 and 1116, probedwith pGML1, respectively

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