Cre Reporter Assay for Translocation (CRAfT): a tool for the study of protein translocation into host cells.

Many pathogenic bacteria introduce virulence proteins, also called effector proteins, into host cells to accomplish infection. Such effector proteins are often translocated into host cells by bacterial type III (T3SS) or type IV secretion systems (T4SS). To better understand the molecular mechanisms underlying virulence, it is essential to identify the effector proteins and determine their functions.

Genome Sequence of the Octopine-Type Agrobacterium tumefaciens Strain Ach5.

We have sequenced the complete genome of the plant pathogen Agrobacterium tumefaciens strain LBA4213, a derivative of the wild-type strain A. tumefaciens Ach5 and the ancestor of A. tumefaciens strain LBA4404 used in genetic engineering.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana.

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis.

Ehrlichia chaffeensis tandem repeat proteins and Ank200 are type 1 secretion system substrates related to the repeats-in-toxin exoprotein family.

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host-pathogen interactions including DNA binding and a network of protein-protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E.

Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain.

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection.

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells.

Yeast (Saccharomyces cerevisiae).

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic organisms. This species has enabled a detailed study of the (genetic) requirements for Agrobacterium-mediated DNA transformation. For instance research with this yeast has led to the recognition that the transforming DNA molecules integrate into the eukaryotic chromosomes either by homologous recombination, which is the preferred pathway in S.

Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells.

The Brucella suis type IV secretion system assembles in the cell envelope of the heterologous host Agrobacterium tumefaciens and increases IncQ plasmid pLS1 recipient competence.

Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related alpha(2)-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex.

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium.

Several human pathogens and the plant pathogen Agrobacterium tumefaciens use a type IV secretion system for translocation of effector proteins into host cells. How effector proteins are selected for transport is unknown, but a C-terminal transport signal is present in the proteins translocated by the A. tumefaciens VirB/D4 type IV secretion system.

Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1.

Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell.

VirD4-independent transformation by CloDF13 evidences an unknown factor required for the genetic colonization of plants via Agrobacterium.

Agrobacterium uses a mechanism similar to conjugation for trans-kingdom transfer of its oncogenic T-DNA. A defined VirB/VirD4 Type IV secretion system is responsible for such a genetic transfer. In addition, certain virulence proteins as VirE2 can be mobilized into host cells by the same apparatus.

Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3.

Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae.

Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens.

Insertional mutagenesis is a powerful tool for the isolation of novel mutations. The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants. To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S.