LbNrt RNA silencing in the mycorrhizal symbiont Laccaria bicolor reveals a nitrate-independent regulatory role for a eukaryotic NRT2-type nitrate transporter.

Fungal nitrogen metabolism plays a fundamental role in function of mycorrhizal symbiosis and consequently in nutrient cycling of terrestrial ecosystems. Despite its global ecological relevance the information on control and molecular regulation of nitrogen utilization in mycorrhizal fungi is very limited. We have extended the nitrate utilization RNA silencing studies of the model mycorrhizal basidiomycete, Laccaria bicolor, by altering the expression of LbNrt, the sole nitrate transporter-encoding gene of the fungus. Here we report the first nutrient transporter mutants for mycorrhizal fungi. Silencing of LbNrt results in fungal strains with minimal detectable LbNrt transcript levels, significantly reduced growth capacity on nitrate and altered symbiotic interaction with poplar. Transporter silencing also creates marked co-downregulation of whole Laccaria fHANT-AC (fungal high-affinity nitrate assimilation cluster). Most importantly, this effect on the nitrate utilization pathway appears independent of extracellular nitrate or nitrogen status of the fungus. Our results indicate a novel and central nitrate uptake-independent regulatory role for a eukaryotic nitrate transporter. The possible cellular mechanisms behind this regulation mode are discussed in the light of current knowledge on NRT2-type nitrate transporters in different eukaryotes.

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.

fHANT-AC genes of the ectomycorrhizal fungus Laccaria bicolor are not repressed by l-glutamine allowing simultaneous utilization of nitrate and organic nitrogen sources.

In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The expression profile of fHANT-AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT-AC as also N starvation and growth on urea and l-asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT-AC genes was detected in mycelia grown on variable concentrations of l-glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT-AC is not signalled via l-glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA-silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT-AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT-AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed.

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA - triggering in the mycorrhizal fungus Laccaria bicolor.

pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two-step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium-mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65-76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T-DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T-DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol-1,4,5-triphosphate 5-phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungus Laccaria bicolor.

Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extraradical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. Besides the optimized use in L. bicolor, general consideration was taken to build a vector system with maximum compatibility with other homobasidiomycetes and different transformation techniques.

Toxicological assessment of Penicillium nalgiovense strains for use as starter cultures in the manufacture of dry fermented sausages.

The use of fungal starter strains in the casing of dry fermented sausages allows standardization of the manufacturing process and ensures consumer safety. Penicillium nalgiovense is normally used for this purpose. Even though this species is reported as safe with respect to the production of the most common mycotoxins, its safety may be strain specific. The aim of the present work was to assess the toxicogenic potential of nine P. nalgiovense strains isolated from dry fermented sausages that were previously suitable as starters. The strains were tested for toxicity on brine shrimp larvae and the human cell line MCF7, for mutagenicity in the Ames test, and for antibacterial activity against gram-positive and gram-negative bacteria. According to our results, several P. nalgiovense strains were positive in more than one bioassay. Therefore, it is important to use different toxicological assays when characterizing strains intended for food use. Strains S1-2 and S14-4, which belong to biotypes 6 and 5, respectively, were nontoxigenic under the conditions tested. Overall, strain S1-2 of P. nalgiovense proved to be best suited as a starter in dry fermented sausage manufacture because in addition of being nontoxicogenic it produces white conidia, which is a desirable feature.

RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus.

Mycorrhizal symbioses are a rule in nature and may have been crucial in plant and fungal evolution. Ectomycorrhizas are mutualistic interactions between tree roots and soil fungi typical of temperate and boreal forests. The functional analysis of genes involved in developmental and metabolic processes, such as N nutrition, is important to understand the ontogeny of this mutualistic symbiosis. RNA silencing was accomplished in the model mycorrhizal fungus Laccaria bicolor by Agrobacterium-mediated gene transfer. Promoter-directed expression of double-stranded RNA with a partial coding sequence of the Laccaria nitrate reductase gene resulted in fungal transgenic strains strongly affected in growth with nitrate as N source in a medium with high concentration of an utilizable C source. The phenotype correlated with a clear reduction of the target gene mRNA level and this effect was not caused by homologous recombination of the T-DNA in the nitrate reductase locus. Transformation with the hairpin sequence resulted in specific CpG methylation of both the silenced transgene and the nitrate reductase encoding gene. The methylation in the target gene was restricted to the silencing trigger sequence and did not represent the entire genomic DNA in the dikaryon suggesting that the epigenetic changes accompanying RNA silencing affected only the transformed nucleus. Mycorrhization experiments of Populus with strongly silenced fungal strains revealed a systematic inhibition of symbiosis under mycorrhization conditions (C starvation) and nitrate as N source compared with the wild type. This inhibition of mycorrhization was reversed by an organic N source only utilizable by the fungus. These observations would indicate that the plant may be capable of monitoring and detecting the nutritional status of a potential symbiont avoiding the establishment of an unsatisfactory interaction. A probable control mechanism conducted by the plant would inhibit symbiosis when the metabolic profile of the fungal partner is not proper and mutual benefit from the symbiotic structure cannot be assured. Our results are the first report showing that the alteration of expression of a fungal gene impairs mycorrhization. Moreover, this work is the first demonstration of RNA silencing in mycorrhizal fungi and clearly shows that gene knock-down is a powerful tool for further functional genomic studies in mycorrhizal research.

T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium-mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T-DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T-DNA right border (RB)-genomic DNA junctions in Escherichia coli. Fifty-one Agrobacterium-transformed fungal strains, picked up at random from a larger collection of T-DNA tagged strains (about 500), were analysed. Sixty-nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.

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.