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polynucleotide vaccination. This suggests that the technology may be generally applicable. Thomas Braciale is correct in pointing out that the mechanism of antigen presentation after the injection of plasmid DNA has not been elucidated. However, based on our recent studies, it is not necessary to propose that antigen production by nonmuscle cells is required for the generation of immune responses. Expression of influenza nucleoprotein (NP) in muscle cells, achieved by transplantation of transfected myoblasts, was sufficient to generate both antibodies and cytotoxic T cells (CTLs) (J.B. Ulmer et al., unpublished). It is not yet known whether muscle cells can function as antigen-presenting cells under these circumstances, or whether secreted NP was internalized, processed and presented by specialized macrophages or dendritic cells4. In either case, uptake of DNA by nonmuscle cells would not be obligatory. While polynucleotide vaccination has not been directly shown to lead

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to persistence of the viral antigen, injection of DNA encoding luciferase resulted in long-term expression by muscle cellss. Potential reasons for this include the longevity of myocytes and a possible lack of an appropriate CTL epitope in this protein. In the case of NP, hightiter antibodies and CTLs persisted in mice for at least 13 months after a single injection of DNA 6. As Braciale points out, antigen expressed by actively dividing cells would be unlikely to persist because of cell turnover and CTLmediated lysis. These results, however, suggest that antigen expression by muscle cells persists for a very long time after DNA injection, and that at least some NP-expressing muscle cells can evade destruction by CTLs. Finally, several theoretical safety considerations are associated with polynucleotide vaccination, and these will be examined carefully. Two. key issues are the potential integration of plasmid DNA into chromosomes and the generation of anti-DNA antibodies. Work pub-

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lished to date has not detected either integration s or anti-DNA antibodies 7 after DNA injection. Although there is no a priori reason to believe that these will occur or that chronic expression of antigen will lead to immune dysregulation and autoimmune injury, such safety studies need to be carried out. Jeffrey B. Ulmer, John J. Donnelly and Margaret A. Liu Dept Virus and Cell Biology, Merck Research Laboratories, 16-3 West Point, PA 19486, USA References

1 Wang, B. et al. (1993)Proc. Natl Acad. $ci. USA 90, 4156-4160 2 Rhodes,G.H. et al. AIDS Res. Hum. Retroviruses (in press) 3 Cox,G.J.M. et al. (1993)J. Virol. 67, 5664-5667 4 Rock,K.L.et al. (1993)J. Immunol. 150, 438-446 5 Wolff,J.A. et al. (1992)Hum. Mol. Genet. 1,363-369 6 Yankauckas,M. et al. DNA Cell Biol. (in press) 7 Jiao, S. et al. (1992)Hum. Gene Ther. 3, 21-33

New insights on T-DNA transfer Charles H. Shaw 'n the eternal summer of A.A. Milne's T h e H o u s e at P o o h . C o r n e r , Winnie the Pooh discovered the phenomenon 'Poohsticks'l: 'That's funny,' said Pooh. 'I dropped it on the other side,' said Pooh, 'and it came out on this side! I wonder if it would do it again?' 'Poohsticks' is a game that involves dropping sticks on the upstream side of a bridge and observing their appearance on the downstream side. Thus, like T-DNA transfer from A g r o b a c t e r i u m t u m e f a c i e n s to plant cells, the uncertainty is not over the initial and final events, but what happens between. A

clutch of recent papers 2-s offer some ripples of understanding. Agrobacterium

tumefaciens

causes crown gall tumour on plants by transfer to the plant chromosome of a part of the Ti plasmid, which is known as T-DNA. The transfer is mediated by the products of the virulence (vir) region of the Ti plasmid. Wounded plants release chemoattractant phenolics (such as acetosyringone) that cause the vir genes to be induced, through the actions of VirA and VirG. The VirD1 topoisomerase and VirD2 site-specific endonuclease act in C.H. Shaw is in the Dept of Biological Sciences, University of Durham, South Road, Durham, UK DH1 3LE.

concert to nick the T-DNA at its ends, which are delineated by 25 bp repeats, and generate a single T-strand 6. VirD2 has at least two additional functions: (1) it becomes tightly bound to the 5' end of the T-strand, forming a relaxation intermediate 2, and (2) it may act as a pilot protein because it has two separate nuclear localization signals in the amino- and carboxyterminal halves. The T-strand is stabilized into an elongated T-complex by the binding of VirE2 [possibly assisted by VirE1 (Ref. 7)], a singlestranded DNA-binding protein that also appears to be involved in nuclear localization s. The 11 products of the virB locus are thought to form a transmembrane pore,

© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00

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through which the T-complex may leave the bacterial celP. Thus the creation of the T-complex is understood in outline and the structure of the final T-DNA in the plant chromosome is well known. Where the uncertainty lies is over the exact method by which the T-complex leaves the bacterial cell, enters the plant cell and becomes integrated into the plant DNA. Using sequence information and subcellular fractionation, the location and possible functions of the virB products have been inferred. Berger and Christie have now confirmed by mutational analysis that the nucleotide-binding domain of VirB4 is essential for virulence3. Interestingly, like VirB11, which has ATPase and kinase activities, VirB4 has no signal-peptide or membrane-localization sequences; however, both proteins fractionate with the cytoplasmic membrane. Thus VirB4 and VirB11 may be associated with the inner face of the membrane and may be involved in activation of the T-complex for transfer. Koukolikova-Nicola et al. have investigated the roles of the products of the virD gene4. VirD3 appears to have no discernible role in T-DNA transfer, while VirD4, which appears to be localized to the inner membrane, may form part of the T-complex export apparatus. By inserting a viral genome multimer into the T-DNA, they could differentiate between T-DNA transfer and integration. During 'agroinfection', the viral genome escapes on T-DNA transfer, which leads to infection even in Agrobacterium mutants incapable of integration. By this method they showed that the carboxy-terminal half of VirD2 was not required for T-DNA transfer, but was required for integration and stable transformation, possibly because of the stronger nuclear localization signals near the carboxyl terminus. In Agrobacterium, virF is something of an enigma. It is absent from some Ti plasmids and appears to be essential for virulence only towards certain plant species, such as Nicotiana glauca. Avirulent mutants of virF can be comp-

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VirA q ~ ,

|

I-~~M Periplasm I I Cytoplasm

j / , pH

I

/~

<10-7M

I--I "~'~'L~) ~q~"" (~cetosyringone) ~

~ _ _ ~

~-SM

VirA

H474 ~

Inductionof virgenes

Chemotaxis

Fig. 1. Hypothetical scheme for phenolic sensing by Agrobacterium tumefaciens showing the major proteins involved: VirA (represented by a line connecting distinct blocked domains), VirG (shaded oval), two putative phenolic-binding proteins (shaded ovals with question marks) and ChvE (rectangle). ChvE is a periplasmic glucose-binding protein that interacts with a domain of VirA to enhance the induction of virgenes. VirA is also a pH sensor. In response to phenolics, the cytoplasmic domain of VirA is phosphorylated at histidine 474 (H474) and the phosphate is transferred to aspartate 52 (D52) of VirG; both events are required for chemotaxis and the induction of virgenes. However, the periplasmic domain of VirA is involved in chemotaxis but not in the induction of virgenes. Structural and concentration differences in the ligands required suggest that these processes require the intercession of distinct phenolic-binding proteins. Thus, at low concentrations (10-5M), acetosyringone may also be bound by a lower-affinity binding protein, which interacts with the transmembrane and cytoplasmic domains of VirA to trigger a much higher level of phosphorylation. This produces sufficient phospho-VirG to mediate binding to the vir box and thus the induction of the virgenes.

lemented by co-infection with helper strains to restore virulence. This property is shared with virE mutants, suggesting that VirF must be transported from the bacterial cell to perform its function. Regensburg-Tu'/nk and Hooykaas have now shown that transgenic N. glauca plants expressing VirF are converted into susceptible hosts for Agrobacterium strains that have defects in virF (Ref. 5). T-DNA uptake is more efficient in the VirF-expressing transgenic plants. Unfortunately, database searches have failed to provide a pointer to what VirF might do in plants 9 (C.H. Shaw, unpublished). Other

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vir products also function in planta: VirE2 and VirD2 specify nuclear localization and VirD2 has some sequence similarity to DNA ligase 7. Thus, a solution to the question of how the T-DNA becomes integrated may not be far away. In 'Poohsticks' the signal to start the game was Rabbit shouting 'Now '1. What is the signal that initiates T-DNA transfer? Three recent papers offer some intriguing insights into this question 1°-12.The sensing of wound phenolics involves phosphorylation of a membrane protein, VirA, and transfer of the phosphate to a cytoplasmic transcriptional activator, VirG; both

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these events are required for chemotaxis and vir induction 11. The periplasmic domain of VirA is required for chemotaxis n and tumorigenesis n, but not for vir induction 12a3. However, phenolics may not interact directly with VirA at all, as a number of distinct chromosomally encoded periplasmic and membrane phenolic-binding proteins (PBPs) have been identified 1°. A hypothesis to explain how the VirA-G system mediates two responses to the same ligand is outlined in Fig. 1, which also shows the role of VirA in sensing pH and sugars 7. Sensing differing phenolic concentrations requires two PBPs with different ligand affinities. The high-affinity PBP (possibly periplasmic) interacts with the periplasmic domain of VirA, while the low-affinity PBP (possibly membrane-bound) associates with the cytoplasmic linker domain, activating and stabilizing VirA. The

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high-affinity PBP triggers a low level of phosphorylation, and the limited amount of phospho-VirG released has a higher affinity for the chemotaxis pathway than it does for inducing the vir genes, thus mediating chemotaxis at low phenolic concentrations. At higher ligand concentrations, the lowaffinity PBP triggers a greater degree of phosphorylation, resulting in sufficient phospho-VirG being formed to participate in significant induction of the vir genes. So what is the future for Agrobacterium research, and how can the mystery of T-complex export and integration be solved? Perhaps Eeyore had the right ideal: Tigger and Eeyore went off together, because Eeyore wanted to tell Tigger How to Win at Poohsticks, which you do by letting your stick drop in a twitchy sort of way, if you understand what I mean, Tigger.

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References

1 Milne,A.A. (1928) The House at Pooh Comer, Methuen 2 Filichkin,S.A.and Gelvin,S.B. (1993) Mol. Microbiol. 8, 915-926 3 Berger,B.R. and Christie,P.J. (1993) J. Bacteriol. 175, 1723-1734 4 Koukolikova-Nicola,Z. et aL (1993) J. Bacteriol. 175, 723-731 5 Regensburg-Tu'ink,A.J.G. and Hooykaas,P.J.J. (1993) Nature 363, 69-71 6 Zambryski,P. (1992) Annu. Rev. Plant Physiol. 43,465-490 7 Winans,S.C. (1992) Microbiol. Rev. 56, 12-31 8 Citovsky,V. et al. (1992) Science 256, 1802-1805 9 Melchers,L. et al. (1990) Plant Mol. Biol. 14, 249-259 10 Lee,K. et al. (1992) Proc. Natl Acad. Sci. USA 89, 8666-8670 11 Palmer,A.C.V. and Shaw,C.H. (1992) J. Gen. Microbiol. 138, 2509-2514 12 Chang, C-H. and Winans, S.C. (1992) J. Bacteriol. 174, 7033-7039 13 Melchers,L.S. etal. (1989) EMBO]. 8, 1919-1925

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