Finding a way to the nucleus

https://doi.org/10.1016/j.mib.2009.11.003Get rights and content

Agrobacterium species transfer single-strand DNA and virulence effector proteins to plants. To understand how Agrobacterium achieves interkingdom horizontal gene transfer, scientists have investigated how the interaction of bacterial effector proteins with host proteins directs T-DNA to the plant nucleus. VirE2, a single-strand DNA binding protein, likely plays a key role in T-DNA nuclear targeting. However, subcellular trafficking of VirE2 remains controversial, with reports of both cytoplasmic and nuclear localization. The recent discovery that phosphorylation of the VirE2 interacting protein VIP1 modulates both nuclear targeting and transformation may provide a solution to this conundrum. Novel experimental systems that allow tracking of VirE2 as it exits Agrobacterium and enters the plant cell will also aid in understanding virulence protein/T-DNA cytoplasmic trafficking.

Introduction

Agrobacterium species, ‘nature's genetic engineer’, are well-known mediators of interkingdom horizontal gene transfer. These phytopathogens cause neoplastic growths on host plants (crown gall and hairy root disease), but are best known as agents for generating transgenic plants [1]. Agrobacterium transports single-strand transferred DNA (T-DNA, Box 1) to plants through a bacterial type IV protein secretion system (T4SS). Processing of T-DNA is initiated by VirD2, which nicks the tumor inducing (Ti) plasmid at T-DNA border repeat sequences. During this cleavage reaction, VirD2 covalently attaches to single-strand T-DNA (termed the ‘T-strand’) at its 5′ end. VirD2 is thought to pilot T-strands through the T4SS, into the plant cell, and to the nucleus. VirD2/T-strands are not the only molecules transferred to the plant via the T4SS. Other transferred Agrobacterium effector proteins include VirD5, VirE2, VirE3, and VirF (for reviews, see [2, 3, 4•]). Transfer of effector proteins to host cells through a T4SS is also important for animal and human pathogenesis by a number of bacteria, including Helicobacter, Brucella, Bordetella, Bartonella, and Legionella species [5].

In addition to VirD2, of special importance for plant genetic transformation is VirE2, a single-strand DNA binding protein. VirE2 binds cooperatively to single-strand DNA in vitro [6, 7, 8] and is hypothesized to bind to T-strands in the plant, thus protecting the DNA from nucleolytic destruction [9, 10]. The complex of T-strands covalently linked to VirD2 and coated by VirE2 molecules is termed the ‘T-complex’ [11]. Although there are strong genetic and in vitro binding data indicating the existence of the T-complex, such a complex has not been demonstrated in plants.

Interaction of VirD2/T-strands and VirE2 with other secreted Agrobacterium virulence effector proteins and plant proteins likely generates ‘super-T-complexes’ that are responsible for subcellular trafficking of T-strands from the plant cell wall and membrane through the cytoplasm, into the nucleus, and to chromatin, thus facilitating T-DNA integration into the plant genome. VirD2 interacts with several plant cyclophilins [12, 13], all tested importin α isoforms [12, 14, 15••], the kinase CAK2Ms [12], a TATA box binding protein [12], and the protein phosphatase PP2C [16]. Interaction with importin α and phosphorylation by PP2C are important for nuclear targeting of VirD2, whereas interaction with CAK2Ms and the TATA box binding protein may be important for targeting of VirD2/T-strands to chromatin [12]. VirE2 also interacts with several importin α isoforms [15••], as well as with two VirE2 interacting proteins VIP1 and VIP2 [17]. Interaction of VIP1 with histones and nucleosomes may mediate targeting of T-strands to plant chromatin [18•, 19]. VIP2 may facilitate T-DNA integration into the genome: vip2 mutant Arabidopsis plants support transient but not stable Agrobacterium-mediated transformation [20]. Because VirE2 molecules likely ‘coat’ T-strands and also interact with plant proteins that help effect nuclear targeting, the subcellular localization of VirE2 is crucial to understanding cytoplasmic trafficking of T-strands. Figure 1 presents several models describing the role of VirE2 in targeting T-strands to the nucleus. These models, and the data supporting them, are discussed below.

Section snippets

Nuclear targeting of important Agrobacterium virulence effector proteins

Both VirD2 and VirE2 contain nuclear localization signal (NLS) sequences that interact with importin α proteins. The bipartite VirD2 NLS sequence near the carboxy-terminus can direct affixed reporter proteins to the nucleus in plant, animal, and yeast cells [21, 22, 23, 24, 25, 26]. Thus, it is likely that VirD2 helps direct covalently linked T-strands to the plant nucleus. The subcellular localization of VirE2 remains, however, controversial. Several reports demonstrated nuclear localization

VIP1 protein and T-strand nuclear targeting

Resolution of these conflicting subcellular localization results may come from a better understanding of the role played by VIP1 protein in the transformation process. VIP1, which interacts with VirE2, is a b-ZIP category transcription factor normally involved in MAP kinase-mediated plant defense signaling [32]. VIP1 is a phospho-protein, and the phosphorylation of VIP1 by MPK-3 causes it to relocalize from the cytoplasm to the nucleus [33••]. Nuclear localization of VIP1 is essential for

Limitations of previous studies involving VirE2 subcellular localization

Additional difficulties in resolving these conflicting VirE2 subcellular localization results and the role of VirE2 in nuclear targeting of T-strands derive both from the techniques used to introduce VirE2 into plant cells and from the different cellular systems employed by the various research groups. In all of the studies published to date, large quantities of VirE2 were either introduced into the cells (by microinjection or direct uptake of proteins into permeabilized cells) or synthesized

Novel assays of VirE2 cellular trafficking

In order to resolve the problems of overexpressing VirE2 directly in plant cells, it would be necessary to track VirE2 as it exits Agrobacterium, enters the plant cell, and interacts with T-strands and plant proteins. However, this presents a number of biological and technical problems. Because only small amounts of VirE2 protein likely enter the plant cell following infection, it may be necessary to tag VirE2 to increase the sensitivity of detection. However, tagging at the C-terminus would

Alternatives to VirE2

Although most Agrobacterium strains encode VirE2 and require this protein for high levels of virulence, some A. rhizogenes strains lack both virE2 and virE1 [40] (which interacts with VirE2 in Agrobacterium and functions as a VirE2 chaperone [41•, 42]). Rather, the Ri-plasmids of these strains encode two proteins termed GALLS-FL (full-length GALLS protein) and GALLS-CT (C-terminal region of GALLS protein). GALLS can substitute for VirE1 and VirE2 in A. tumefaciens [43]. Other than the T4SS and

Conclusions

Many nucleic acids enter a cell from external sources, whether from bacteria or viruses. For many of these nucleic acids, the final destination is the nucleus where they either replicate or integrate into the host genome. Targeting of nucleic acids to the nucleus involves associated proteins that may derive from the invading organism, the host, or both. When Agrobacterium transfers T-DNA to host cells, it also transfers several effector proteins that aid in protecting the DNA and targeting it

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I thank Drs Walt Ream and Lan-Ying Lee for their critical reading of this manuscript, and Ms Mei-Jane Fang, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, for confocal microscopy work. Work in the author's laboratory is funded by the US National Science Foundation, the Biotechnology Research and Development Corporation, the US Department of Energy through the Corporation for Plant Biotechnology Research, and Dow AgroSciences.

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