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Arbuscular mycorrhizal fungi and bacteria: how to construct prokaryotic DNA-free genomic libraries from the Glomales
FEMS Microbiology Letters 170 (1999) 425^430
Arbuscular mycorrhizal fungi and bacteria: how to construct prokaryotic DNA-free genomic libraries from the Glomales M. Hosny b
a;b
, D. van Tuinen a , F. Jacquin a , P. Fuëller b , B. Zhao V. Gianinazzi-Pearson a , P. Franken b; *
1;a
,
a Laboratoire de Phytoparasitologie, INRA-CNRS, CMSE/INRA, BV 1540, 21034 Dijon Ceèdex, France Max-Planck-Institut fuër terrestrische Mikrobiologie and Laboratorium fuër Mikrobiologie, Philipps-Universitaët, Karl-von-Frisch-StraMe, 35043 Marburg, Germany
Received 24 November 1998; accepted 1 December 1998
Abstract Spores of various arbuscular mycorrhizal fungal isolates were analyzed for DNA of prokaryotic origin by amplification of the 16S rRNA. This shows that the presence of bacteria is not restricted to certain taxa within the Glomales, but distributed over all genera. Further experiments revealed, however, that, although single Glomus mosseae spores did not contain bacteria, samples of a number of spores were still contaminated with prokaryotes. In order to obtain genomic libraries from two arbuscular mycorrhizal fungi nearly free of clones of prokaryotic sequences, DNA extracted from spores was purified on CsCl gradients and used for library construction. Polymerase chain reaction analysis with primers for rRNA genes showed that the libraries contained if at all, only very low amounts of clones originated from bacteria. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Arbuscular mycorrhizal fungi; Bacteria-like organism ; Gigaspora rosea; Glomus mosseae; Scutellospora castanea; Genomic library
1. Introduction Arbuscular mycorrhizal (AM) fungi of the order Glomales (Zygomycota) form a mutualistic symbiosis with the roots of most land plants [1,2]. In their natural environment, however, they interact not only
* Corresponding author. Tel.: +49 (6421) 178300; Fax: +49 (6421) 178309; E-mail: [email protected] 1 Present address: College of Life Science and Technology, Huazhong Agricultural University, Wuahn, 430070 Hubei, China.
with plant roots, but are also colonized by organisms belonging to the soil micro£ora [3,4]. Contamination by other eukaryotes can be avoided by hand-picking of clean, healthy-looking spores under the binocular and surface sterilization, but this is more di¤cult for bacteria. Moreover, bacteria-like organisms (BLOs) were reported over 25 years ago to be present inside the spores of AM fungi [5] and the genus Burkholderia is now known to be an obligate endophyte which is present in high numbers during all life stages of Gigaspora margarita [6,7]. Extracts from even pure looking fungal biomass can, therefore, be accompanied by and probably contain substantial amounts of material of prokaryotic origin. For this
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 7 7 - 1
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reason, it is not surprising that van Buuren et al. [8] found about one-third of clones in a genomic library of G. margarita to be of prokaryotic origin and that a genomic clone from a library previously obtained for Glomus mosseae [9] contained sequences of a bacterial operon (Buëtehorn and Franken, unpublished). This raises obvious problems for screening such libraries to isolate fungal genes. Here we present spore analyses of di¡erent AM fungi for BLOs and in particular Burkholderia, and describe a procedure for eliminating bacterial and enriching fungal DNA during the establishment of genomic libraries of AM fungi.
2. Materials and methods 2.1. Fungal material Sixteen fungal isolates were obtained from the European Banque of Glomales (BEG) and one from the Laboratoire de Phytoparasitologie (LPA) collection. Spores were isolated from pot cultures of onion (Allium cepa L.) or clover (Trifolium repens L.) plants by wet-sieving and Percoll gradient [10], except for Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG12). For this isolate, four di¡erent plants (Rubus idaeus L., Lotus corniculatus L., Allium cepa L. and Allium porrum L.) were used and sievings of fungal material were lacerated in a Waring blender for 1 min at low speed, to release the chlamydospores from the sporocarps before loading the Percoll gradient. Spores were controlled under a binocular microscope for their purity and collected with forceps into Eppendorf tubes (one to three spores per tube with PCR bu¡er for PCR analysis or, 1000 spores per tube with sterile water for genomic library construction). 2.2. DNA puri¢cation and library construction Spores were washed ¢ve times in TE bu¡er and incubated overnight at 37³C with lysozyme in TE (1 mg ml31 ). Spores were washed again in TE bu¡er and DNA was extracted following the method of Zeèzeè et al. [11]. After removing proteins with 1 vol. phenol^chloroform and DNA precipitation in 2.5 M ammonium acetate and 1 vol. isopropanol, DNA
puri¢cation was carried out on a CsCl gradient (1.7 density) [12]. DNA was recovered under UV light, precipitated again with isopropanol and quanti¢ed by spectrophotometry at 260 nm. Five hundred nanograms of BglII partially digested nuclear DNA of G. mosseae were dephosphorylated with calf intestine phosphatase (Gibco-BRL) and added to 1 Wg lambdaDASH II vector arms (Stratagene) pre-digested with BamHI and XhoI. DNA of Scutellospora castanea Walker (BEG 1) was digested with BglII/ BamHI and cloned into the vector lambdaEMBL3 (Appligene), while for Gigaspora rosea Nicolson and Schenck (BEG 9) the vector lambdaZAPII (Stratagene) was used as described before [9]. Vector arms and genomic DNA were incubated in 5 Wl volume overnight at 4³C in the presence of 3 units T4 DNA ligase (Stratagene). After control of the ligation reaction by agarose gel electrophoresis, the DNA was incubated with packaging extract (Packaging III Plus, Stratagene). Cells of the Escherichia coli XL1blue MRA (P2) strain (Stratagene) were infected with the resulting phages and plated for titration and ampli¢cation of the library following the instructions of the supplier. 2.3. PCR PCR assays for ribosomal DNA (rDNA) were carried out using four di¡erent sets of primer, ITS1/ ITS4 [13] or LR1/NDL22 [14] for amplifying the fungal ITS and 25S rDNA, F27/1492R for the prokaryotic 16S rDNA [15] and a speci¢c primer pair for the 16S DNA of the genus Burkholderia [7]. Spores were crushed directly in the bu¡er for PCR ampli¢cation and 50 ng extracted DNA or 1 Wl of the phage libraries were used as template after complete denaturing for 10 min at 100³C. The PCR reactions were conducted in 20 Wl volume with 0.5 U of Taq polymerase (Gibco-BRL), 200 WM dNTPs and 1 WM of each primer in the bu¡er of the supplier of the enzyme. The ampli¢cations were done for 90 s at 93³C followed by 30 cycles with 30 s denaturation at 93³C, 1 min annealing at 50³C and 1 min elongation at 72³C. Ampli¢cation products were analyzed by electrophoresis on an 1% agarose gel and subsequent staining with ethidium bromide. Each PCR experiment was conducted at least three times giving identical results.
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427
3. Results and discussion One to three spores of each of the 17 tested fungi (Table 1) were crushed, and PCR was carried out using a primer pair speci¢c for Burkholderia 16S rDNA, or universal primers for the small subunit (SSU) of the prokaryotic rDNA cluster or the eukaryotic 25S rDNA. While the eukaryotic 25S rDNA was ampli¢ed in all cases, the prokaryotic primers gave ampli¢cation products only for ten isolates (Table 1). Amongst those, the SSU of the genus Burkholderia was only detected in S. castanea, S. gregaria and Gig. margarita. BLOs belonging to this prokaryotic genus have been identi¢ed before in the latter species [7]. The remaining seven isolates seemed to contain other bacteria. The pattern of distribution of bacteria did not follow the taxonomy of the Glomales. In each genus, isolates were present which contained or did not contain prokaryotic DNA. This DNA might be derived from BLOs present in the cytoplasm of the spores or from bacteria which colonize the spore wall. The function of these prokaryotes is not clear, but several examples are reported where certain bacterial strains enhance presymbiotic AM fungal development or colonization of roots [16^19]. To ¢nd out if these bacteria are the same as those closely linked to the AM fungal spores will be an interesting future task and can Table 1 Presence (+) or absence (3) of prokaryotic DNA in spores of glomalean fungi Species
Isolate
Burkholderia
16S
Scutellospora castanea Gigaspora rosea Glomus geosporum Glomus mosseae Acaulospora laevis Glomus claroideum Gigaspora candida Glomus caledonium Glomus coronatum Glomus ¢stulosum Glomus ¢stulosum Acaulospora scrobiculata Gigaspora margarita Scutellospora heterogama Glomus versiforme Scutellospora gregaria Acaulospora lacunosa
BEG 1 BEG 9 BEG 11 BEG 12 BEG 13 BEG 14 BEG 17 BEG 20 BEG 22 BEG 23 BEG 31 BEG 33 BEG 34 BEG 35 BEG 47 LPA 48 BEG 78
+ 3 3 3 3 3 3 3 3 3 3 3 + 3 3 + 3
+ 3 + 3 + 3 + 3 + + 3 3 + + 3 + +
Fig. 1. PCR ampli¢cation on genomic DNA of Glomus mosseae. Total genomic DNA from spores (T) and DNA fractions after CsCl2 gradient (E and P) served as template for PCR ampli¢cations using primer pairs speci¢c for the eukaryotic ITS region (A) or the prokaryotic 16S small subunit (B), both of the ribosomal rRNA gene cluster. Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
probably help to formulate e¡ective inocula for application in plant production systems. Spores from G. mosseae were isolated from pot cultures of di¡erent host plants. Although no bacterial DNA was detected in single spores of this isolate, DNA extracts of samples with 100 spores or more gave prokaryotic SSU ampli¢cation products (Fig. 1B, lane T), probably due to bacteria attached to the surface of the spores [20]. Similar results were obtained for Gig. rosea. It was therefore decided to eliminate bacterial DNA before constructing a genomic library, using a method for enrichment of nuclear DNA. After extraction, DNA was puri¢ed on a continuous CsCl gradient and two fractions were obtained as distinct £uorescent bands in the gradient. Two micrograms of DNA was obtained from one fraction (E), using about 50 000 spores, while 0.9 Wg was extracted from the other (P). In contrast, between 0.3 and 0.4 Wg total DNA was isolated directly from 1000 spores (T). This indicates a loss of material by a factor of 6 during puri¢cation. Agarose gel electrophoresis revealed that the DNA in all sam-
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Fig. 2. PCR ampli¢cation on genomic libraries of Glomus mosseae. Primer pairs speci¢c for the eukaryotic ITS region or the prokaryotic 16S small subunit were used for ampli¢cations with phage solutions of libraries established from total spore DNA (lane 1) or from CsCl-enriched DNA as templates (lane 2). As negative control, water was used (lane 3). Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
ples was not degraded and of high molecular weight (data not shown). In order to monitor DNA enrichment, PCR was conducted on similar amounts of DNA from T, E or P fractions using primers speci¢c for the prokaryotic 16S rDNA or the eukaryotic ITS region. Ampli¢cation products were obtained with both primer pairs when total DNA was used as template (Fig. 1A,B, lane T). The E fraction gave high amounts of an ampli¢cation product with the primers speci¢c for the eukaryotic ITS region (Fig. 1A, lane E) while the prokaryotic 16S rDNA could not be detected (Fig. 1B, lane E). The opposite was obtained for the P fraction from the CsCl gradient (Fig. 1A,B, lane P), where signal intensity was high for the 16S rDNA and not detectable with eukaryotic primers.
Fig. 3. PCR analysis of Scutellospora castanea. PCR ampli¢cations were carried out on genomic DNA from spores (lanes 2^4) or the library established from CsCl gradient-enriched DNA (lane 5 and 6) using primer pairs speci¢c for Burkholderia (lane 2), the prokaryotic 16S rDNA (lanes 3 and 5) or the eukaryotic 25S region (lanes 4 and 6). As negative control, water was used (lane 1). Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
These results clearly indicate enrichment of eukaryotic DNA and elimination of prokaryotic DNA from the E fraction, which was consequently used to establish a fungal genomic library. Results from the P fraction show also that this protocol can be used to enrich prokaryotic DNA from AM fungal spores which may be useful for a molecular analysis of these highly specialized organisms. The enriched nuclear DNA from G. mosseae spores was partially digested and cloned into the vector lambda DASH II. A total number of 100 000 clones were obtained and the library was ampli¢ed for further use (Table 2). In order to estimate the degree of contamination of the library with clones of prokaryotic origin, PCR was conducted directly on the library using the same primer pairs as above for the two DNA fractions. For compar-
Table 2 Genomic libraries of AM fungi Species
Isolate
Lambda vector
Number of clones
Average size of inserts
Times genome size
Scutellospora castanea Gigaspora rosea Glomus mosseae
BEG 1 BEG 9 BEG 12
EMBL ZAPII DASH
400 000 350 000 100 000
13 kb 4 kb 13 kb
6 2.2 6.7
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ison, the genomic library of G. mosseae which was previously established directly from spore DNA [9] was included in this experiment. The results in Fig. 2 show that in both cases, the eukaryotic primer pair gave a PCR fragment for the ITS region of similar intensity. In contrast, the PCR fragment corresponding to prokaryotic 16S rDNA was only clearly visible with phages from the library derived from spore DNA (Fig. 2, lane 1). The average size of the inserts from 20 randomly picked clones of the enriched library was around 13 kb (Table 2) which results in an amount of 1.3U108 nt or 7 pg of fungal DNA distributed over the 100 000 independent clones. Based on calculation of the genome size of AM fungi belonging to the genus Glomus [21], the library covers about three times the genome of G. mosseae and is therefore representative. A single copy gene has already been cloned successfully (Requena et al., unpublished). This protocol has also been applied to S. castanea, the spores of which contain BLO-like organisms including Burkholderia, and Gig. rosea. About 10 000 spores, isolated by wet-sieving and Percoll gradient separation, were used for each fungus. Nuclear DNA was extracted, enriched by CsCl gradient separation and cloned into lambda vectors (Table 2). Fig. 3 illustrates the e¤ciency of the protocol for S. castanea. Whereas bacterial (lane 3), and more speci¢cally Burkholderia (lane 2), DNA could be detected in spore extracts, neither (lane 5) were ampli¢ed from the genomic library. In conclusion, the presented protocol circumvents the problem of contamination of AM fungal genomic libraries by DNA from endophytic or other bacteria associated with spores. This method for genomic cloning can surely be recommended for di¡erent AM fungi than those presented here. It may also be useful for obtaining libraries of BLOs in order to study the function of these prokaryotes in glomalean spores. In addition, the protocol could be extended to genomic library construction in other situations where a prokaryotic and an eukaryotic organism live in close association.
Acknowledgments This work was supported by the DFG (SFB 395)
429
and the BEG-net EU Concerted Action (Contract B104-CT97-2225). Information from the BEG can be obtained at the internet address: http:// wwwbio.ukc.ac.uk/beg/
References [1] Morton, J.B. and Benny, G.L. (1990) Revised classi¢cation of arbuscular mycorrhizal fungi (Zygomycetes) : a new order, Glomales, two new suborders, Glomineae and Gigasporineae, and two new families, Acaulosporaceae and Gigasporaceae, with an emendation of Glomaceae. Mycotaxon 37, 471^ 491. [2] Newman, E.I. and Reddell, P. (1987) The distribution of mycorrhizas among families of vascular plants. New Phytol. 106, 745^751. [3] Lee, P.-J. and Koske, R.E. (1994) Gigaspora gigantea: parasitism of spores by fungi and actinomycetes. Mycol. Res. 98, 458^466. [4] Perotto, R. and Bonfante, P. (1997) Bacterial associations with mycorrhizal fungi: close and distant friends in the rhizosphere. Trends Microbiol. 5, 496^501. [5] Mosse, B. (1970) Honey-coloured, sessile Endogone spores: II. Changes in ¢ne structure during spore development. Arch. Microbiol. 74, 129^145. [6] Scannerini, S. and Bonfante, P. (1991) Bacteria and bacterialike objects in endomycorrhizal fungi. In: Symbiosis as a Source of Evolutionary Innovation (Margulis, L. and Fester, R., Eds.), pp. 273^278. MIT Press, Cambridge, MA. [7] Bianciotto, V., Bandi, C., Minerdi, D., Sironi, M., Tichy, H.V. and Bonfante, P. (1996) An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria. Appl. Environ. Microbiol. 62, 3005^3010. [8] Van Buuren, M., Lanfranco, L., Longato, S., Harrison, M. and Bonfante, P. (1997) Construction and characterisation of genomic libraries from endomycorrhizal fungi. In: Conference Proceedings of the COST-Action 8.21: Gene expression in arbuscular mycorrhizas. Torino. [9] Franken, P. and Gianinazzi-Pearson, V. (1996) Phage cloning of ribosomal RNA genes from the arbuscular mycorrhizal fungi Glomus mosseae and Scutellospora castanea. Mycorrhiza 6, 167^173. [10] Hosny, M., Dulieu, H. and Gianinazzi-Pearson, V. (1996) A simple and rapid method for collecting Glomales spores from soil. In: Mycorrhizas in Integrated Systems. From Genes to Plant Development (Azcon-Aguilar, C. and Barea, J.M., Eds.), pp. 541^542. European Commission, EUR 16 728, Luxembourg. [11] Zeèzeè, A., Dulieu, H. and Gianinazzi-Pearson, V. (1994) DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus, Scutellospora castanea. Mycorrhiza 4, 251^254. [12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 1888 pp. Cold Spring Harbor Press, Cold Spring Harbor, NY.
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[13] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In : PCR Protocols (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., Eds.), pp. 315^322. Academic press, New York. [14] Van Tuinen, D., Jaquot, E., Zhao, B., Gollotte, A. and Gianinazzi-Pearson, V. (1998) Characterization of root colonization pro¢les by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR. Mol. Ecol. 7, 103^111. [15] Lane, D.J. (1991) 16S/23S rRNA sequencing. In : Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M., Eds.), pp. 115^147. John Wiley and Sons, New York. [16] Azcon-Aguilar, C., Diaz-Rodriguez, R.M. and Barea, J.M. (1986) E¡ect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Trans. Br. Mycol. Soc. 21, 337^340.
[17] Von Alten, H., Lindemann, A. and Schoënbeck, F. (1993) Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria. Mycorrhiza 2, 167^173. [18] Gryndler, M., Vejsadova, H., Vosaètka, M. and Catska, V. (1995) In£uence of bacteria on vesicular-arbuscular mycorrhizal infection of maize. Folia Microbiol. 40, 95^99. [19] Requena, N., Jiminez, I., Toro, M. and Barea, J.M. (1997) Interactions between plant-growth-promoting rhizobacteria (PGPR), arbuscular mycorrhizal fungi and Rhizobium spp. in the rhizosphere of Anthyllis cytisoides, a model legume for revegetation in mediterranean semi-arid ecosystems. New Phytol. 136, 667^677. [20] Filippi, C., Bagnoli, G., Citernesi, A.S. and Giovannetti, M. (1998) Ultrastructural spatial distribution of bacteria associated with sporocarps of Glomus mosseae. Symbiosis 24, 1^12. [21] Hosny, M., Gianinazzi-Pearson, V. and Dulieu, H. (1998) Nuclear DNA content of 11 fungal species of the Glomales. Genome 41, 422^428.
FEMSLE 8571 13-1-99
Arbuscular mycorrhizal fungi and bacteria: how to construct prokaryotic DNA-free genomic libraries from the Glomales M. Hosny b
a;b
, D. van Tuinen a , F. Jacquin a , P. Fuëller b , B. Zhao V. Gianinazzi-Pearson a , P. Franken b; *
1;a
,
a Laboratoire de Phytoparasitologie, INRA-CNRS, CMSE/INRA, BV 1540, 21034 Dijon Ceèdex, France Max-Planck-Institut fuër terrestrische Mikrobiologie and Laboratorium fuër Mikrobiologie, Philipps-Universitaët, Karl-von-Frisch-StraMe, 35043 Marburg, Germany
Received 24 November 1998; accepted 1 December 1998
Abstract Spores of various arbuscular mycorrhizal fungal isolates were analyzed for DNA of prokaryotic origin by amplification of the 16S rRNA. This shows that the presence of bacteria is not restricted to certain taxa within the Glomales, but distributed over all genera. Further experiments revealed, however, that, although single Glomus mosseae spores did not contain bacteria, samples of a number of spores were still contaminated with prokaryotes. In order to obtain genomic libraries from two arbuscular mycorrhizal fungi nearly free of clones of prokaryotic sequences, DNA extracted from spores was purified on CsCl gradients and used for library construction. Polymerase chain reaction analysis with primers for rRNA genes showed that the libraries contained if at all, only very low amounts of clones originated from bacteria. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Arbuscular mycorrhizal fungi; Bacteria-like organism ; Gigaspora rosea; Glomus mosseae; Scutellospora castanea; Genomic library
1. Introduction Arbuscular mycorrhizal (AM) fungi of the order Glomales (Zygomycota) form a mutualistic symbiosis with the roots of most land plants [1,2]. In their natural environment, however, they interact not only
* Corresponding author. Tel.: +49 (6421) 178300; Fax: +49 (6421) 178309; E-mail: [email protected] 1 Present address: College of Life Science and Technology, Huazhong Agricultural University, Wuahn, 430070 Hubei, China.
with plant roots, but are also colonized by organisms belonging to the soil micro£ora [3,4]. Contamination by other eukaryotes can be avoided by hand-picking of clean, healthy-looking spores under the binocular and surface sterilization, but this is more di¤cult for bacteria. Moreover, bacteria-like organisms (BLOs) were reported over 25 years ago to be present inside the spores of AM fungi [5] and the genus Burkholderia is now known to be an obligate endophyte which is present in high numbers during all life stages of Gigaspora margarita [6,7]. Extracts from even pure looking fungal biomass can, therefore, be accompanied by and probably contain substantial amounts of material of prokaryotic origin. For this
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 7 7 - 1
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M. Hosny et al. / FEMS Microbiology Letters 170 (1999) 425^430
reason, it is not surprising that van Buuren et al. [8] found about one-third of clones in a genomic library of G. margarita to be of prokaryotic origin and that a genomic clone from a library previously obtained for Glomus mosseae [9] contained sequences of a bacterial operon (Buëtehorn and Franken, unpublished). This raises obvious problems for screening such libraries to isolate fungal genes. Here we present spore analyses of di¡erent AM fungi for BLOs and in particular Burkholderia, and describe a procedure for eliminating bacterial and enriching fungal DNA during the establishment of genomic libraries of AM fungi.
2. Materials and methods 2.1. Fungal material Sixteen fungal isolates were obtained from the European Banque of Glomales (BEG) and one from the Laboratoire de Phytoparasitologie (LPA) collection. Spores were isolated from pot cultures of onion (Allium cepa L.) or clover (Trifolium repens L.) plants by wet-sieving and Percoll gradient [10], except for Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG12). For this isolate, four di¡erent plants (Rubus idaeus L., Lotus corniculatus L., Allium cepa L. and Allium porrum L.) were used and sievings of fungal material were lacerated in a Waring blender for 1 min at low speed, to release the chlamydospores from the sporocarps before loading the Percoll gradient. Spores were controlled under a binocular microscope for their purity and collected with forceps into Eppendorf tubes (one to three spores per tube with PCR bu¡er for PCR analysis or, 1000 spores per tube with sterile water for genomic library construction). 2.2. DNA puri¢cation and library construction Spores were washed ¢ve times in TE bu¡er and incubated overnight at 37³C with lysozyme in TE (1 mg ml31 ). Spores were washed again in TE bu¡er and DNA was extracted following the method of Zeèzeè et al. [11]. After removing proteins with 1 vol. phenol^chloroform and DNA precipitation in 2.5 M ammonium acetate and 1 vol. isopropanol, DNA
puri¢cation was carried out on a CsCl gradient (1.7 density) [12]. DNA was recovered under UV light, precipitated again with isopropanol and quanti¢ed by spectrophotometry at 260 nm. Five hundred nanograms of BglII partially digested nuclear DNA of G. mosseae were dephosphorylated with calf intestine phosphatase (Gibco-BRL) and added to 1 Wg lambdaDASH II vector arms (Stratagene) pre-digested with BamHI and XhoI. DNA of Scutellospora castanea Walker (BEG 1) was digested with BglII/ BamHI and cloned into the vector lambdaEMBL3 (Appligene), while for Gigaspora rosea Nicolson and Schenck (BEG 9) the vector lambdaZAPII (Stratagene) was used as described before [9]. Vector arms and genomic DNA were incubated in 5 Wl volume overnight at 4³C in the presence of 3 units T4 DNA ligase (Stratagene). After control of the ligation reaction by agarose gel electrophoresis, the DNA was incubated with packaging extract (Packaging III Plus, Stratagene). Cells of the Escherichia coli XL1blue MRA (P2) strain (Stratagene) were infected with the resulting phages and plated for titration and ampli¢cation of the library following the instructions of the supplier. 2.3. PCR PCR assays for ribosomal DNA (rDNA) were carried out using four di¡erent sets of primer, ITS1/ ITS4 [13] or LR1/NDL22 [14] for amplifying the fungal ITS and 25S rDNA, F27/1492R for the prokaryotic 16S rDNA [15] and a speci¢c primer pair for the 16S DNA of the genus Burkholderia [7]. Spores were crushed directly in the bu¡er for PCR ampli¢cation and 50 ng extracted DNA or 1 Wl of the phage libraries were used as template after complete denaturing for 10 min at 100³C. The PCR reactions were conducted in 20 Wl volume with 0.5 U of Taq polymerase (Gibco-BRL), 200 WM dNTPs and 1 WM of each primer in the bu¡er of the supplier of the enzyme. The ampli¢cations were done for 90 s at 93³C followed by 30 cycles with 30 s denaturation at 93³C, 1 min annealing at 50³C and 1 min elongation at 72³C. Ampli¢cation products were analyzed by electrophoresis on an 1% agarose gel and subsequent staining with ethidium bromide. Each PCR experiment was conducted at least three times giving identical results.
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427
3. Results and discussion One to three spores of each of the 17 tested fungi (Table 1) were crushed, and PCR was carried out using a primer pair speci¢c for Burkholderia 16S rDNA, or universal primers for the small subunit (SSU) of the prokaryotic rDNA cluster or the eukaryotic 25S rDNA. While the eukaryotic 25S rDNA was ampli¢ed in all cases, the prokaryotic primers gave ampli¢cation products only for ten isolates (Table 1). Amongst those, the SSU of the genus Burkholderia was only detected in S. castanea, S. gregaria and Gig. margarita. BLOs belonging to this prokaryotic genus have been identi¢ed before in the latter species [7]. The remaining seven isolates seemed to contain other bacteria. The pattern of distribution of bacteria did not follow the taxonomy of the Glomales. In each genus, isolates were present which contained or did not contain prokaryotic DNA. This DNA might be derived from BLOs present in the cytoplasm of the spores or from bacteria which colonize the spore wall. The function of these prokaryotes is not clear, but several examples are reported where certain bacterial strains enhance presymbiotic AM fungal development or colonization of roots [16^19]. To ¢nd out if these bacteria are the same as those closely linked to the AM fungal spores will be an interesting future task and can Table 1 Presence (+) or absence (3) of prokaryotic DNA in spores of glomalean fungi Species
Isolate
Burkholderia
16S
Scutellospora castanea Gigaspora rosea Glomus geosporum Glomus mosseae Acaulospora laevis Glomus claroideum Gigaspora candida Glomus caledonium Glomus coronatum Glomus ¢stulosum Glomus ¢stulosum Acaulospora scrobiculata Gigaspora margarita Scutellospora heterogama Glomus versiforme Scutellospora gregaria Acaulospora lacunosa
BEG 1 BEG 9 BEG 11 BEG 12 BEG 13 BEG 14 BEG 17 BEG 20 BEG 22 BEG 23 BEG 31 BEG 33 BEG 34 BEG 35 BEG 47 LPA 48 BEG 78
+ 3 3 3 3 3 3 3 3 3 3 3 + 3 3 + 3
+ 3 + 3 + 3 + 3 + + 3 3 + + 3 + +
Fig. 1. PCR ampli¢cation on genomic DNA of Glomus mosseae. Total genomic DNA from spores (T) and DNA fractions after CsCl2 gradient (E and P) served as template for PCR ampli¢cations using primer pairs speci¢c for the eukaryotic ITS region (A) or the prokaryotic 16S small subunit (B), both of the ribosomal rRNA gene cluster. Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
probably help to formulate e¡ective inocula for application in plant production systems. Spores from G. mosseae were isolated from pot cultures of di¡erent host plants. Although no bacterial DNA was detected in single spores of this isolate, DNA extracts of samples with 100 spores or more gave prokaryotic SSU ampli¢cation products (Fig. 1B, lane T), probably due to bacteria attached to the surface of the spores [20]. Similar results were obtained for Gig. rosea. It was therefore decided to eliminate bacterial DNA before constructing a genomic library, using a method for enrichment of nuclear DNA. After extraction, DNA was puri¢ed on a continuous CsCl gradient and two fractions were obtained as distinct £uorescent bands in the gradient. Two micrograms of DNA was obtained from one fraction (E), using about 50 000 spores, while 0.9 Wg was extracted from the other (P). In contrast, between 0.3 and 0.4 Wg total DNA was isolated directly from 1000 spores (T). This indicates a loss of material by a factor of 6 during puri¢cation. Agarose gel electrophoresis revealed that the DNA in all sam-
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Fig. 2. PCR ampli¢cation on genomic libraries of Glomus mosseae. Primer pairs speci¢c for the eukaryotic ITS region or the prokaryotic 16S small subunit were used for ampli¢cations with phage solutions of libraries established from total spore DNA (lane 1) or from CsCl-enriched DNA as templates (lane 2). As negative control, water was used (lane 3). Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
ples was not degraded and of high molecular weight (data not shown). In order to monitor DNA enrichment, PCR was conducted on similar amounts of DNA from T, E or P fractions using primers speci¢c for the prokaryotic 16S rDNA or the eukaryotic ITS region. Ampli¢cation products were obtained with both primer pairs when total DNA was used as template (Fig. 1A,B, lane T). The E fraction gave high amounts of an ampli¢cation product with the primers speci¢c for the eukaryotic ITS region (Fig. 1A, lane E) while the prokaryotic 16S rDNA could not be detected (Fig. 1B, lane E). The opposite was obtained for the P fraction from the CsCl gradient (Fig. 1A,B, lane P), where signal intensity was high for the 16S rDNA and not detectable with eukaryotic primers.
Fig. 3. PCR analysis of Scutellospora castanea. PCR ampli¢cations were carried out on genomic DNA from spores (lanes 2^4) or the library established from CsCl gradient-enriched DNA (lane 5 and 6) using primer pairs speci¢c for Burkholderia (lane 2), the prokaryotic 16S rDNA (lanes 3 and 5) or the eukaryotic 25S region (lanes 4 and 6). As negative control, water was used (lane 1). Dashes at the left indicate the 500-bp, the 1-kb and the 2-kb fragment of the 1-kb ladder used as size marker.
These results clearly indicate enrichment of eukaryotic DNA and elimination of prokaryotic DNA from the E fraction, which was consequently used to establish a fungal genomic library. Results from the P fraction show also that this protocol can be used to enrich prokaryotic DNA from AM fungal spores which may be useful for a molecular analysis of these highly specialized organisms. The enriched nuclear DNA from G. mosseae spores was partially digested and cloned into the vector lambda DASH II. A total number of 100 000 clones were obtained and the library was ampli¢ed for further use (Table 2). In order to estimate the degree of contamination of the library with clones of prokaryotic origin, PCR was conducted directly on the library using the same primer pairs as above for the two DNA fractions. For compar-
Table 2 Genomic libraries of AM fungi Species
Isolate
Lambda vector
Number of clones
Average size of inserts
Times genome size
Scutellospora castanea Gigaspora rosea Glomus mosseae
BEG 1 BEG 9 BEG 12
EMBL ZAPII DASH
400 000 350 000 100 000
13 kb 4 kb 13 kb
6 2.2 6.7
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ison, the genomic library of G. mosseae which was previously established directly from spore DNA [9] was included in this experiment. The results in Fig. 2 show that in both cases, the eukaryotic primer pair gave a PCR fragment for the ITS region of similar intensity. In contrast, the PCR fragment corresponding to prokaryotic 16S rDNA was only clearly visible with phages from the library derived from spore DNA (Fig. 2, lane 1). The average size of the inserts from 20 randomly picked clones of the enriched library was around 13 kb (Table 2) which results in an amount of 1.3U108 nt or 7 pg of fungal DNA distributed over the 100 000 independent clones. Based on calculation of the genome size of AM fungi belonging to the genus Glomus [21], the library covers about three times the genome of G. mosseae and is therefore representative. A single copy gene has already been cloned successfully (Requena et al., unpublished). This protocol has also been applied to S. castanea, the spores of which contain BLO-like organisms including Burkholderia, and Gig. rosea. About 10 000 spores, isolated by wet-sieving and Percoll gradient separation, were used for each fungus. Nuclear DNA was extracted, enriched by CsCl gradient separation and cloned into lambda vectors (Table 2). Fig. 3 illustrates the e¤ciency of the protocol for S. castanea. Whereas bacterial (lane 3), and more speci¢cally Burkholderia (lane 2), DNA could be detected in spore extracts, neither (lane 5) were ampli¢ed from the genomic library. In conclusion, the presented protocol circumvents the problem of contamination of AM fungal genomic libraries by DNA from endophytic or other bacteria associated with spores. This method for genomic cloning can surely be recommended for di¡erent AM fungi than those presented here. It may also be useful for obtaining libraries of BLOs in order to study the function of these prokaryotes in glomalean spores. In addition, the protocol could be extended to genomic library construction in other situations where a prokaryotic and an eukaryotic organism live in close association.
Acknowledgments This work was supported by the DFG (SFB 395)
429
and the BEG-net EU Concerted Action (Contract B104-CT97-2225). Information from the BEG can be obtained at the internet address: http:// wwwbio.ukc.ac.uk/beg/
References [1] Morton, J.B. and Benny, G.L. (1990) Revised classi¢cation of arbuscular mycorrhizal fungi (Zygomycetes) : a new order, Glomales, two new suborders, Glomineae and Gigasporineae, and two new families, Acaulosporaceae and Gigasporaceae, with an emendation of Glomaceae. Mycotaxon 37, 471^ 491. [2] Newman, E.I. and Reddell, P. (1987) The distribution of mycorrhizas among families of vascular plants. New Phytol. 106, 745^751. [3] Lee, P.-J. and Koske, R.E. (1994) Gigaspora gigantea: parasitism of spores by fungi and actinomycetes. Mycol. Res. 98, 458^466. [4] Perotto, R. and Bonfante, P. (1997) Bacterial associations with mycorrhizal fungi: close and distant friends in the rhizosphere. Trends Microbiol. 5, 496^501. [5] Mosse, B. (1970) Honey-coloured, sessile Endogone spores: II. Changes in ¢ne structure during spore development. Arch. Microbiol. 74, 129^145. [6] Scannerini, S. and Bonfante, P. (1991) Bacteria and bacterialike objects in endomycorrhizal fungi. In: Symbiosis as a Source of Evolutionary Innovation (Margulis, L. and Fester, R., Eds.), pp. 273^278. MIT Press, Cambridge, MA. [7] Bianciotto, V., Bandi, C., Minerdi, D., Sironi, M., Tichy, H.V. and Bonfante, P. (1996) An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria. Appl. Environ. Microbiol. 62, 3005^3010. [8] Van Buuren, M., Lanfranco, L., Longato, S., Harrison, M. and Bonfante, P. (1997) Construction and characterisation of genomic libraries from endomycorrhizal fungi. In: Conference Proceedings of the COST-Action 8.21: Gene expression in arbuscular mycorrhizas. Torino. [9] Franken, P. and Gianinazzi-Pearson, V. (1996) Phage cloning of ribosomal RNA genes from the arbuscular mycorrhizal fungi Glomus mosseae and Scutellospora castanea. Mycorrhiza 6, 167^173. [10] Hosny, M., Dulieu, H. and Gianinazzi-Pearson, V. (1996) A simple and rapid method for collecting Glomales spores from soil. In: Mycorrhizas in Integrated Systems. From Genes to Plant Development (Azcon-Aguilar, C. and Barea, J.M., Eds.), pp. 541^542. European Commission, EUR 16 728, Luxembourg. [11] Zeèzeè, A., Dulieu, H. and Gianinazzi-Pearson, V. (1994) DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus, Scutellospora castanea. Mycorrhiza 4, 251^254. [12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 1888 pp. Cold Spring Harbor Press, Cold Spring Harbor, NY.
FEMSLE 8571 13-1-99
430
M. Hosny et al. / FEMS Microbiology Letters 170 (1999) 425^430
[13] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In : PCR Protocols (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., Eds.), pp. 315^322. Academic press, New York. [14] Van Tuinen, D., Jaquot, E., Zhao, B., Gollotte, A. and Gianinazzi-Pearson, V. (1998) Characterization of root colonization pro¢les by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR. Mol. Ecol. 7, 103^111. [15] Lane, D.J. (1991) 16S/23S rRNA sequencing. In : Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M., Eds.), pp. 115^147. John Wiley and Sons, New York. [16] Azcon-Aguilar, C., Diaz-Rodriguez, R.M. and Barea, J.M. (1986) E¡ect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Trans. Br. Mycol. Soc. 21, 337^340.
[17] Von Alten, H., Lindemann, A. and Schoënbeck, F. (1993) Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria. Mycorrhiza 2, 167^173. [18] Gryndler, M., Vejsadova, H., Vosaètka, M. and Catska, V. (1995) In£uence of bacteria on vesicular-arbuscular mycorrhizal infection of maize. Folia Microbiol. 40, 95^99. [19] Requena, N., Jiminez, I., Toro, M. and Barea, J.M. (1997) Interactions between plant-growth-promoting rhizobacteria (PGPR), arbuscular mycorrhizal fungi and Rhizobium spp. in the rhizosphere of Anthyllis cytisoides, a model legume for revegetation in mediterranean semi-arid ecosystems. New Phytol. 136, 667^677. [20] Filippi, C., Bagnoli, G., Citernesi, A.S. and Giovannetti, M. (1998) Ultrastructural spatial distribution of bacteria associated with sporocarps of Glomus mosseae. Symbiosis 24, 1^12. [21] Hosny, M., Gianinazzi-Pearson, V. and Dulieu, H. (1998) Nuclear DNA content of 11 fungal species of the Glomales. Genome 41, 422^428.
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