RESUMEN
The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.
Asunto(s)
Proteínas de Arabidopsis , Proteínas Bacterianas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas , Factores de Virulencia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidad , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular , ADN de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas Asociadas a Fase-S , Proteínas Ligasas SKP Cullina F-box , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
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.
Asunto(s)
Agrobacterium tumefaciens/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Integrasas/metabolismo , Canales Iónicos , Transporte de Proteínas , Proteínas Virales , Factores de Virulencia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidad , Arabidopsis/genética , Arabidopsis/microbiología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Resistencia a Medicamentos , Integrasas/genética , Kanamicina/farmacología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , VirulenciaRESUMEN
The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here. Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid. The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids. Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.
Asunto(s)
Agrobacterium tumefaciens/genética , Arginina/análogos & derivados , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Factores de Virulencia , Arginina/genética , Elementos Transponibles de ADN , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Análisis de Secuencia de ADNRESUMEN
Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plasmids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.
Asunto(s)
Agrobacterium tumefaciens/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Plásmidos/genética , Factores de Virulencia , Agrobacterium tumefaciens/patogenicidad , Secuencia de Bases , ADN Bacteriano/genética , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Plantas Tóxicas , Nicotiana/microbiología , Virulencia/genéticaRESUMEN
We have used a modified CaMV 35S promoter to direct the expression of chimaeric genes encoding human serum albumin (HSA) in transgenic potato and tobacco plants. To secrete the protein, either the human prepro-sequence or the signal sequence from the extracellular tobacco protein PR-S was used. We demonstrate secretion of HSA with both types of signal sequences in transgenic leaf tissue and in suspension cultures. HSA produced in transgenic potato plants was purified to chromatographic homogeneity. N-terminal amino acid sequence analysis revealed that the processing of the precursor protein was dependent on the type of signal sequence. Expression of the human preproHSA gene lead to partial processing of the precursor and secretion of proHSA. Fusion of HSA to the plant PR-S presequence resulted in cleavage of the presequence at its natural site and secretion of correctly processed HSA that is indistinguishable from the authentic human protein.
Asunto(s)
Plantas/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Albúmina Sérica/biosíntesis , Transfección , Secuencia de Aminoácidos , Secuencia de Bases , Quimera/genética , Humanos , Datos de Secuencia Molecular , Plantas/metabolismo , Plantas Tóxicas , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
For transformation of sunflower (Helianthus annuus L. cv. Zebulon), shoot apical meristems were dissected from seeds and cocultivated with a disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS- and NPTII-activity. The influence of the media conditions, the time of cocultivation and the stage of the developing seed on shoot development and meristem transformation was analysed. Transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by assays for GUS and NPTII. GUS-positive shoots were rooted on rockwool and transferred to soil. Transformation of shoot meristem cells occurred at low frequencies. Chimaeric expression of the two genes was observed in transformed plants. Integration of the foreign DNA in the sunflower genome was confirmed with the polymerase chain reaction.