Alice Beijersbergen

Bio: Alice Beijersbergen is an academic researcher from Leiden University. The author has contributed to research in topics: Agrobacterium tumefaciens & Ti plasmid. The author has an hindex of 6, co-authored 6 publications receiving 2039 citations.

Agrobacterium tumefaciens-mediated transformation of filamentous fungi.

Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.

Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae.

Abstract: 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 Atumefaciens 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 Scerevisiae 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 Scerevisiae 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

The Virulence System of Agrobacterium Tumefaciens

Abstract: The gram-negative soil bacterium Agrobacterium tumefaciens causes the plant disease crown gall. This disease is characterized by the formation of tumors or crown galls at wound sites of infected dicotyledonous plants (for recent reviews see Kado, 1991; Winans, 1992; Zambryski, 1992; Hooykaas and Schilperoort, 1992). During tumor induction Agrobacterium attaches to the plant and transfers part of its tumor inducing (Ti) plasmid (Fig.l) to plant cells at wound sites. The transferred or T-DNA, which is surrounded by 24 basepair (bp) imperfect direct repeats or border repeats, becomes integrated in the plant cell nuclear DNA. Upon expression of the genes located on the T-DNA, proteins are produced involved in the production of the plant hormones auxin (IAA) and cytokinin (isopentenyl-AMP). These hormones cause the tumorous phenotype, characterized by the ability of the cells to proliferate unlimited and autonomously in the absence of added phytohormones. The T-DNA also encodes synthases catalyzing the formation of opines. These opines are mostly built from an amino acid and a sugar. Based on the kind of opines produced, Agrobacterium strains are classified as octopine, nopaline, succinamopine and leucinopine strains. The opines formed in the tumors are metabolized by the agrobacteria which induced tumor formation, but not by most other soil organisms. Thus, by genetic manipulation of plant cells Agrobacterium creates a favorable niche for itself, a process also called “genetic colonization”.

Conjugative transfer by the virulence system of Agrobacterium tumefaciens

Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the transferred DNA (T-DNA), to plant cells during tumor induction. Expression of this T-DNA in plant cells results in their transformation into tumor cells. There are similarities between the process of T-DNA transfer to plants and the process of bacterial conjugation. Here, the T-DNA transfer machinery mediated conjugation between bacteria. Thus, products of the Vir region of the Ti plasmid of Agrobacterium tumefaciens, normally involved in transfer of DNA from bacteria to plants, can direct the conjugative transfer of an IncQ plasmid between agrobacteria.

Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Abstract: Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

Agrobacterium tumefaciens-mediated transformation of filamentous fungi.

Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.

Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants

Abstract: The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

Auxin and Plant-Microbe Interactions

Abstract: Microbial synthesis of the phytohormone auxin has been known for a long time. This property is best documented for bacteria that interact with plants because bacterial auxin can cause interference with the many plant developmental processes regulated by auxin. Auxin biosynthesis in bacteria can occur via multiple pathways as has been observed in plants. There is also increasing evidence that indole-3-acetic acid (IAA), the major naturally occurring auxin, is a signaling molecule in microorganisms because IAA affects gene expression in some microorganisms. Therefore, IAA can act as a reciprocal signaling molecule in microbe-plant interactions. Interest in microbial synthesis of auxin is also increasing in yet another recently discovered property of auxin in Arabidopsis. Down-regulation of auxin signaling is part of the plant defense system against phytopathogenic bacteria. Exogenous application of auxin, e.g., produced by the pathogen, enhances susceptibility to the bacterial pathogen.

The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58

Abstract: The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.