Mycorrhizal roots of the deciduous trees European beech ( Fagus sylvatica (L.)) and Sessile oak ( Quercus petraea (MattuschkaLiebl.)) and the conifers Norway spruce ( Picea abies (L.) H. Karst.) and European larch ( Larix decidua (Mill.)) associated with the ectomycorrhizal fungi matt bolete ( Xerocomus pruinatus (Fries 1835)) or bay bolete ( X. badius (Fries 1818)) were analysed with respect to the occurrence of dihydrolipoyl dehydrogenase (EC 1.8.1.4) allozymes. In root tissues of the two deciduous trees , two gene loci could be visualized after cellulose acetate electrophoresis while three loci were expressed in root tissues of the two coniferous species. The two fungal species and further ectomycorrhizal fungi expressed exclusively one dihydrolipoyl dehydrogenase gene. In Xerocomus pruinatus and X. badius , the dihydrolipoyl dehydrogenase gene consists of 1460 bp and 1370 bp, respectively, including five introns each consisting of 52 bp. Their DNA sequences correspond to 70 to 90% to other fungal dihydrolipoyl dehydrogenase genes. One monomer of the dimeric dihydrolipoyl dehydrogenase enzyme consists of 486 (X. pruinatus) or 454 (X. badius) amino acids which sum up to a molecular mass of 55 kDa (X. pruinatus), respectively 52 kDa (X. badius). The number of positively charged amino acid residues makes 79 (X. pruinatus) and 68 (X. badius) and the number of negatively charged amino acid residues was calculated to make 46 (X. pruinatus) and 48 (X. badius); isoelectric points make 9.99 (X. pruinatus) and 9.68 (X. badius). Calculated three dimensional structures reveal a short NADH binding site being part of a larger FAD-binding site and a binding/dimerization domain.
Most European forest trees form at their root tips, a symbiosis with ectomycorrhizal fungi belonging to the ascomycota, basidiomycota or mitosporic fungi [
Mycorrhizal samples and fruiting bodies were collected from European beech (Fagus sylvatica (L.)), Sessile oak (Quercus petraea (MattuschkaLiebl.)), Norway spruce (Picea abies (L.) H. Karst.) and European larch (Larix decidua (Mill.)) growing in pure stands at the south-side of the Taunus Mountains situated on the southern part of the state of Hesse, Germany (
The collected mycorrhizae were put in marked plastic bags and transported at 4˚C in a cooled box to the lab where they were put in ice water and cleaned from adhering soil and humus particles under a microscope. Then, mycorrhizae of the same species were put in 1.5 ml Safelock Eppendorf tubes and stored at −20˚C.
The matt bolete (Xerocomus pruinatus (Fr. & Hoek)) and the bay bolete (X. badius) belonging to the Basidiomycetes are forming mycorrhizae with fine roots of conifers and deciduous trees [
The cap of the fruiting body of X. pruinatus can reach a diameter of 10 cm. Its
Forest district | Wiesbaden-Chausseehaus | Königstein | ||
---|---|---|---|---|
Near the city of | Taunusstein | Glashütten | Königstein | |
GPS | N50˚ 07.875' | N50˚ 07.875' | N50˚ 13.508' | N50˚ 12.623' |
E08˚ 10.382' | E08˚ 10.705' | E08˚ 24.018' | E08˚ 25.992' | |
Tree species | Norway spruce (Picea abies) | European beech (Fagus sylvatica) | Sessile oak (Quercus petraea) | European larch (Larix decidua) |
Sampling period | Apr-June/ Sept-Nov 2006-2010 | Apr-June/ Sept-Nov 2006-2010 | Sep-Oct 2009 June-July 2010 | Sept. - Nov. 2005 June 2010 |
shape is at first hemispherical, later convex to flattened. Its colour varies from light brown to greyish or dark brown to sometimes olivaceous or reddish brown to almost black. The cap is dry, velvety or finely dusted. The stipe is cylindrical to almost club-shaped, sometimes hardly swollen in its lower part. The yellow stipe downwards gradually gets a reddish colour. Stipe and pale yellow flesh and tubes are bluing when bruised or injured. The diameter and the shape of the cap of the fruiting body of X. badius are similar to that of X. pruinatus. The colour of the cap of X. badius varies from dark reddish brown to chestnut brown to dark brick. The cap is smooth when dry, but distinctly viscid under wet weather. The stipe of the fruiting body is cylindrical, spindle-shaped or almost club-shaped and often tapered towards the base. Tubes and flesh of X. badius are whitish or yellowish and turn blue when injured (cf. http://boletales.com/genera/xerocomus/x-pruinatus/).
Native proteins were extracted from mycorrhizal roots associated with X. pruinatus or X. badius, non mycorrhizal fine roots of seedlings, mycorrhizal roots separated into root tissues and enclosing hyphae, and fruiting bodies. Mycorrhizae were separated into hyphae and central root-tissues under an enlargement of 25 × fixing a mycorrhizal root put in ice water with a fine tweezers and separating the outer hyphae with a needle or a preparation forceps [
Cellulose acetate gels (Titan III, 7.6 cm × 7.6 cm, Helena Laboratories, Beaumont, Texas) were swollen under about 8˚C for 20 min in electrophoresis buffer which consisted of: 0.05 M Tris, 0.001 M Na2-EDTA, 0.001 M MgCl2 and 0.18 M maleic acid, pH 7.8 (modified according to [
Immediately after electrophoresis dihydrolipoyl dehydrogenase activities were visualized covering gels with an agar overlay. The staining solution consisted of 1 ml 0.1 M Tris-HCl buffer, pH 8.5, 1.5 ml NADH solution (3 mg/ml), 5 drops DCIP (2,6-dichlorphenol-indophenol solution, 3 mg/ml) and 5 drops MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromid solution, 10 mg/ml) [
The decolorized gels were photographed and put on a transmitted light plate to note the visible enzyme bands. Gels were then dried over night between several layers of dry tissue and then stored in welded polyethylene pockets.
Total DNA was extracted according to [
To identify mycorrhizal samples and fruiting bodies, the multicopy internal transcribed spacer (ITS) region of their ribosomal DNA (rDNA) was amplified and sequenced. The rDNA repeats, comprising the 18S rRNA gene, the ITS-1-spacer, the 5.8S rRNA, the ITS-2-spacer and the 28S rRNA gene, was amplified using the primer pair ITS1 [
ITS 1 (White et al. 1990): | 5’-TCCGTAGGTGAACCTGCGG-3’ |
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ITS1F (Gardes und Bruns 1993): | 5’-CTTGGTCATTTAGAGGAAGTAA-3’ |
ITS4 (White et al. 1990): | 5’-TCCTCCGCTTATTGATATGC-3’ |
ITS 4B (Gardes und Bruns 1993): | 5’-CAGGAGACTTGTACACGGTCCAG-3’ |
Total RNA was extracted from mycorrhizal roots or fruiting bodies using the “NucleoSpinÒ RNA-Plant”-Kit, by Machery and Nagel (Düren, Germany). An amount of 50 to 100 mg of fresh material was homogenized in a 1.5 ml Eppendorf Safe tube with a micropistil under liquid nitrogen. The resulting homogenate was pipetted on ice into a microcentrifuge tube and 350 µl “RAP”-buffer (guanidine-HCl lysis buffer) and 3.5 µl 2-mercaptoethanol added, vortexing the mixture. The resulting lysate was pipetted on a “NucleoSpinÒ”-filter inserted into a collecting tube and then centrifuged for 1 min at 11,000 × g at room temperature. The filtrate was transferred into a microcentrifuge tube, 350 µl ethanol (70%) added and the mixture five times pipetted up and down. The resulting lysate was loaded on a “NucleoSpinÒ-RNA-Plant” column and the unit centrifuged for 1 min at 11,000 × g, to bind the total RNA (and DNA) to the silica membrane. Then the column was placed into a new collecting tube, 350 µl “Membrane Desalting” buffer added to the column and the unit centrifuged for 1 min at 11,000 × g, to desalt the membrane. Afterwards, 95 µl DNase reaction mixture were applied onto the silica membrane of the column and the unit incubated at room temperature for 15 min to digest the bound DNA. Then, the silica membrane was washed adding 200 µl “RA2” buffer to the column and centrifuging the unit for 1 min at 11,000 × g. Afterwards, the column was placed into a new collecting tube adding 600 µl “RA3” solution to the column and centrifuging the unit for 1 min at 11,000 × g. The flow-through was discarded and the column placed in the collecting tube again. Then, 250 µl “RA3” buffer was added and the unit centrifuged for 2 min at 11,000 × g. After that the column was put into a nuclease-free 1.5 ml microcentrifuge tube. Total RNA was eluted from the membrane by adding 60 µl RNase-free water followed by a 1 min lasting centrifugation at 11,000 × g.
A first strand cDNA was synthesized by use of “The First Strand cDNA Synthesis-Kit” of Fermentas (St. Leon-Rot, Germany), annealing an oligo(dT) primer to the poly(A) tail of mRNAs. Into a microcentrifuge tube 14 µl of purified total RNA and 1 µl of oligo(dT) primer (100 pmol) were added, the mixture briefly vortexed and centrifuged for 2 sec at 11,000 × g. The RNA-primer mix was denatured at 70˚C for 5 min and then placed on ice. Then, 5 µl M-“MulV-5x RTase-buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2), 2 µl dNTP-mix (10 mM), 1 µl “Ribbolock-RNAse inhibitor” and 1 µl diethyl pyrocarbonate (DEPC)-water were added, and the mixture incubated for 60 min at 42˚C. A 10 minute incubation at 70˚C terminated the reaction.
The DNA sequence of the enzyme NADH diaphorase of Xerocomus badius and X. pruinatus associated with European beech or Norway spruce was amplified by use of the primer pair P1 and P2 (
Primers were purchased from Eurofins MWG Operon (Ebersberg, Germany). The used Primers were deduced from partial cDNA sequences published at the Genbank NCBI for the basidiomycetes Ustilago maydis, Cryptococcus neoformans, Laccaria bicolor and the diaphorase sequence of the two basidiomycetes Xerocomus badius and Xerocomus pruinatus gained via genome sequencing [
Sequencing of PCR products and genome-sequencing of the fungi Xerocomus badius and X. pruinatus via Illumina HiSeq 2000 (Illumina 2006, San Diego, California, USA) was done by GENterprise-Genomics (Mainz, Germany). For fungal identification, BLAST searches were carried out against the public sequence databases NCBI (http://www.ncbi.nlm.nih.gov/) and UNITE (http://unite.ut.ee). Sequences were assigned to matching species names when the BLAST matches showed identities higher than 97% and scores higher than 900 bits.
P1 | Primer-fw1: | 5’-CTT CGG TCA CAC GTA TCC T-3’ |
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Primer-rev1: | 5’-CTC GCT GAG TGT GGG CTA-3’ | |
P2 | Primer-fw2: | 5’-CCA GTG ACA CCA CTT ACA-3’ |
Primer-rev2: | 5’-TGA GTG TGG GCT AGA ATA GA-3’ | |
Dia1-fw: | 5’-G(AG)T TGA GGC (AC)AA GAA C(AG)T-3’ |
The name suggested by UNITE, a curated database for ectomycorrhizal fungi [
Separation of native proteins extracted from mycorrhizae of European beech by Cellulose Acetate electrophoresis resulted in up to seven isozymes of the enzyme dihydrolipoyl dehydrogenase. The various isozymes were adjoined to four gene loci A, B’, B and C (
In order to find out the affiliation of dihydrolipoyl dehydrogenase allozymes to tree roots and fungal hyphae respectively, ectomycorrhizae from European beech, Sessile oak, Norway spruce and European larch associated with the fungus Xerocomus pruinatus were investigated as well as rhizomorphae and fruiting bodies of that fungus. Additionally, non mycorrhizal root tips of European beech were analysed. It results that allozymes at locus C exclusively stem from the fungus X. pruinatus (
also observed for the ectomycorrhizal fungi Lactarius spp., Paxillus involutus, Russula ochroleuca and Xerocomus badius in association with the same four host trees (
The loci A, B’ and B are belonging to each of the four host trees. The deciduous trees European beech and Sessile oak are expressing loci A and B whereas the conifers Norway spruce and European larch possess in addition the active gene locus B’ (
In extracts of the ectomycorrhizal fungi Boletus edulis, Laccaria amethystina, Russula ochroleuca, Tylopilus felleus, Xerocomus badius and Xerocomus pruinatus only one active dihydrolipoyl dehydrogenase enzyme and one corresponding enzyme gene was observed. We assume that the enzyme is part of the two mitochondrial enzyme complexes pyruvate dehydrogenase (EC 1.2.4.1) and alpha-ketoglutarate dehydrogenase (EC 1.2.4.2). In contrast to these results we conclude the presence of two active dihydrolipoyl dehydrogenase genes within the deciduous tree species European beach and Sessile oak. Here, each of the two mitochondrial enzyme complexes pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase may contain a slightly differing form of the enzyme dihydrolipoyl dehydrogenase. The two conifers Norway spruce and European larch which function as hosts for ectomycorrhizal fungi are expressing three dihydrolipoyl dehydrogenase gene loci. Provided the corresponding enzyme forms result from two, respectively three different enzyme loci, upon the evolution of the deciduous trees from conifers one of the corresponding enzyme genes may have been silenced. Histochemical stainings that served to visualize dihydrolipoyl
dehydrogenase activities showed that the enzyme was more active in hyphae of Xerocomus badius than in those of X. pruinatus. Kinetic analyses lead to corresponding results. Differing activities were also observed between the ectomycorrhizal species Cenococcum geophilum, Scleroderma citrium, Paxillus involutus and Pisolitus tinctorius [
The DNA of Xerocomus pruinatus and X. badius was analyzed by use of “The Next-Generation-Illumina sequencing-method” (Solexa/Illumina, Berlin, (performed by GENterprise-Genomics, Mainz University). After the genome had been sequenced, localized primers were deduced as described in chapter 2.11 in order to amplify the gene sequence of the dihydrolipoyl dehydrogenase gene.
The full length of the dihydrolipoyl dehydrogenase gene has a length of 1631bp (cDNA: 1370 bp + (5 Introns = 261 bp) in Xerocomus badius and 1721 (cDNA: 1460 bp + 261 bp) in X. pruinatus (cf. sequences listed at the Appendix). The DNA sequences of the dihydrolipoyl dehydrogenase gene isolated from Xerocomus pruinatus and X. badius resemble those of other fungi deposited at the NCBI-gene bank to 70% to 78% (
Five introns, each having a length of 52 bp, could be localized comparing the full gene length with that of the cDNA length (
The gene sequences of the dihydrolipoyl dehydrogenase enzymes existing within the ectomycorrhizal fungi Boletus edulis, Laccaria amethystina, Paxillus
Xerocomus pruinatus | % similarity compared to | Xerocomus badius | % similarity compared to | ||
---|---|---|---|---|---|
X. pruinatus | 99 | X. pruinatus | 90 | ||
X. badius | 90 | X. badius | 98 | ||
Cryptococcus neoformans | 78 | Cryptococcus neoformans | 78 | ||
Coprinus cinerea | 75 | Ustilago maydis | 76 | ||
Laccaria bicolor | 75 | Coprinus cinerea | 74 | ||
Cryptococcus gatti | 71 | Laccaria bicolor | 74 | ||
Ustilago maydis | 70 | Cryptococcus gatti | 71 |
Intron number | Sequence range | Size (bp) |
---|---|---|
1 | 295 - 347 | 52 |
2 | 485 - 538 | 53 |
3 | 985 - 1037 | 52 |
4 | 1172 - 1224 | 52 |
5 | 1691 - 1743 | 52 |
involutus and Russula ochroleuca also include five 52 bp long introns located at the regions of the Xerocomus gene. These observations are in accordance with reports concerning the gene structure of the basidiomycetes Cryptococcus gatti, Cryptococcus neoformans, Coprinus cinerea, Ustilago maydis and Laccaria bicolor and the ascomycetes Candida albicans, C. orthopsilosis, Mycosphaerella graminicola and Trichophytum rubrum deposited at the Gene Bank (NCBI). The coding sequence of the gene of X. pruinatus deviates at 144 positions from that of X. badius. Besides the single nucleotide polymorphisms, the X. pruinatus gene contains a 48 bp long sequence at the positions 200 to 248 that could not be proved for the DNA and cDNA sequences of the gene from X. badius. Altogether, the two gene sequences deviate at 192 positions, which makes 11%. The number of single nucleotide polymorphisms of the five introns of X. badius and X. pruinatus sum up to 74 bp, corresponding to a deviation of 28.5%. Consequently, the nucleotide deviations in the five intron areas are about three times higher than those within the coding regions. The host trees European beech and Norway spruce did not influence the dihydrolipoyl dehydrogenase gene sequences in the two Xerocomus species.
The cDNA sequences of the dihydrolipoyl dehydrogenases from the two Xerocomus species served to determine their amino acid sequences (
The number of positively charged amino acid residues (Arg and Lys) within the enzyme of X. pruinatus makes 79 while it makes 68 in X. badius. The number of negatively charged amino acids (Asp and Glu) makes 46 in X. pruinatus and 48 in X. badius. Molecular weights and isoelectric points were determined by use of the software ExPASy-“Protparam” (https://web.expasy.org/protparam/) (
The length of cDNA of the dihydrolipoyl dehydrogenase gene of the basidiomycete Coprinopsis cinerea makes 1527 bp, corresponding to 494 amino acids [
mass makes 51558 Da [
that its amino acid sequence contains four functional domains: an NADH domain within a larger FAD domain, a central domain and a dimerization domain at its C-terminal end [
Altogether calculations resulted in 27.4 alpha helices, 24.8% beta strand structures, 11.9% beta loops and 35.9% other windings SWISS-Model [
Species | Nucleotides (bp) | Amino acids | Molecular weight (Da) | calculated isoelectric point | |
---|---|---|---|---|---|
DNA | cDNA | ||||
Xerocomus pruinatus | 1721 | 1460 | 486 | 55537.6 | 9.99 |
Xerocomus badius | 1631 | 1370 | 454 | 52163.5 | 9.68 |
Schirkonyer, U. and Rothe, G.M. (2018) Affiliation of Dihydrolipoyl Dehydrogenase Allozymes in Mycorrhizae of European Forest Trees and Characterization of the Enzyme of the Matt Bolete (Xerocomus pruinatus) and the Bay Bolete (X. badius). Open Journal of Ecology, 8, 356-377. https://doi.org/10.4236/oje.2018.86022