Mycorrhizae

46. Molecular cloning and characterization of phosphate transporters from arbuscular mycorrhizal associations.

Maria J. Harrison, H. Liu, I.E. Maldonado-Mendoza and M. L. van Buuren. The Samuel Roberts Noble Foundation, Plant Biology Division, 2510 Sam Noble Pkwy, Ardmore, Oklahoma 73401.

Arbuscular mycorrhizal (AM) fungi are soil borne fungi that form mutualistic associations with the majority of species of land plants. The fungi are obligate symbionts and colonize the cortex of the plant root in order to obtain carbon from their plant hosts. The association is also beneficial for the plant as the fungi assist the plant with the aquisition of phosphate and other mineral nutrients from the soil. Our aim is to investigate the molecular mechanisms underlying phosphate transport in the symbiosis. Towards this goal we have isolated cDNA clones encoding phosphate transporters from roots of M. truncatula colonized with the AM fungus Glomus versiforme. cDNA clones from both the fungus and the plant were obtained and both encode proteins that are predicted to contain 12 membrane spanning domains, a secondary structure that is typical of membrane transporters from prokaryotes and eukaryotes. The cDNAs were expressed in yeast where they were able to complement a yeast phosphate transport mutant, therefore indicating that the encoded proteins are functional phosphate transporters. Phosphate transporters have been cloned previously from Neurospora and Saccharomyces. The Glomus transporter shares 48% identity at the amino acid level with the PHO84 gene product from Saccharomyces and 45% with the PHO-5 gene product from Neurospora. The Glomus and Medicago proteins are less similar and share approximately 37% amino acid identity. The Glomus phosphate transporter gene is expressed in the external hyphae which extend out of the root into the soil, a location which is consistent with a role in phosphate uptake in the mycorrhizal symbiosis. The expression of the Medicago transporter gene is currently under investigation.

47. Development of DNA markers for the identification of VA mycorrhizal fungal strains.

James E. Jurgenson, Jodee R. Foote, Fai Yee, Cindy Boyd and Jason Abbas, Department of Biology, University of Northern lowa,Cedar Falls, Iowa 50614.

AM mycorrhizal funai are obligate symbiotes of higher plants. Although they are thought to be plant parasites it is clear that they have a mutualistic relationship with their host. The plant benefits because the fungi can provide a better supply of inorganic nutrients than the plant can acquire on its own. AM fungi have been difficult to study because of their requirement for a host plant for growth. Consequently specific host fungal associations have not been easily substantiated. In order to more closely examine this and other fungal/host relationships we are developing DNA based detection methods which use the DNA Polymerase Chain Reaction (PCR) to detect the presence of specific AM species in field plant and soil samples. Random amplification of polymorphic DNA analysis was used to generate DNA fragments that are unique to isolates of several arbuscular mycorrhizal fungi. Sequence analysis of these fragments allowed generation of primer pairs, and subsequent specific identification of these genomes even in the presence of competing genomic DNA's. This approach can be used to specifically detect the presence of these fungi in mixtures of spores or infected roots.

48. Cloning of differentially expressed novel transcription factor like gene from early stages of ectomycorrhizal interaction of Laccaria bicolor and Pinus resinosa.

S.J. Kim¹, S.T. Hiremath², Jun Zheng² and G.K. Podila¹. ^lDept. of Biological Sciences, Michigan Technological University, Houghton, MI 49931, ²USDA-NEFES, Delaware, OH 43015.

We have cloned several cDNA clones that are differentially expressed from ectomycorrhizal fungus Laccaria bicolor in response to its host red pine. DDRT-PCR has been used to identify and isolate partial cDNA clones from L. bicolor, after various time points of interaction with red pine seedlings, in an in vitro system we have developed in our lab. Full length cDNAs of these differentially expressed clones were obtained using a combination of novel 5' and 3' RACE technique. The differential expression of these genes in response to red pine seedlings has been confirmed through Northerin blot analysis of RNA samples isolated from various time points of interaction between the L. bicolor and red pine seedlings. Genbank data base searches have indicated that one of the clones isolated from very early stage of interaction belongs to a novel class of transcription factors. It is now possible to isolate symbiosis related genes from ectomycorrhizal interactions using the DDRT-PCR methods. Details of this novel transcription factor and importance of its expression in the very early stages of ectomycorrhizal interaction between L. bicolor and red pine will be presented.

49. Signalling in the ectomycorrhizal symbiosis. The tryptophan betaine, hypaphorine, produced by Pisolithus tinctorius stimulates the expression of an auxin-regulated gene in roots of Eucalyptus globulus.

Frederic Lapeyrie, Thierry Beguiristain, Uwe Nehls & Francis Martin. Equipe de Microbiologie Forestiere, I.N.R.A, Centre de Nancy, 54280 Champenoux, France.

The development of the ectomycorrhizal association between Eucalyptus globulus and Pisolithus tinctorius induces dramatic changes in gene expression in both symbionts.

Hypaphorine, the betaine of tryptophan, is the major indolic compound which accumulates in P. tinctorius hyphae. Hypaphorine is accumulated in P. tinctorius tissues as soon as hyphae are in contact with host plant root surface. Such accumulation is controlled by specific root diffusible molecules, it could be induced through a membrane, but non-host plants are inactive.

The fungal hypaphorine had no IAA like activity on E. globulus root elongation and ramification, instead, a strong reduction of root hairs elongation was recorded. Furthermore the up-regulation of the level of EgPar transcripts was observed in roots incubated in the presence of either Pisolithus acellular extracts or hypaphorine. The EgPar gene expressed at a low level in roots and shoots of eucalypt seedling is amongst the identified symbiosis-regulated genes, the steady-state level of EgPar transcripts being drastically up-regulated in roots during the early stages of ectomycorrhiza development. It shows a high homology with auxin-induced genes from tobacco and Arabidopsis.

The latter data indicates that the fungal hypaphorine is able to trigger gene expression of the host plant and may act as an auxin derivative in eucalypt roots. This is the first report of an alteration of the host plant gene expression by a diffusible signal from an ectomycorrhizal fungus.

50. Phylogeny and population structure of the asexual mycorrhizal fungus Cenococcum geophilum Fr.

Katherine F. LoBuglio and John W. Taylor, Department of Plant Biology, University of California, Berkeley, CA 94720.

The phylogenetic relationship of the asexual mycorrhizal fungus Cenococcum geophilum Fr. among ascomycetes was examined by phylogenetic analysis of nucleotide sequence data from the 18S ribosomal RNA genic region. A specific focus of this study was to test the hypothesis that the genus Elaphomyces is the closest sexual relative of Cenococcum geophilum. DNA parsimony and distance analysis of the sequence data separated C. geophilum and Elaphomyces on distant clades when 44 additional genera of ascomycetes were included in the phylogenetic analyses. Cenococcum was positioned as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothidiales, and strongly supported Elaphomyces to be of Plectomycete origin. Among the sexual Ascomycetes examined no close sexual relative to C. geophilum was identified. Currently, we are examining the hypothesis that natural populations of C. geophilum are clonal and not recombining. Ten polymorphic loci present in two population sites have been identified by a PCR-SSCP strategy. Population genetic analysis of the multilocus genotypes obtained will measure levels of effective linkage between the loci (linkage disequilibrium) and determine if C. geophilum has a clonal or recombining population structure.

51. Rapid typing of Tuber borchii mycorrhizae by PCR amplification with specific primers.

Antonietta Mello, Lilia Garnero, Sabina Longato, Silvia Perotto and Paola Bonfante. Centro di Studi sulla Micologia del Terreno - CNR - and Dipartimento di Biologia Vegetale, Università, V.le Mattioli 25, 10125 Torino, Italy

Truffles are ascomycetous fungi that form ectomycorrhizae with the roots of trees such as oak, poplar, willow and hazel, and some shrubs such as Cistus. Truffle fruitbodies are usually identified on the basis of the structure of the peridium and gleba, the size and shape of their spores and asci, and wall ornamentation. However, all these features are lost during the symbiotic phase. Identification of truffles during their symbiotic phase is a main step in applied research on species of commercial value. We are therefore developing molecular typing methods based on PCR amplification.

DNA isolated from fruitbodies, mycelia and mycorrhizae of T. borchii was amplified with the universal primers pair ITS1/ITS4. RFLP analysis of the amplified ITS region of the ribosomal genes allowed discrimination of T. borchii from other truffle species on all DNA sources. The ITS region of T. borchii was then cloned and sequenced to design specific primers. The primers pair TBA/TBB was shown to be specific for this species after testing several fungal and plant species. Interestingly, they also amplified DNA from ancient herbarium samples. In conclusion, we have developed quick and reliable methods to identify T. borchii during all phases of the life cycle. The next step will be the construction of primers specific for T. magnatum, a species of extreme economic value.

52. Carbon allocation in ectomycorrhiza: Identification and first characterisation of monosaccharide transporters. U. Nehls, A. Wiese, R. Hampp, Universitat Tubingen, Physiol. Okol. der Pflanzen, Auf der Morgenstelle 1, D 72076 Tubingen, Germany

An ectomycorrhiza is a symbiotic organ formed between some soil fungi and the fine roots of woody plants. One important feature of ectomycorrhizal function is the conversion of plant carbohydrates into amino acids by the fungus, and their reimport into the plant. The main transport form of carbohydrates in plants, sucrose, cannot be utilized directly by ectomycorrhizal fungi. We thus assume that a plant apoplastic acid invertase cleaves sucrose to hexoses, and both, plant cortical cells and fungal hyphae compete for hexose uptake.

In this study, primers, designed against conserved regions of known fungal and plant monosaccharide transporters, were used to amplify cDNA fragments from P.abies/ A. muscaria mycorrhiza. These PCR fragments in turn, were used to isolate one fungal and one plant full length cDNA clone from a cDNA library. The deduced protein sequence of the P. abies cDNA (PaMST-1) revealed high homology to a Saccharum H+/monosaccharide transporter. PaMST-1 was mainly expressed in stem and roots. Its expression was slightly reduced in roots by ectomycorrhiza formation. The A. muscaria transporter (AmMST-1) gene codes for an open reading frame showing best homology to a N. crassa H+/monosaccharide transporter. The AmMST-1 gene was expressed at a basal level in all fungal hyphe. Nevertheless, its expression was significantly enhanced in symbiosis. An enhancement of AmMST-1 expression, comparable to that found in mycorrhiza, was also obtained in mycelia grown in suspension culture at increased glucose concentrations. This could be used to estimate a threshold concentration of monosaccharides at the root/fungus interface of ectomycorrhiza.

Molecular and functional diversity of ericoid mycorrhizal fungi.

Perotto S. and Paola Bonfante, Dipartimento di Biologia Vegetale and CSMT- CNR, V.le Mattioli 25, 10125 Torino, Italy
Ericaeous plants are widespread on the globe and colonize acidic substrates ranging from and sandy soils to humid mor-humus substrates. Our aims are to investigate whether this wide variety of environments also corresponds to genetic and functional diversity of the associated fungal symbionts. We have investigated the nuclear rDNA genes (ITS and 18S regions) of several ericoid isolates collected worldwide, in order to investigate their relationships. The results obtained so far suggest that the number of fungal species known to form ericoid mycorrhiza is probably greater than previously thought. An interesting feature revealed by amplification with universal primers and sequencing of the amplified DNA fragments is the occurrence, in the 18S rDNA of most ericoid isolates, of several Group I introns, an event rarely reported in fungi and in some algae. For several isolates, insertion of these elements at specific positions in the 18S genes is optional in different rDNA repeats, Moreover, specific insertions can be present or absent in isolates belonging to the same species and geographically very. In conclusion, the occurrence of Group I introns in the rDNA genes further increases the genetic diversity already observed in ericoid fungi with other methods (Perotto et at, 1996). The behaviour of these introns in ericoid fungi seems to be quite similar to the situation observed in lichen-forming fungi (Gargas et al, 1995), another symbiotic group of fungi. However, the functional significance of Group I introns for both symbiosis remains to be elucidated.
Gargas A, DePriest PT, Taylor J-W. Mol Biol Evol, 12: 208-215, 1995
Perotto S, Actis-Perino E, Peruginin I, Bonfante P. Mol Ecol, 5: 123-131, 1996