RESUMO
Genome assembly and annotation using short-paired reads is challenging for eukaryotic organisms due to their large size, variable ploidy and large number of repetitive elements. However, the use of single-molecule long reads improves assembly quality (completeness and contiguity), but haplotype duplications still pose assembly challenges. To address the effect of read length on genome assembly quality, gene prediction and annotation, we compared genome assemblers and sequencing technologies with four strains of the ectomycorrhizal fungus Laccaria trichodermophora. By analysing the predicted repertoire of carbohydrate enzymes, we investigated the effects of assembly quality on functional inferences. Libraries were generated using three different sequencing platforms (Illumina Next-Seq, Mi-Seq and PacBio Sequel), and genomes were assembled using single and hybrid assemblies/libraries. Long reads or hybrid assemby resolved the collapsing of repeated regions, but the nuclear heterozygous versions remained unresolved. In dikaryotic fungi, each cell includes two nuclei and each nucleus has differences not only in allelic gene version but also in gene composition and synteny. These heterokaryotic cells produce fragmentation and size overestimation of the genome assembly of each nucleus. Hybrid assembly revealed a wider functional diversity of genomes. Here, several predicted oxidizing activities on glycosyl residues of oligosaccharides and several chitooligosaccharide acetylase activities would have passed unnoticed in short-read assemblies. Also, the size and fragmentation of the genome assembly, in combination with heterozygosity analysis, allowed us to distinguish homokaryotic and heterokaryotic strains isolated from L. trichodermophora fruit bodies.
Assuntos
Genoma , Laccaria , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , HaplótiposRESUMO
For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.
Assuntos
Proteínas de Fluorescência Verde/genética , Laccaria/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Agrobacterium/genética , Basidiomycota/genética , Núcleo Celular/genética , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Laccaria/metabolismo , Proteínas Luminescentes/metabolismo , Microrganismos Geneticamente Modificados , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Transformação Genética , Proteína Vermelha FluorescenteRESUMO
Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.
Assuntos
Agrobacterium/genética , Laccaria/genética , Mutagênese Insercional , Micorrizas/genética , Sequência de Bases , Sítios de Ligação/genética , Southern Blotting , DNA Bacteriano/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Genoma Fúngico/genética , Análise de Sequência de DNA , Simbiose , Transformação GenéticaRESUMO
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.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Laccaria/genética , Micorrizas/genética , RNA Fúngico/genética , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte de Ânions/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Laccaria/metabolismo , Micorrizas/metabolismo , Transportadores de Nitrato , Nitratos/metabolismo , Nitrogênio/metabolismo , Populus/microbiologia , Interferência de RNA , RNA Fúngico/antagonistas & inibidores , RNA Fúngico/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Simbiose/fisiologiaRESUMO
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.
Assuntos
Agrobacterium tumefaciens/fisiologia , Laccaria/fisiologia , Agrobacterium tumefaciens/metabolismo , Laccaria/metabolismoRESUMO
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.
Assuntos
Técnicas de Silenciamento de Genes/métodos , Laccaria/genética , Micorrizas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Silenciamento de Genes/instrumentação , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Sequências Repetidas Invertidas , Laccaria/fisiologia , Micorrizas/fisiologia , Nitrato Redutases/genética , Nitrato Redutases/metabolismo , RNA Interferente Pequeno/química , Simbiose , Transformação Genética , Árvores/microbiologia , Árvores/fisiologiaRESUMO
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.
Assuntos
Técnicas de Silenciamento de Genes , Inativação Gênica , Vetores Genéticos , Laccaria/genética , RNA Interferente Pequeno/genética , Antifúngicos/farmacologia , Cinamatos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Nitrato Redutases/antagonistas & inibidores , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Seleção Genética , Transcrição GênicaRESUMO
Fue evaluada la actividad de extractos acuosos y etánolicos de cinco especies de basidiomicetos comestibles; Armillariella polymyces (Silip en Q´ eqchi´), Cantharellus lateritius (Anacate), Laccaria amethystina (sombrerito, sombrero de Xara, monja), Lactarius deliciosus (Shara amarilla, amacaria, cabeza de xara) Pleurotus ostreatus (Hongo ostra, hongo blanco), sobre la proliferación de linfocitos y la activación del sistema de complemento. El efecto sobre la linfoproliferación, fue medido evaluando la viabilidad celular de linfocitos humanos que fueron enfrentados a diferentes concentraciones de extracto acuoso y etanólico de cada basidiomiceto. Los resultados obtenidos mostraron actividad inhibitoria inespecífica (ya que no se encontró efecto de dosis-respuesta)...
Assuntos
Agaricales , Basidiomycota , Laccaria , Linfócitos , PleurotusRESUMO
Se caracterizó el crecimiento miceliar in vitro de una cepa nativa de Laccaria bicolor (196,1997), recolectada en Huehuetenango, Guatemala, en medio Melin-Norkrans Modificado (MMN) con cuatro niveles de pH; 4.5, 5.5, 6.5 y 7.0, incubando las placas durante 40 días a 26 centígrados. Se determinó que el mayor diámetro de las colonias se obtuvo en el pH 7.0. La morfología de las colonias varió según los tratamientos; con pH 4.5 y 5.5 la cepa produjo acúmulo hifales, mientras que con pH 6.5 y 7.0 las colonias fueron lisas y fibrilosas. Microscópicamente se observaron hifas de 2.0-4.0...
Assuntos
Agaricales , Meios de Cultura , Concentração de Íons de Hidrogênio , Laccaria , MicélioRESUMO
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.