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The Six Functions of Agrobacterium Vire2

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The Six Functions of Agrobacterium Vire2 Commentary The six functions of Agrobacterium VirE2 Doyle V. Ward and Patricia C. Zambryski* Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 grobacterium tumefaciens is a soil- Aborn pathogen with the unique ability to genetically transform plants. Agrobac- terium infects plant wound sites, causing crown gall disease. This agriculturally sig- nificant disease results from the transfer of a single-stranded (ss) segment (the T-strand) of the bacterium’s tumor- inducing plasmid to the host cell (re- viewed in refs. 1–3; Fig. 1). On integration into the host genome, genes encoded by the T-strand direct the synthesis of plant growth hormones, resulting in tumorous proliferation of plant cells. T-strand genes also cause the plant to produce opines, compounds that represent a major carbon and nitrogen source uniquely metabolized by the bacterium. Thus, Agrobacterium has evolved a mechanism to genetically engi- neer host cells to create a favorable niche for itself. This natural ability has been coopted by researchers who use Agrobac- terium to engineer plants for agricultural and research purposes. Fig. 1. The six functions of VirE2. The vir regulon, encoding the major loci virA-E and virG-H, is expressed Of great interest is the mechanism that on detection of plant wound signals. VirD2 and VirD1 liberate the T-strand and VirD2 remains covalently transfers DNA across the inner and outer bound to the 5Ј end. 1: VirE2 coats the T-strand, protects it from degradation, and maintains it in a bacterial membranes and the plasma transportable conformation. 2: VirE2 associates with VirE1, required for VirE2 export. 3: VirE2 exits membrane of the host. Although the spe- Agrobacterium via the type IV exporter independently, or as part of the T-complex. Alternatively, VirE2 may exit by an alternate pathway (pink; ref. 4). 4: VirE2 forms a pore in the plant plasma membrane cific mechanics are unknown, it is gener- allowing passage of the T-complex and coats the T-strand in the plant cytoplasm. 5: VirD2 and VirE2 ally assumed that a type IV exporter trans- interact with plant cytoplasmic chaperones (RocA, Roc4, and CypA). Other factors (AtKAP␣, VIP1) may COMMENTARY fers the T-strand across the bacterial target the T-complex to the nucleus. 6: VirE2 interacts with nuclear factors (VIP2) that mediate interaction membranes in a process analogous to con- with chromatin and facilitate integration of the T-strand. jugation. Agrobacterium is the prototypi- cal example of a pathogen using a type IV exporter. Homologs of the exporter pro- VirE2 binds the T-strand cooperatively, VirE2 from Agrobacterium extracts and teins are involved in bacterial conjugation, without sequence specificity, and protects immunogold labeling of VirE2 ‘‘strings’’ in and are virulence proteins in several im- it from degradation (8–10). VirE2 and situ (8, 14). There is evidence, however, portant human pathogens including Bor- VirD2 contain nuclear localization se- that VirE2 can enter the plant cell inde- detella pertussis (whooping cough), Bru- quences (NLS) that promote nuclear pendent of the T-strand. First, coinfection cella suis (brucellosis), Helicobacter pylori uptake of the T-complex (11, 12). with two Agrobacterium strains, one lack- (gastric ulcers), Legionella pneumophila VirE2:ssDNA complexes microinjected ing T-strand but containing VirE2, the (Legionnaire’s disease), and Rickettsia into plant cells give rise to nuclear accu- other lacking VirE2 but containing T- prowazekii (epidemic typhus) (5, 6). mulation of the ssDNA that can be strand DNA, lead to successful plant The biophysical studies of Dumas et al. blocked by nuclear import inhibitors (13). transformation (15). Neither strain alone is capable of transformation. Second, (7) reported in this issue are surprising VirE2 may also assist nuclear uptake of Agrobacterium lacking VirE2 transforms because they suggest that a single protein, the T-complex by keeping the T-strand in transgenic plants that express the VirE2 an unfolded state (9). VirE2 forms a membrane channel that protein (11), demonstrating that VirE2 is transfers the T-strand through the plant There is little doubt that VirE2 associ- not required for T-strand export. Finally, plasma membrane. This result has impli- ates with the T-strand in the plant cell; VirE2 export can be inhibited without cations for our understanding of Agrobac- however, there is controversy over affecting T-strand export (16, 17). terium mediated transformation and, po- whether this also occurs in the bacterium. tentially, for delivery of DNA in gene VirE2’s strong cooperative T-strand bind- therapy. ing and abundance suggest association in See companion article on page 485. VirE2 is one of the most abundant Vir Agrobacterium, as does the observed co- *To whom reprint requests should be addressed. E-mail: proteins. Fig. 1 summarizes its functions. immunoprecipitation of the T-strand with [email protected]. PNAS ͉ January 16, 2001 ͉ vol. 98 ͉ no. 2 ͉ 385–386 Downloaded by guest on October 2, 2021 Given the functions already attributed way. Legionella dotB mutants (dotB is ho- VIP1, a bZIP protein, promotes nuclear to VirE2, few would have predicted a mologous to Agrobacterium virB11) are import of VirE2. VIP2 is homologous to direct role in T-strand transfer. VirE2 is defective in pore insertion (21). Whereas Drosophila Rga, a protein thought to me- hydrophilic with no predicted membrane it is unlikely that the Legionella pore trans- diate interaction between chromatin pro- spanning domains, yet fractionation ex- fers DNA, type IV exporters in other teins and transcription complexes (28, 32). periments detect a small but significant bacterial pathogens may insert pore pro- VIP2 may promote intranuclear transport portion of VirE2 in the bacterial outer teins in the membranes of their hosts. or T-strand integration. Similar factors membrane and periplasm (8). This obser- The results of Dumas et al. (7) may may be required for efficient transforma- vation prompted Dumas et al. (7) to in- assist development of gene therapy tech- tion of non-plant organisms. vestigate the significance of VirE2 mem- nologies. VirE2 mediated transformation In summary, VirE2 performs an unusu- brane association. In vitro biophysical may avoid problems inherent in the use of ally large number of functions (their Fig. approaches demonstrated that VirE2 in- viral delivery systems. Once transported 1). Dumas et al. (7) report a sixth function: teracts with lipids and, interestingly, forms across the plasma membrane of the recip- VirE2 forms channels in lipid bilayers. large, anion-selective, voltage-gated chan- ient cell, however, additional factors may Thus, VirE2 may insert in the plant plasma membrane and facilitate passage nels selective for transport of ssDNA. be required for nuclear uptake and inte- of the T-strand (Fig. 1, function 4). Addi- Formation of pores large enough to gration of the DNA. These factors may be tional work will be required to confirm allow passage of ssDNA may have dele- lacking in non-plant hosts. For example, this proposed function in vivo, and it is terious effects in plant cells containing VirD2 contains an NLS that allows its critical to test whether these results can be VirE2. Voltage gating in vitro suggests that nuclear import in animal and yeast cells ͞ repeated with larger molecules the size of the opening closing of the channels may (23–26). Although VirE2 localizes to plant the T-strand. Interesting questions arise be regulated in vivo. Whereas gating may nuclei (11, 13, 27), it does not exhibit from their results. How might VirE2, after moderate the effects of pores formed in nuclear import in intact animal cells (25, membrane insertion, ‘‘uninsert’’ so as to plant cells, it may be the role of the specific 26, 28). None of these experiments ad- participate in nuclear import? Does VirE1 chaperone, VirE1, to prevent the forma- dress integration of the DNA in the host also participate in the insertion process? tion of pores in Agrobacterium. VirE1 is genome. It is likely that specific host fac- The resourcefulness of the VirE2 protein required for export of VirE2 from tors are required at this critical step. is remarkable, performing multiple criti- Agrobacterium and may inhibit T-strand Plant proteins have been identified in cal functions at several points in T-strand binding (18–20). Arabidopsis thaliana that physically inter- transfer. Will future research determine a A pore-forming protein may also be act with VirD2 and VirE2 (28). AtKAP␣, seventh function? Perhaps, after evolving exported by a L. pneumophila type IV which belongs to a protein family known six functions, virE2 rested. exporter (21). Legionella multiplies in hu- to mediate nuclear import (29), interacts man macrophages inside a specialized with the VirD2 NLS, but not with VirE2 We thank V. Citovsky for critical comments and phagosome that Legionella is able to ma- (30). Three isoforms of cyclophilins, personal communications. The authors ac- nipulate and thus inhibit phagosome– RocA, Roc4, and CypA, also interact with knowledge the support of a National Science Foundation Postdoctoral Research Fellowship lysosome fusion (22). Insertion of a pore- VirD2 and may act as VirD2 chaperones in Microbiology (to D.V.W.) and the Novartis forming protein in the membrane of the in the plant cell (31). Two VirE2 interact- Agriculture Discovery Institute (to the Depart- phagosomal compartment may interfere ing proteins, VIP1 and VIP2, have been ment of Plant and Microbial Biology at the with its targeting in the endocytic path- identified (1). When expressed in yeast, University of California, Berkeley). 1. Tzfira, T. & Citovsky, V. (2000) Mol. Plant Pathol. 12. Howard, E. A., Zupan, J. R., Citovsky, V. & 23. Relic, B., Andjelkovic, M., Rossi, L., Nagamine, 1, 201–212. Zambryski, P. C. (1992) Cell 68, 109–118. Y. & Hohn, B. (1998) Proc. Natl. Acad. Sci. USA 2.
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