Biomedical subjects

Lan-Ying Lee

Publications and source records attributed to Lan-Ying Lee.

6 recordsLinked to original sources

PubMed2006

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

Biological Assay↗

PubMed2006

Integration of genes into the chromosome of Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens has been widely used to transform numerous plant species. Frequently, investigators want to place more than one gene into Agrobacterium in order to manipulate various bacterial functions during plant genetic transformation. These genes are frequently brought into the bacterium by multiple plasmids. It is difficult to maintain several plasmids in the same bacterium without the use of a variety of antibiotics. However, the use of some antibiotics is not feasible for certain Agrobacterium strains. Also, too many different antibiotics in the culture medium generally results in slow growth of Agrobacterium. Therefore, it is to one's advantage to place genes of interest onto the bacterial chromosome to eliminate the use of multiple antibiotics during bacterial growth. This chapter describes in detail a method to integrate a gene of interest into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome. The integrated gene is stably maintained as single copy per cell without the need for selection.

Agrobacterium tumefaciens↗

PubMed2004

Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of agrobacterium transformation.

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.

Arabidopsis↗

PubMed2004

Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of IncQ plasmids between Agrobacterium cells and the establishment of IncQ plasmids in plant cells.

The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.

Agrobacterium tumefaciens↗

PubMed2003

The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA.

The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability. Four ATP-dependent ligases, designated DNA ligases I-IV, have been identified in higher eukaryotes, and each one has distinct functions. In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining. Recently, an Arabidopsis thaliana orthologue of the yeast and mammalian DNA ligase IV gene was found and termed AtLIG4. Here we describe the isolation and functional characterisation of a plant line with a T-DNA insertion in the AtLIG4 gene. Plants homozygous for the T-DNA insertion did not display any growth or developmental defects and were fertile. However, mutant seedlings were hypersensitive to the DNA-damaging agents methyl methanesulfonate and X-rays, demonstrating that AtLIG4 is required for the repair of DNA damage. Recently, we showed that a yeast lig4 mutant is deficient in Agrobacterium T-DNA integration. However, using tumorigenesis and germline transformation assays, we found that the plant AtLIG4 mutant is not impaired in T-DNA integration. Thus, in contrast to yeast, DNA ligase IV is not required for T-DNA integration in plants.

Arabidopsis↗

PubMed2003

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Agrobacterium tumefaciens↗