RESUMO
The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.
Assuntos
Agrobacterium tumefaciens/metabolismo , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Integrases/metabolismo , Canais Iônicos , Transporte Proteico , Proteínas Virais , Fatores de Virulência , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidade , Arabidopsis/genética , Arabidopsis/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Resistência a Medicamentos , Integrases/genética , Canamicina/farmacologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/metabolismo , VirulênciaRESUMO
We analyzed 29 T-DNA inserts in transgenic Arabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.
Assuntos
Agrobacterium tumefaciens/genética , Arabidopsis/metabolismo , DNA Bacteriano/metabolismo , Arabidopsis/genética , Sequência de Bases , Primers do DNA , DNA Bacteriano/química , DNA de Plantas/química , DNA de Plantas/metabolismo , DNA de Cadeia Simples/metabolismo , Vetores Genéticos , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Plasmídeos , Reação em Cadeia da Polimerase , Transfecção/métodosRESUMO
Agrobacterium tumefaciens transfers part of its tumour-inducing (Ti) plasmid, the transferred or T-DNA, to plants during tumourigenesis. This represents the only example of naturally occurring trans-kingdom transfer of genetic material. Here we report that A.tumefaciens can transfer its T-DNA not only to plant cells, but also to another eukaryote, namely the yeast Saccharomyces cerevisiae. The Ti plasmid virulence (vir) genes that mediate T-DNA transfer to plants were found to be essential for transfer to yeast as well. Transgenic S.cerevisiae strains were analysed for their T-DNA content. Results showed that T-DNA circles were formed in yeast with precise fusions between the left and right borders. Such T-DNA circles were stably maintained by the yeast if the replicator from the yeast 2 mu plasmid was present in the T-DNA. Integration of T-DNA in the S.cerevisiae genome was found to occur via homologous recombination. This contrasts with integration in the plant genome, where T-DNA integrates preferentially via illegitimate recombination. Our results thus suggest that the process of T-DNA integration is predominantly determined by host factors.
Assuntos
DNA Bacteriano , Plasmídeos , Rhizobium/genética , Saccharomyces cerevisiae/genética , Transformação Bacteriana , Fatores de Virulência , Proteínas de Bactérias/genética , Sequência de Bases , Endodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , Genes Bacterianos , Genes Fúngicos , Dados de Sequência Molecular , Recombinação Genética , Rhizobium/patogenicidade , Fatores de Transcrição/genética , VirulênciaAssuntos
Agrobacterium tumefaciens/genética , Plasmídeos , Transformação Genética , Agrobacterium tumefaciens/efeitos dos fármacos , Agrobacterium tumefaciens/crescimento & desenvolvimento , Antibacterianos/farmacologia , Meios de Cultura , Eletroporação/métodos , Técnicas Genéticas , Indicadores e Reagentes , SoluçõesRESUMO
To find VirG proteins with altered properties, the virG gene was mutagenized. Random chemical mutagenesis of single-stranded DNA containing the Agrobacterium tumefaciens virG gene led with high frequency to the inactivation of the gene. Sequence analysis showed that 29% of the mutants contained a virG gene with one single-base-pair substitution somewhere in the open reading frame. Thirty-nine different mutations that rendered the VirG protein inactive were mapped. Besides these inactive mutants, two mutants in which the vir genes were active even in the absence of acetosyringone were found on indicator plates. A VirG protein with an N54D substitution turned out to be able to induce a virB-lacZ reporter gene to a high level even in the absence of the inducer acetosyringone. A VirG protein with an I77V substitution exhibited almost no induction in the absence of acetosyringone but showed a maximum induction level already at low concentrations of acetosyringone.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Acetofenonas/farmacologia , Agrobacterium tumefaciens/patogenicidade , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , DNA de Cadeia Simples/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Mutagênicos , Fases de Leitura Aberta/genética , Tumores de Planta/microbiologia , Plantas/microbiologia , Mutação Puntual , Seleção Genética , Transcrição GênicaRESUMO
Octopine and nopaline strains of Agrobacterium tumefaciens were found to differ in virulence on Nicotiana glauca. This difference is due to the absence of a functional virF locus, which is necessary for efficient tumorigenesis on N. glauca, from the nopaline Ti plasmids. Genetic studies and DNA sequence analysis of the virF locus revealed that virF embraces one open reading frame coding for a hydrophilic protein with a molecular mass of 22,437 Da. Transcription of virF is directed from left to right, towards the T region, and is strongly induced by the phenolic compound acetosyringone. We established that virA and virG, two genes known to be essential for induction of the vir regulon, are necessary for acetosyringone-induced virF expression, implying that virF is a member of this vir regulon. Agrobacterium virF mutants can be complemented for tumor induction by co-infection with avirulent Agrobacterium 'helper' strains. We found that such 'helper' strains must express not only the virF gene but also the vir operons virA, virB, virD and virG.
