RESUMO
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
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...