RESUMO
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
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Basidiomycota/genética , DNA Bacteriano/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Basidiomycota/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Farmacorresistência Fúngica/genética , Genes Sintéticos , Fleomicinas/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Schizophyllum/genética , Seleção GenéticaRESUMO
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.
El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Basidiomycota/genética , DNA Bacteriano/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Basidiomycota/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Farmacorresistência Fúngica/genética , Genes Sintéticos , Reação em Cadeia da Polimerase , Fleomicinas/farmacologia , Regiões Promotoras Genéticas/genética , Seleção Genética , Schizophyllum/genéticaRESUMO
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.