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Host Proteins Guide Agrobacterium-Mediated Plant Transformation

neckardt{at}aspb.org

Agrobacterium tumefaciens is widely used for genetic transformation of plants because of its natural ability to transfer foreign DNA into the host plant genome. It is also a significant plant pathogen that causes crown gall disease on several agronomically important species, including grape vines, stone fruit, and nut trees (DeCleene and DeLey, 1976). After infection of a host plant, the bacterium transfers several virulence (Vir) proteins and a segment of DNA (called transfer DNA or T-DNA) from its tumor-inducing plasmid into the plant cell. With the help of Vir proteins, the T-DNA is then translocated into the host cell nucleus and integrated into the host genome, eliciting crown gall disease as oncogenes present within the T-DNA direct the uncontrolled proliferation of crown gall tumors. For use as a genetic tool, the oncogenes are removed such that genetic transformation can be effected without the development of disease. Despite the importance and widespread use of Agrobacterium-mediated transformation as a genetic tool, the mechanisms of T-DNA transfer into the plant cell and integration into the host genome are not well understood. Increased understanding of these processes will likely pave the way for improvements in transformation technology (Gelvin, 2003).

We are beginning to have a reasonable understanding of the key factors involved in the regulation of T-DNA nuclear import and genomic integration (reviewed in Zupan et al., 2000; Gelvin, 2003). T-DNA enters the plant cell bound to VirD2, an endonuclease that is involved in the cleavage of the T-DNA from the tumor-inducing plasmid, resulting in the production of the single-stranded T-DNA molecule covalently bound to VirD2 at the 5' end. Several other Vir proteins are transferred into the plant cell along with the T-DNA, including VirE2, which subsequently binds to and coats the T-DNA along its length. VirD2 and VirE2 contain nuclear localization signal (NLS) sequences and are likely involved in mediating nuclear import of the T-DNA complex through interactions with Arabidopsis proteins, including VIP1 and possibly KAP-. VIP1 binds VirE2, and antisense inhibition of VIP1 expression has been correlated with a reduction in nuclear targeting of VirE2 (Tzfira et al., 2001). AtKAP- belongs to a family of proteins known as importins, which are known to be involved in nuclear translocation of proteins containing NLS sequences. Ballas and Citovsky (1997) showed that AtKAP- binds VirD2 in vitro in an NLS-dependent manner, but in vivo evidence for a role for this protein in nuclear import of T-DNA is lacking.

After nuclear import, bound VirD2 and the coat of VirE2 and VIP1 proteins are removed before (or during) T-DNA integration into the host genome. Tzfira et al. (2004) have shown that bacterial VirF, which localizes to the plant cell nucleus along with the T-DNA complex, is an F-box protein that interacts with VIP1 and destabilizes both VIP1 and VirE2, presumably by targeting them for proteasomal degradation. VirF also interacts with the Arabidopsis Skp1 homolog ASK1 and thus may form an SCF (Skp1-cullin-F-box) complex with ASK1 (Schrammeijer et al., 2001). Tzfira et al. (2004) found that VirF did not interact with or destabilize VirD2, which suggests that VirD2 may remain bound to the T-DNA until a later stage. There is evidence that VirD2 plays an additional role in guiding the efficiency or precision of genomic integration (Mysore et al., 1998; Tinland et al., 2000).

By contrast, far less is known about the mechanism and regulation of T-DNA and Vir protein transport into the plant cell. The bacterium uses a Type IV secretion system (T4SS) to transfer the T-DNA complex and Vir proteins across the double membrane of the bacterial envelope (reviewed in Cascales and Christie, 2003). The bacterial T4SS is a complex of subunits encoded by 11 virB genes and virD4. There are two main structural components to the complex: a membrane-associated transporter complex that spans the bacterial double membrane and a closely associated T-pilus, which is an exocellular filamentous appendage. Bacterial pili perform various functions associated with mediating bacterial adhesion to specific targets in the environment. The T-pilus is structurally and functionally similar to the F-pilus, which mediates cell–cell adhesion and transfer of DNA during bacterial conjugation (Zupan et al., 2000; Cascales and Christie, 2004). The T-pilus is essential for T-DNA transfer to a foreign host and for Agrobacterium virulence, but its precise function, along with the mechanism of T-DNA and Vir protein transport across the plant plasma membrane, remains unknown. In this issue of The Plant Cell, Hwang and Gelvin (pages 3148–3167) report on the identification of four Arabidopsis proteins that interact with the main T-pilus protein, VirB2, and show that the presence of these proteins is required for efficient transformation.

The authors used a yeast two-hybrid assay to identify four Arabidopsis proteins that interact with VirB2: three related proteins of previously unknown function named VirB2-Interacting (BTI) proteins (BTI1-3) and a Ras-related small GTPase, AtRAB8. Further tests in yeast showed that all four proteins interact specifically with VirB2 and not with any other Agrobacterium Vir proteins tested. The BTI proteins were found to interact with each other and with AtRAB8 in vitro. Experiments using antisense RNA and RNA interference directed against each of the BTI proteins and AtRAB8, and overexpression of BTI proteins, further suggested that all four proteins have activity associated with efficient transformation. Interestingly, a transient increase in the levels of BTI proteins was observed immediately after Agrobacterium infection. Finally, localization of green fluorescent protein:BTI fusion proteins with confocal microscopy showed that the BTI proteins preferentially localize to the periphery of root cells in transgenic plants (see figure). The authors hypothesize that the three BTI proteins and AtRAB8 interact with the T-pilus in vivo and are involved in the initial interaction of Agrobacterium with plant cells.

View larger version (92K): [in this window] [in a new window]   Arabidopsis BTI Proteins Accumulate at the Plant Cell Periphery and Interact with the Major Agrobacterium T-Pilus Protein VirB2. The figure shows a confocal microscopic image of a transgenic Arabidopsis root expressing a BTI1:GFP fusion protein.

The work of Hwang and Gelvin provides some of the first information on the black box surrounding the function of the T-pilus and factors involved in regulating transport of the T-DNA complex across the plant plasma membrane. It remains unknown whether the T-pilus forms a conduit through which T-DNA and Vir proteins are transported into the plant cell or whether it merely serves to make first contact with the plant cell surface and plays some other role in the formation of a transport complex across the plasma membrane (Gelvin, 2003). The identification of these four plant proteins that localize to the plant cell periphery and specifically interact with VirB2 should help to guide further investigation of this question.

In addition, knowledge of plant proteins involved in the regulation of Agrobacterium-mediated transformation may lead to improvements in engineering of plant transformation systems and the development of this valuable tool in recalcitrant species. Using another approach, Nam et al. (1999) initiated a project that has led to the identification of numerous T-DNA insertional mutants of Arabidopsis that are resistant to Agrobacterium-mediated transformation (rat). More than 125 rat mutants have been identified that disrupt various steps in the transformation process, ranging from the initial attachment of bacteria to plant roots to nuclear import and T-DNA integration (Zhu et al., 2003). It is evident that successful transformation requires the participation and assistance of numerous host proteins.

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Plant Proteins That Interact with VirB2, the Agrobacterium tumefaciens Pilin Protein, Mediate Plant Transformation

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Plant Cell 2004 16: 3148-3167. [Abstract] [Full Text]  

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