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VirB/D4-Dependent Protein Translocation from Agrobacterium into Plant Cells

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Science  03 Nov 2000:
Vol. 290, Issue 5493, pp. 979-982
DOI: 10.1126/science.290.5493.979

Abstract

The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.

The Gram-negative soil bacteriumAgrobacterium tumefaciens causes crown gall disease on plants. During the infection process, a segment of the bacterial tumor-inducing (Ti) plasmid, the T region, is transferred to recipient plant cells, ultimately resulting in phytohormone overproduction (1–3). Transfer of the T region occurs as a single-stranded DNA-protein complex, resembles conjugation in many ways, and is mediated by a set of Virulence (Vir) proteins, which are encoded by the Ti plasmid. Transport requires the 11 VirB proteins, which constitute the proposed channel, and the coupling factor VirD4 (4, 5). Agrobacteriumstrains carrying mutations in virE2 or virF are avirulent on (certain) plants; however, tumors are formed upon coinoculation of these vir mutants with an avirulent helper strain, which lacks the T region but contains a complete virregion (6). Initially, it was thought that this “extracellular” complementation was due to the secretion of an essential enzyme or metabolite in the medium. However, it later became apparent that both the single-stranded DNA binding protein VirE2 and the F-box protein VirF have a function within the plant cell during tumor formation, given that virE2 and virFmutants can incite tumors on transgenic plants that produce VirE2 or VirF, respectively (7, 8). Both transferred DNA (T-DNA) transport and extracellular complementation require an intact VirB/D4 transport system; therefore, we hypothesized that the transport machinery might mediate transport of proteins besides that of the nucleoprotein T complex (7). However, until now it could not be formally excluded that the Vir proteins only move into the plant cell as part of a complex with the T strand.

We used the site-specific recombinase Cre from bacteriophage P1 [for review, see (9)] to detect translocation of a functional Cre enzyme by its fusion to VirE2 or VirF from Agrobacteriuminto recipient plant cells. To this end, we selected transgenicArabidopsis thaliana C24 line 3043 (10,11) (Fig. 1), in which a lox-flanked (floxed) DNA segment prevents expression of a neomycin phosphotransferease (nptII) marker gene. The introduction of a T-DNA coding for Cre recombinase (11) in this plant line led to efficient deletion of the floxedDNA, resulting in the fusion of the 35S promoter region to the nptII gene as visualized by resistance to kanamycin [1.3 ± 0.6 kanamycin-resistant (Kmr) calli per root explant; Fig. 2B].

Figure 1

Schematic representation of a Cre-mediated excision event in pSDM3043 (11), leading to reconstruction of a functional lox-nptII translational fusion. a and b are the primer binding sites. LB and RB, left and right T-DNA border sequences; npt, neomycin phosphotransferase;bar, bialaphos resistance gene; pDE35S, promoter region of the 35S transcript of cauliflower mosaic virus with a double enhancer sequence. Arrows indicate lox sites.

Figure 2

Root explants ofArabidopsis line 3043 on medium containing kanamycin, 3 to 4 weeks after cocultivation with (A) LBA1100 expressing Cre, (B) LBA1100 harboring a T-DNA vector expressing Cre, (C) LBA1149 expressing the Cre::VirE2 fusion protein, or (D) LBA2561 expressing the NLS::Cre::VirFΔ42N fusion protein. (E) PCR analysis with primers a and b (Fig. 1) on Kmr shoots obtained after cocultivation with strains shown in (C) (lanes 1 to 4) and (D) (lanes 6 to 9) shows excision and original target fragments. Lane 5, control PCR on DNA from target line 3043. Scale bar, 1 cm.

Next, to assay for protein transport, Cre recombinase was expressed under control of the vir induction system inAgrobacterium, either alone or as an NH2-terminal or COOH-terminal fusion with VirF or VirE2, respectively (12). Expression of the fusion proteins inAgrobacterium was confirmed by Western (immunoblot) analysis (13). Recombination activity of the fusion proteins was detected by their ability to perform an excision event in plasmid pSDM3043, which was introduced into the relevantAgrobacterium strains (13). After cocultivation of root explants (14) of Arabidopsis line 3043 with disarmed Agrobacterium strain LBA1100 (15) (wild-type vir, lacking T-DNA) harboring nontransmissable plasmid pRL662 (12) expressing the Cre recombinase alone, we detected no or sometimes a single survivor on medium containing kanamycin (on average one callus per 600 explants;Fig. 2A). This small number of Kmr background calli was also obtained upon cocultivation of plant line 3043 withAgrobacterium strains not expressing Cre. We can therefore conclude that bacterially expressed Cre recombinase is not transferred to plant cells from Agrobacterium.

However, when Cre was fused to the NH2-terminal region of the Vir protein (Cre::VirE2; NLS::Cre::VirF), but not when fused to the COOH-terminus (VirE2::Cre; NLS::VirF::Cre), cocultivation with plant line 3043 was followed by a more efficient recovery of Kmr calli (9 ± 2 calli per 100 explants for Cre::VirF; 6 ± 2 calli per 100 explants for Cre::VirE2; Fig. 2C). There was no consistent difference when the strain expressed wild-type VirE2 or VirF protein in addition to the fusion proteins {LBA1100 compared with LBA1149 [virE2::Tn3HoHo1(15)] or LBA2561 [ΔvirF(16)]}. Apparently, the transport channel functions such that both wild-type Vir proteins and the Cre::Vir fusion proteins are transferred efficiently and concurrently.

Polymerase chain reaction (PCR) analysis (17) was performed on Kmr shoots to show that resistance was indeed caused by translational fusion of the 35S promoter region to the nptII coding region because of Cre activity. A PCR reaction with primers annealing in the 35S promoter region (Fig. 1; primer a) and the nptII sequence (primer b) resulted in amplification of a 0.7-kb fragment, diagnostic for excision, whereas the expected 2.3-kb fragment was detected in DNA samples from the original plant line 3043. The amplification of target DNA fragments in DNA samples from the Kmr shoots, besides the excision fragments, shows that Cre-mediated recombination occurred in cells of the original homozygous target line 3043 (Fig. 2E).

In summary, Agrobacterium can deliver the Cre recombinase into plant cells, resulting in detectable excision events, but only when expressed as a fusion protein attached to the NH2-terminus of VirE2 or VirF. This implies that the COOH-termini of VirF and VirE2 need to be free to allow transport, possibly because important (transport) signals are located there. Cocultivation of 3043 root explants with Agrobacteriumstrains expressing a Cre::VirF fusion lacking 42 NH2-terminal amino acids of VirF (18) (NLS::Cre::VirFΔ42N) resulted in an increase in the number of Kmr-resistant calli (54 ± 23 calli per 100 explants; Fig. 2D). This shows that the domain responsible for transport is not located in this NH2-terminal region. In fact, this region does contain an F box (19), which might confer instability on the protein in plant cells or lead to retention of the protein in the cytoplasm through its binding with F-box–interacting proteins. Therefore, deletion of this domain might indirectly lead to an enhanced nuclear delivery. Additional evidence for a COOH-terminally located transport signal was obtained by using a larger 166–amino acid NH2-terminal deletion (18) of VirF (NLS::Cre::VirFΔ166N). When fused to Cre and expressed in Agrobacterium, the remaining 37 COOH-terminally located amino acids were sufficient for obtaining Kmr calli with similar efficiency as NLS::Cre::VirFΔ42N (40 Kmr calli ± 7 per 100 root explants). Thus, we conclude that a transport signal is present in this small region. Close inspection of this area and comparison with that of VirE2 revealed the presence of a common motif of three amino acids (Arg-Pro-Arg).

Next, we examined which specific virulence functions were necessary for protein transport. Given that removal of the 42 NH2-terminal amino acids of VirF resulted in about fivefold higher frequencies of excision after cocultivation, we transferred plasmids harboring NLS::cre::virFΔ42Nas well as Cre::virE2 into the virmutants LBA1142 (virA), LBA1143 (virB4), LBA1144 (virB7), LBA1145 (virG), LBA1146 (virC2), LBA1147 (virD2), LBA1148 (virD4), and LBA1150 (virD1) (15). Additionally,NLS::cre::virFΔ42N was introduced into LBA1149 (virE2) and Cre::virE2 into LBA2561 (virF). After cocultivation of 3043 root explants with strains carrying transposon insertions invirA, virG, virB4, virB7,virD1, virD2, and virD4, no Kmr calli were selected (Table 1), indicating that the expression of the affected genes is essential for transport of both NLS::Cre::VirFΔ42N and Cre::VirE2. In contrast, in a virC2 mutant, protein transfer was not inhibited and calli were obtained at high efficiency. Furthermore, the VirE2 protein was apparently not essential for transport of VirF and VirF was not necessary for transport of VirE2. To rescue the distal functions of the virD operon in the mutants LBA1147 and LBA1150, we expressed VirD3 and VirD4 in trans in these strains. As expected, this resulted in restoration of fusion protein transport (Table 1), showing that VirD1 and VirD2 are not essential. Thus, protein translocation depends on the VirA/VirG regulatory system, necessary for the expression of the other virulence genes, and otherwise on the VirB/D4 proteins that are known to form a putative transport channel and a coupling factor, respectively.

Table 1

Efficiency of transfer of Cre::virE2 and NLS::Cre::virFΔ42N fusion proteins from differentAgrobacterium mutants (derived from LBA 1100) in representative experiments. The number of Kmr calli was estimated 3 weeks after cocultivation with target root explants. Two Petri dishes were used per strain in each experiment. Transposon insertion mutations may affect other downstream-located genes in the operon. ND, not determined.

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In summary, we show directly that the Agrobacterium VirB/D4 transport system mediates the transfer of VirE2 and VirF proteins into plant cells independently of T-DNA transport. These data support the earlier suggestion based on extracellular complementation experiments that VirE2 and the VirD2–T-DNA nucleoprotein can be transported separately and form T complexes in the plant cell. On the basis of sequence comparison, the Agrobacterium VirB/D4 transport system was classified recently within a family of eubacterial transport systems, referred to as type IV secretion systems (4,5, 20). Members include structures used by broad host range conjugative plasmids for DNA transfer, but also the Ptl transporter of the human pathogen Bordetella pertussis(21), which uses it for the secretion of proteinaceous pertussis toxins in human cells. Here, we show that a system that is involved in translocation of nucleoprotein complexes has also kept the ability to introduce monomeric proteins into recipient cells, adding a body of evidence to our earlier proposal that DNA delivery systems have evolved from protein secretion systems (7). This is in line with our finding that the coupling factor VirD4, of which homologs were speculated to be the interface between the relaxosome of conjugative structures and the transport apparatus, is also an essential component for protein transport.

We propose that NH2-terminal fusions to either (parts of) VirF or VirE2 might deliver functional proteins across kingdom boundaries, for purposes in which proteins are required in recipient cells only transiently. A system based onAgrobacterium may be functional for plants, yeast (22), and fungi (23). The similarity between family members of eubacterial type IV secretion systems suggests that an approach similar to the one described here forAgrobacterium may also be used for the delivery of fusion proteins in mammalian cells by derivatives of the relevant pathogens that are attenuated in virulence.

  • * To whom correspondence should be addressed. E-mail: hooykaas{at}rulbim.leidenuniv.nl

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