Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3′-phosphoadenosine 5′-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.     

Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor

Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants. Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation. The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells. Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots. Northern analysis showed that expression was detectable in L bicolor 48 h after interaction as well as in the established mycorrhizal tissue. Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans.     

Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor

An in vitro system for a Laccaria bicolor×Pinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT–PCR). The DDRT–PCR identified several cDNAs that are differentially expressed as early as 6 h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein–protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6 h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.     

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.     

Mechanisms for the development of genetically variable mycorrhizal mycelia in the ectomycorrhizal fungus Laccaria bicolor.

An in vitro study investigated mechanisms for the development of genetically variable mycorrhizal mycelia for Laccaria bicolor. Seedlings of jack pine (Pinus banksiana) grown nonaseptically in an autoclaved soil substrate were given different L. bicolor inoculum treatments. These included (i) a dikaryotic mycelium genotype (D); (ii) D and basidiospores collected from one group of five sporophores (T1); (iii) D and basidiospores collected from 10 sporophores, two from each of five different groups (T5); (iv) T1 alone; (v) T5 alone; and (vi) a noninoculated control. Dikaryotic mycelial inoculum was provided at the time of sowing, while basidiospore inoculum was added at 10 weeks after seed germination. Sporophore formation was induced after 20 weeks of growth, and dikaryotic cultures were isolated from their tissue. Seedlings were harvested, and growth and mycorrhization were assessed. Levels of both were generally lower for T1-treated seedlings, compared with seedlings receiving D, while levels for T5-treated seedlings were intermediate. Sporophore genotype variability was assessed for inoculum treatments by using the isoenzymatic marker leucine aminopeptidase. The greatest genetic variability was seen with the basidiospore treatments T1 and T5, with up to four leucine aminopeptidase patterns per seedling. The mixed treatments D plus T1 and D plus T5 produced most frequently, but not exclusively, the inoculated dikaryon genotype. After isoenzyme results were assessed, variable sporophore isolates of mixed treatments were analyzed with randomly amplified polymorphic DNA and PCR mitochondrial DNA markers to determine if they were formed by dikaryon-monokaryon crosses between the inoculated dikaryon and monosporous mycelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of five microsatellite loci in an ectomycorrhizal fungus Laccaria laccata

Laccaria laccata is an early successional ectomycorrhizal fungus. We isolated five polymorphic microsatellite loci from L. laccata using a dual‐suppression polymerase chain reaction technique. The number of alleles per locus ranged from three to six. The observed and expected heterozygosities ranged from 0.269 to 0.462, and 0.249 to 0.775, respectively. These microsatellite markers would be valuable molecular tools for population genetic studies of L. laccata.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.     

Occurrence and distribution of endobacteria in the plant-associated mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Fluorescence in situ hybridization, associated with confocal laser scanning microscopy or epifluorescence microscopy with deconvolution system, has allowed the detection of a community of intracellular bacteria in non-axenic samples of the ectomycorrhizal fungus Laccaria bicolor S238N. The endobacteria, mainly alpha-proteobacteria, were present in more than half of the samples, which consisted of ectomycorrhizae, fungal mats and fruit bodies, collected in the glasshouse or in the forest. Acridine orange staining suggests that the endobacteria inhabit both live and dead fungal cells. The role of these endobacteria remains to be clarified.     

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.     

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole-genome sequence assemblies

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens

Most boreal and temperate forest trees form a mutualistic symbiosis with soil borne fungi called ectomycorrhiza (ECM). In this association both partners benefit due to nutrient exchange at the symbiotic interface. Laccaria bicolor is the first mycorrhizal fungus with its genome sequenced thus making possible for the first time to analyze genome scale gene expression profiles of a mutualistic fungus. However, in order to be able to take full advantage of the genome sequence, reverse genetic tools are needed. Among them a high throughput transformation system is crucial. Herein we present a detailed protocol for genetic transformation of L. bicolor by means of Agrobacterium tumefaciens with emphasis on critical steps affecting the success and efficiency of the approach.