Paul J. J. Hooykaas

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

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.

Agrobacterium-mediated transformation as a tool for functional genomics in fungi.

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.

The Bases of Crown Gall Tumorigenesis

The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region.

Abstract One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ− phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the TL + TR region. Most of the TR region, present as TR-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the TR region together with a small part of the adjacent TL region, which is always present as TL-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoe and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.

Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens

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.

Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination.

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens–mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

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. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosine‐phosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4‐dependent type IV secretion (T4S) system. Yeast two‐hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl‐interactor 1 (Abi‐1), an adaptor protein that interacts with Abl‐1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl‐1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti‐AnkA antibody, Abl‐1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl‐1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular α‐proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl‐1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.

Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration

Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T‐DNA), derived from its tumour‐inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T‐DNA also to monocotyledonous plants, yeasts and fungi. The T‐DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T‐DNA recipient to demonstrate that the non‐homologous end‐joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T‐DNA into the host genome. We discovered a minor pathway for T‐DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T‐DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.

Maintenance of Embryonic Auxin Distribution for Apical-Basal Patterning by PIN-FORMED–Dependent Auxin Transport in Arabidopsis

Molecular mechanisms of pattern formation in the plant embryo are not well understood. Recent molecular and cellular studies, in conjunction with earlier microsurgical, physiological, and genetic work, are now starting to define the outlines of a model where gradients of the signaling molecule auxin play a central role in embryo patterning. It is relatively clear how these gradients are established and interpreted, but how they are maintained is still unresolved. Here, we have studied the contributions of auxin biosynthesis, conjugation, and transport pathways to the maintenance of embryonic auxin gradients. Auxin homeostasis in the embryo was manipulated by region-specific conditional expression of indoleacetic acid-tryptophan monooxygenase or indoleacetic acid-lysine synthetase, bacterial enzymes for auxin biosynthesis or conjugation. Neither manipulation of auxin biosynthesis nor of auxin conjugation interfered with auxin gradients and patterning in the embryo. This result suggests a compensatory mechanism for buffering auxin gradients in the embryo. Chemical and genetic inhibition revealed that auxin transport activity, in particular that of the PIN-FORMED1 (PIN1) and PIN4 proteins, is a major factor in the maintenance of these gradients.

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system.

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1‐11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T‐DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild‐type strain that formed only uninfected tumour‐like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.