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Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005? 200557615221531Original ArticleAgrobacterium colonization of unwounded plantsA. Brencic, E. R. Angert and S. C. Winans Molecular Microbiology (2005) 57(6), 1522–1531 doi:10.1111/j.1365-2958.2005.04763.x Unwounded plants elicit Agrobacterium vir gene induction and T-DNA transfer: transformed plant cells produce opines yet are tumour free Anja Brencic, Esther R. Angert and Stephen C. Winans* Department of Microbiology, Cornell University, Ithaca, NY 14853, USA. Summary Agrobacterium tumefaciens is well known to cause crown gall tumours at plant wound sites and to benefit from this plant association by obtaining nutrients called opines that are produced by these tumours. Tumourigenesis requires expression of the vir regulon in response to chemical signals that are thought to be released from wound sites. Here, we examine chemical interactions between A. tumefaciens and unwounded plants. To determine whether unwounded plants can release significant amounts of vir gene inducers, we constructed an A. tumefaciens strain carrying a PvirB–gfp fusion. This fusion was strongly induced by co-culture with tobacco seedlings that have been germinated without any intentional wounding. The release of phenolic vir gene inducers was confirmed by GC/MS analysis. We also constructed a strain containing the gfp reporter located on an artificial T-DNA and expressed from a plant promoter. A. tumefaciens efficiently transferred this T-DNA into cells of unwounded plants in the absence of exogenous vir gene inducers. Many cells of seedlings colonized by the bacteria also produced octopine, which was detected using a Pocc-gfp reporter strain. This indicates transfer of the native T-DNA. However, these transformed plant cells did not form tumours. These results suggest that successful colonization of plants by A. tumefaciens, including T-DNA transfer and opine production, does not require wounding and does not necessarily cause cell proliferation. Transformation of plant cells without inciting tumours may represent a colonization strategy for this pathogen that has largely been overlooked. Accepted 8 June, 2005. *For correspondence. E-mail scw2@ cornell.edu; Tel. (+1) 607 255 2413; Fax (+1) 607 255 3904. © 2005 Blackwell Publishing Ltd Introduction Many pathogens of plants or animals can colonize their hosts in several different ways with a range of consequences. In particular, many can colonize their hosts for extended periods of time without causing any clinical manifestations. For example, Mycobacterium tuberculosis can infect the human lung in the absence of symptoms, and colonization causes disease only in a subset of those infected (Parrish et al., 1998; Stewart et al., 2003; Monack et al., 2004). Helicobacter pylori, species of Neisseria, Staphylococcus, Streptococcus, and many other bacteria that we generally consider to be serious pathogens, can frequently establish long-term infections in humans that are clinically asymptomatic (Monack et al., 2004). Agrobacterium tumefaciens has long been known to induce crown gall tumours at the wound sites of a variety of plants. These tumours can seriously impair the host (Smith and Townsend, 1907), and result from the transfer of oncogenic DNA fragments (T-DNA) from the tumourinducing (Ti) megaplasmid to the chromosomes of plant cells (Binns and Constantino, 1998; Tzfira et al., 2000; Zhu et al., 2000; Zupan et al., 2000; Gelvin, 2003; Tzfira et al., 2004). Some of the transferred genes cause uncontrolled cell division, while others direct the synthesis of bacterial nutrients called opines. A. tumefaciens, however, is not an obligate pathogen, as it can grow vigorously as a saprophyte. It can also colonize plants without causing any symptoms, forming relatively benign biofilms on roots (Matthysse and Kijne, 1998; Matthysse and McMahan, 1998). These commensal interactions between A. tumefaciens and unwounded plants have received relatively little attention, and are the topic of the present study. T-DNA transfer requires products of the vir regulon, whose transcription is induced by specific plant-released phenolic compounds in combination with several other stimuli, including plant-released monosaccharides, acidic pH and temperatures below 30∞C (Heath et al., 1995). These signals are detected by the VirA-VirG two component system, and by the periplasmic sugar binding protein ChvE (Stachel et al., 1985; 1986; Stachel and Zambryski, 1986; Heath et al., 1995). Generally, it is believed that vir gene-inducing signals are released from plants only at wound sites. Phenolics are involved in lignification and Agrobacterium colonization of unwounded plants 1523 healing of the wound, and may also be released from the wound site as antimicrobials (Dixon and Paiva, 1995). However, there are very few if any data suggesting that wounding is required for the production or release of these compounds. In one study, two phenolic inducers, acetosyringone (AS) and a-hydroxyacetosyringone, were released in higher amounts from tobacco leaf discs than from unwounded leaves (Stachel et al., 1985). However, these compounds were first isolated from cultured plant cells or roots, neither of which had been wounded (Stachel et al., 1985; 1986). While the cultured cells and tissues used in those studies may not reflect the exudates of intact plants, it nevertheless seems plausible that unwounded plants might release sufficient vir gene inducers to stimulate the vir regulon. It has long been known that inoculating A. tumefaciens on plants rarely if ever causes tumours unless the plant is wounded at the site of inoculation (14). However, the precise role of wounding in this process is unclear. As described above, plant injury may be important for the release of vir gene inducers. Wounding of plant tissues may also be important to compromise any physical barriers that might block T-DNA transfer. The waxy cuticle that coats plant epidermis may block productive physical contacts between the bacterium and the host cell envelope that are required for T-DNA transfer (Wagner and Matthysse, 1992; Swart et al., 1994; Zhu et al., 2003). However, leaf mesophyll cells and root epidermal cells are not protected by a cuticle, because they must be permeable to gasses, minerals, and water. Indeed, one study showed that T-DNA can be transferred to mesophyll cells of unwounded plants (Escudero and Hohn, 1997), although transfer in that study occurred only when the bacteria were pre-induced with a synthetic phenolic compound. It has been proposed that cell division during wound healing may play a role in tumourigenesis (Kahl, 1982; Binns and Thomashow, 1988). Dividing cells may have less rigid cell walls that can be more easily breached. In addition, DNA replication may facilitate T-DNA integration, and the dividing cells may be more sensitive to the mitogenic effects of the T-DNA encoded phytohormones. Supporting these ideas is the finding that application of exogenous auxin prior to infection stimulates plant cell transformation (Sangwan et al., 1992; Stover et al., 1997; Chateau et al., 2000). Other studies reported that Agrobacterium-induced tumours were limited to the cambium, which is a meristematic tissue and thus predisposed to cell division (Sangwan et al., 1991; Ghorbel et al., 2000). In another study, the apical meristem of maize seedlings was a preferred site for transformation (Shen et al., 1993). Wounding can cause virtually any plant cell to dedifferentiate and become meristematic (Taiz and Zeiger, 2002). This normally occurs during the process of wound healing and is followed by rapid proliferation of cells at the site of © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 injury. Therefore, wounding may be required for differentiated plant cells to begin neoplastic cell division. Wounding may also provide A. tumefaciens physical access to internal meristematic tissues such as the cambium. This study focuses on interactions between A. tumefaciens and unwounded plants. Based on the studies described above, it seemed plausible that unwounded plants could release sufficient amounts of vir gene inducers to cause strong vir expression in agrobacteria colonizing these plants. Furthermore, it seemed plausible that at least some cells of these plants could serve as targets of T-DNA transformation. Wounding might, however, be required for neoplastic proliferation of transformed plant cells, although these cells may be able to produce and release opines that can be consumed by the bacteria. To test these hypotheses, we constructed a strain of A. tumefaciens containing a PvirB–gfp fusion, and used it to assay the induction of vir genes in bacteria co-cultured with tobacco seedlings that had been germinated without any intentional wounding. We also constructed a strain containing a gfp gene expressed from a plant promoter and located within an artificial T-DNA. We used this strain to test whether unwounded plants can be transformed in the absence of exogenously added phenolic compounds. Finally, we used A. tumefaciens strains containing either a PoccQ–gfp or an ooxA–gus reporter, both of which are octopine-inducible, to test for production and release of octopine by transformed tobacco cells. Results Construction and characterization of a P virB–gfp reporter To observe in planta induction of vir genes, we constructed an A. tumefaciens strain (AB601) that contains a wild-type copy of the virB operon and a PvirB–gfp fusion carried in cis. To characterize this fusion, the strain was cultured in AB minimal medium (pH 5.8) in the presence of different concentrations of AS, and fluorescence of bacterial cultures was quantified by flow cytometry. The average fluorescence of all cultures increased with increasing AS concentrations and with time, and the fusion reached its maximal induction after two to 3 days of incubation (Fig. 1A). In the culture where no AS was added, cells were uninduced for the entire length of the experiment (Fig. 1A). Expression of the reporter was abolished by culturing the cells at neutral pH rather than acidic medium or at 37∞C rather than 27∞C (data not shown). For all AS concentrations tested, all cells in a culture eventually became fluorescent, however, each culture showed a broad distribution of fluorescence, and some cells within each culture became fluorescent considerably earlier than others (Fig. 1B). Cells incubated for 12 h with AS appeared to have a bimodal distribution of fluorescence 1524 A. Brencic, E. R. Angert and S. C. Winans vir-inducing compounds at concentrations sufficient to activate the vir regulon. To quantify the in planta vir induction, we periodically sampled six seedlings that had been inoculated as described above and vortexed them in liquid MS medium to recover the bound bacteria. Bacterial samples were then frozen at -80∞C and later assayed for vir gene induction by flow cytometry. Induction of the PvirB–gfp reporter was first detected in the sample obtained on day 3 after inoculation, and was strongest in the sample recovered on the final day (day 8) of the experiment (Fig. 3A). Bacteria in these samples showed a bimodal distribution of fluorescence (Fig. 3B). On day 8, approximately half of the cells showed a fairly uniform level of fluorescence, while the other half remained uninduced. The induced subpopulation by day 8 showed a fluorescence intensity of 100– 200 fluorescence units (Fig. 3B), which is similar to that obtained using AB liquid medium supplemented with 10 mM AS (Fig. 1B, middle column), although induction on plant surfaces required a considerably longer time interval (8 days vs. 2 days). Overall, these results confirm our hypothesis that vir genes of agrobacteria colonizing unwounded tobacco seedling are strongly induced. This was later further substantiated by our ability to detect TDNA transfer under these conditions (see below). Fig. 1. Expression of PvirB–gfp of strain AB601 cultured in AB medium (pH 5.8) in the presence of different AS concentrations. Fluorescence was detected by flow cytometry for 10 000 cells per sample. A. Average fluorescence of samples cultured with 100 mM (), 10 mM (), 1 mM (), or no AS (). B. Histograms showing distribution of fluorescence among individual cells of each sample at different time points during incubation. No fluorescent cells were detected in samples cultured in the absence of AS (data not shown). Analysis of vir-inducing compounds in seedling exudates To identify phenolic inducers released from unwounded seedlings, exudates of three types of plants (tobacco, squash and tomato) were analysed using bioassays (Miller, 1972) and by mass spectroscopy. Induction of the virB–lacZ reporter of A. tumefaciens strain A136(pCH114) intensities, with approximately half of the cells remaining uninduced. At later time points, most cultures showed greater homogeneity of GFP expression (Fig. 1B). Induction of P virB–gfp in bacteria bound to unwounded tobacco seedlings To test whether the vir regulon is induced in the presence of intact whole plants, we inoculated hydroponically germinated tobacco seedlings with strain AB601, and placed them on solidified medium containing MS salts and lacking any carbon source. Care was taken not to injure these seedlings in any way. Periodically, individual seedlings were examined by epifluorescence microscopy for the presence of fluorescent bacteria attached to the surfaces of these seedlings. Bound bacteria began to express GFP approximately 3 days after inoculation (Fig. 2). This was true for root-bound bacteria (Fig. 2A and B), as well as for bacteria colonizing the cotyledons (Fig. 2C and D). These observations show that intact tobacco seedlings release Fig. 2. Induction of PvirB–gfp of strain AB601 on root surfaces (A and B) and cotyledons (C and D) of tobacco seedlings, as detected by fluorescence microscopy 6 days after inoculation. A and C. Bright field images. B and D. Fluorescence images. © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Agrobacterium colonization of unwounded plants 1525 Hydroponically germinated tobacco seedlings were inoculated with strain AB602 and transferred to solidified MS medium. Periodically, individual seedlings were removed and examined by epifluorescence microscopy. After 3–4 days of incubation, we observed fluorescent plant cells, with the predicted distribution of fluorescence within each transformed cell (Fig. 4) (Haseloff et al., 1997; Haseloff, 1999). This distribution was very similar to that of transgenic Arabidopsis thaliana plants expressing the same transgene (Haseloff, 1999). The numbers of fluorescent cells per seedling varied considerably among individual seedlings, but in many seedlings the fluorescent cells were quite numerous. Transformed cells were largely limited to the leaf mesophyll (Fig. 4B and D), although occasionally we also observed transformation of leaf epidermal cells (Fig. 4F), stomatal cells, and root epidermal cells (data not shown). No fluorescent plant cells were observed using a bacterial strain lacking pBIN-mgfp5-ER or using a virG mutant strain. We do not know whether the gfp transgene was integrated into the plant genome. Fig. 3. In planta expression of the PvirB–gfp fusion of strain AB601. Bacteria were recovered from tobacco seedlings at different time points after inoculation, and fluorescence was detected by flow cytometry for 10 000 cells per sample. A. Average fluorescence of samples recovered from seedlings at different time points after inoculation. B. Histograms showing distribution of fluorescence among individual cells of each sample at different time points during incubation. (pCH116) (Chang et al., 1996), was detected with exudates of tobacco and squash, and was highest in the sample from the final day of the experiment (day 15), when activity of tobacco exudates was equivalent to 1– 10 mM AS, while the activity of squash exudates was approximately threefold lower (Table S1). Exudates of tomato exhibited no vir-inducing activity (Table S1). GC/ MS analysis revealed AS in the exudates of tobacco (Fig. S1A and B), while exudates of squash contained two other vir inducers, syringate and vanillate (Fig. S1C–E). Tomato exudates did not contain detectable levels of any known vir gene inducer (data not shown). Transformation of intact tobacco seedlings Based on the above results, it seemed possible that A. tumefaciens could transfer T-DNA to the cells of intact tobacco seedlings without AS pretreatment. To test this, we introduced plasmid pBIN-mgfp5-ER into A. tumefaciens strain R10, resulting in strain AB602. pBIN-mgfp5ER is a binary vector with gfp fused to the 35S CaMV promoter and inserted between the T-DNA borders of the vector (Haseloff et al., 1997). The GFP protein encoded by this plasmid is modified for transport to the endoplasmic reticulum, which tends to be localized near the nuclei and cell periphery (Haseloff et al., 1997). © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Fig. 4. Expression of a T-DNA-localized gfp gene in cells of tobacco seedlings, as viewed by fluorescence microscopy 8 days after inoculation with strain AB602. A and B. Transformed mesophyll cells of a cotyledon. C and D. Shoot apex surrounded by two cotyledons, with transformed cells located in the shoot apex and near the apical meristem. E and F. Edge of a cotyledon with one transformed epidermal cell and numerous transformed cells in the mesophyll layer. G and H. Close-up views of single transformed cells shown in part B. A, C and E. Bright field images. B, D, F, G and H. Fluorescence images. 1526 A. Brencic, E. R. Angert and S. C. Winans The fluorescent tobacco cells described above never initiated rapid cell division that would have resulted in a tumour. In the entire course of this study, tumours were not observed in unwounded plants, regardless of the bacterial strain. In each experiment, some seedlings were left to grow on MS medium for at least 3 weeks. These seedlings did not develop tumours and the great majority lacked any obvious disease symptoms. Detection of octopine by bacteria bound to unwounded seedlings Although the transformed cells detected in this study did not proliferate, it seemed likely that many of them were transformed by the native T-DNA. These cells would express opine synthase genes, and would release opines in amounts that might be sufficient for detection by the colonizing bacteria. To test this, we inoculated seedlings with strain AB603, which is a derivative of strain R10 with a PoccQ–gfp fusion and an intact occ operon carried in cis. Eight days after inoculation, we observed fluorescent bacteria on two out of nine examined seedlings. By day 14, fluorescent cells were observed on virtually all of the seedlings (Fig. 5A), though the induced bacteria were limited to cotyledons and their numbers varied greatly from seedling to seedling. Although the roots were heavily colonized, none of the root-colonizing bacteria were fluorescent (data not shown). This is consistent with the extremely low level of root cell transformation described above. To confirm these results and to quantify the in planta induction of the occQ operon, we inoculated seedlings with strain KYC16, which carries an octopine-inducible ooxA–gusA7 fusion (Cho et al., 1996). This strain detects octopine at 1000-fold lower concentrations than does AB603 (data not shown), probably because AB603 degrades octopine, while KYC16 does not, so the added octopine is not depleted (Cho et al., 1996). At regular intervals, bacteria were recovered from the seedlings by maceration and assayed for b-glucuronidase (Gallagher, 1992). This activity was first detected on day 5 after inoculation, and continued to increase for the remaining 11 days of the experiment (Fig. 5B). The maximal detected activity was similar to the activity obtained when strain KYC16 was cultured in AT medium in the presence of 10–100 nM octopine (Fig. 5C). However, based on our microscopic observations, we predict that many bacteria were not induced, especially those bound to the roots. As the b-glucuronidase assay measures the average gene expression of the entire population, within each sample, activity of the induced subpopulation was probably higher. No Gus activity was detected in the samples processed from uninoculated seedlings (data not shown). These results indicate that cells of these seedlings were trans- Fig. 5. A. Expression of the PoccQ–gfp fusion of strain AB603 during colonization of tobacco seedling cotyledons, as viewed by fluorescence microscopy 8 days after inoculation. The same strain incubated in defined broth was not detectably fluorescent (data not shown). B. GUS specific activity of ooxA–gus of strain KYC16 recovered from tobacco seedlings at different time points after inoculation. Before inoculation of seedlings, KYC16 cells were cultured overnight in AB minimal medium (pH 5.8) with 25 mM AS. Expression is represented in units of b-glucuronidase specific activity (Gallagher, 1992). No Gus activity was detected in the samples processed from uninoculated seedlings (data not shown). C. GUS specific activity of the ooxA–gus of strain KYC16 cultured in AT minimal medium at 27∞C in the presence of 10 mM octopine (), 1 mM (X), 100 nM (D), 10 nM (), or no octopine (). Samples were analysed for b-glucuronidase specific activity as described previously (Gallagher, 1992). formed by the native T-DNA, resulting in the production of octopine, which was detected and presumably catabolized by the bacteria. Discussion To our knowledge, this is the first report of the use of a Pvir–gfp fusion to study the induction of the A. tumefaciens vir regulon, though others have made AS-inducible vir–gfp fusions (Rashkova et al., 2000; Atmakuri et al., 2003). We established that vir genes are induced during colonization of intact tobacco seedlings, and we detected AS in the exudates of axenic tobacco seedlings. We also detected syringate and vanillate in the exudates of squash seedlings. These results unequivocally demonstrate that intentional wounding is not required for the release of vir© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Agrobacterium colonization of unwounded plants 1527 gene inducers, and that these compounds are released from unwounded plants in amounts sufficient for induction of vir genes. Although we cannot rule out the possibility that small wound-like lesions occur during normal plant growth, it may be better to think of vir-gene inducing signals as ‘plant-released’, rather than ‘wound-released’. However, it still needs to be established whether these findings can be extended to other plant species and/or to other stages of growth. When PvirB–gfp-expressing bacteria were treated with AS in broth cultures, at early stages of induction, only a small proportion of them were induced. This could be due to the fact that virA and virG are positively autoregulated, and that increased amounts of VirG enhance induction (Heath et al., 1995). It seems possible that some cells in a population might reach a threshold at which the concentration of VirG is sufficiently high to initiate a positive amplification loop, while other cells in the same population may not reach this threshold until later times. Such a phenomenon could help explain why in one study with cultures of AS-treated agrobacteria, only 10% of the bacteria displayed the virB-encoded pilus (Fullner et al., 1996). Cell to cell variation in the response to environmental stimuli has been described for numerous biological systems (McAdams and Arkin, 1997; 1999). These variations are commonly observed even under the most uniform experimental conditions, such as in well-mixed bacterial cultures, and are thus believed to arise from variations in internal cellular processes rather than from differing microenvironments. The molecular-level phenomena that produce these differences are thought to be rooted in the statistical mechanical behaviour of smallscale chemical systems, where concentrations of reacting species are extremely low and, consequently, stochastic fluctuations in reaction rates are large (McAdams and Arkin, 1997; 1999). We noted a difference in induction kinetics between bacteria cultured in broth with AS and bacteria growing on plant surface. This could be due to the plants releasing inducers only in the presence of bacteria (Stachel et al., 1985), however, this cannot be the only explanation because we have detected these compounds in exudates of axenic plants (Fig. S1, Table S1). It could also be because in the former experiment (Fig. 1A), glucose was provided for bacterial growth and to enhance vir gene induction, while in the latter experiment (Fig. 3), the bacteria obtained all nutrients from their hosts. Furthermore, many of the bacteria growing on seedlings appeared to form microcolonies or biofilms and may have been less metabolically active than planktonic cells. We further showed that A. tumefaciens can transfer TDNA to cells of unwounded tobacco seedlings without pretreatment of the bacteria with AS. T-DNA transfer to cells of non-wounded tobacco has been documented © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 before, however, transformation in that study required that the bacteria be pre-induced with AS (Escudero and Hohn, 1997). Differences in experimental design, such as the age of the seedlings and the fact that in our study seedlings were grown hydroponically until inoculation, must account for the different results of the two studies. Most of the transformed cells described in this study were located within the mesophyll layer of the cotyledons, although some were located in the leaf epidermis. Transformation of these cells might seem surprising given the existence of the waxy cuticle. The study by the Hohn lab showed that plant transformation required opening of stomata (Escudero and Hohn, 1997), suggesting that the bacteria used stomata to enter the apoplast. In our experiments, the stomata were frequently observed to be fully open (data not shown). Thus, the mesophyll and epidermal cells may have been transformed by the bacteria that entered the apoplast, which would not require penetrating the cuticle. Only rarely did we observe transformation of the root epidermal cells. Further evidence that roots were rarely transformed was obtained using a PoccQ–gfp reporter, which was induced in bacteria colonizing the cotyledons, but not in those colonizing roots or root hairs. It is not clear why roots were not more readily transformed. In particular, root hairs lack a cuticle, and the nuclei of root epidermal cells are frequently in the root hair itself, so they should in principle be highly susceptible to transformation. One trivial explanation is that transformed root cells do not express detectable levels of GFP or release octopine at levels sufficient for detection. Although A. tumefaciens is a soil inhabitant, it should not be surprising that these bacteria can colonize leaves, especially those of seedlings, which grow among soil particles, often in very wet conditions. Seedlings are readily colonized by various species of soil bacteria, including Pseudomonas spp., which is one of the most frequent colonizers of leaf surfaces (Andrews and Harris, 2000; Hirano and Upper, 2000). What surprised us, however, was that these bacteria detected signals generally thought to be released only from wound sites, that they were so readily able to transfer T-DNA, and that T-DNA transfer did not cause tumours. Tumours were never observed in the absence of wounding with any of the strains used in this study. Neoplastic proliferation of transformed cells might require wounding and/or might occur only in particular cell types. In the previous study using unwounded plants, cell proliferation was occasionally detected (Escudero and Hohn, 1997). Others, however, have shown that tumours initiate only from meristematic tissues, such as the vascular cambium (Sangwan et al., 1991; Ghorbel et al., 2000). As wounding induces plant cell dedifferentiation and division, it is possible that in tissues that are not intrinsically meristematic, 1528 A. Brencic, E. R. Angert and S. C. Winans the wound response is needed to initiate tumours (Kahl, 1982; Binns and Thomashow, 1988). Another possible explanation is that tumours may occur only when a sufficiently large number of plant cells are transformed in a confined space, analogous to bacterial ‘quorum sensing’. Without exogenous wounding, the number of transformed plant cells might be below this critical threshold. It is also possible that in our experiments, T-DNA did not integrate into the plant chromosome and hence that the amount of plant hormones produced during transient expression of T-DNA genes was not sufficient to initiate cell proliferation. The infected seedlings, however, produced octopine at levels that were readily detectable by the bacteria. This indicates that successful colonization of plants by A. tumefaciens, resulting in opine production by the colonized plants, might occur in the absence of tumours. As described above, phytohormone production might have been too low in transformed cells to initiate their proliferation, or, alternatively, these differentiated cells might not proliferate in response to phytohormones. However, one of these hormones, cytokinin, stimulates transport of nutrients from the rest of the plant to the cells that release it (Davies, 1990), and might therefore enhance the release of nutrients to the bacteria. Transport of nutrients to the transformed cells might also enhance the production of opines. In this study, we have demonstrated that A. tumefaciens can detect vir gene inducers from unwounded seedlings, that it can efficiently transfer T-DNA to the cells of these seedlings, and that these transformed seedlings release octopine but do not show any obvious signs of disease. Taken together, these results suggest that transformation of cells of non-wounded plants could be an entirely new strategy for A. tumefaciens plant colonization that has largely been overlooked, and this discovery could lead to an entirely new paradigm for A. tumefaciens–plant interactions. Based on the results of our study, it is plausible that transformation of plant cells without causing their proliferation could be a common outcome of T-DNA transfer. Studies of this and most other host–pathogen interactions have mainly been focused on catastrophic interactions, while commensal interactions are often overlooked or less emphasized. However, minimizing the harm to the host may be a sound strategy for many pathogens, and commensalism may represent the ideal interaction for both host and parasite (Ewald, 1983). Experimental procedures Strains and plasmids Plasmid pJS202 is a derivative of pVIK165 (Kalogeraki and Winans, 1997), which was designed to construct fusions between bacterial promoters and gfp(S65T). In pSJ202, the gfp gene of pVIK165 was replaced with gfp(S65G, S72A) (Cormack et al., 1996), by digesting pVIK165 with XbaI (which cuts within the multiple cloning site) and MfeI (which cuts within the gfp gene) and introducing an XbaI-MfeI fragment containing gfp(S65G, S72A) from plasmid pKEN (Cormack et al., 1996). To make a PvirB–gfp fusion, a 420 bp DNA fragment that contains the virB promoter was synthesized by polymerase chain reaction (PCR) amplification using primers 5¢-GCAC GAATTCCCGAGCGAAGGTTTTCGC-3¢ and 5¢-GCAGAG CTCCGAATGCCGTGCATCGAGACGG-3¢, and was introduced into pSJ202 as an EcoRI-SacI fragment. The resulting plasmid, pSSC103, was mobilized from Escherichia coli strain S17-ë pir to A. tumefaciens strain R10 by conjugation, where it recombined into the Ti plasmid by Campbell integration, resulting in a PvirB–gfp fusion. In the resulting strain, AB601, the native virB operon is preserved downstream of the integrated plasmid. Strain R10 is a wild-type A. tumefaciens strain with octopine-type Ti plasmid pTiR10 (Sciaky et al., 1978). A strain expressing a PoccQ–gfp fusion was constructed by the same procedure except that a 260 bp occQ-occR intergenic region including the occQ promoter was cloned from pLW131 (Wang and Winans, 1995), into pSJ202 as an EcoRV fragment, resulting in plasmid pRA400. This plasmid was mobilized from E. coli S17-ë pir to A. tumefaciens R10 by conjugation, resulting in strain AB603. AB603 retained the wild-type copy of the occQ operon and a PoccQ–gfp fusion carried in cis. Strain KYC16 is a derivative of strain R10 with Tn5gusA7 integrated at the ooxA gene (Cho et al., 1996). Plasmid pBIN-mgfp5-ER is a binary vector that carries a fusion between the 35S CaMV promoter and gfp, inserted between the T-DNA borders (Haseloff et al., 1997). The plasmid was introduced into A. tumefaciens strain R10 via electroporation, resulting in strain AB602. Agrobacterium tumefaciens reporter strain A136(pCH114) (pCH116) was used in bioassays to test for the presence of vir inducers in plant exudates. The strain expresses virA–, virG– and virB–lacZ (Chang et al., 1996). Bioassays For bioassays of seedling exudates, seeds of tobacco (Nicotiana tabacum cv. Samsun NN), tomato (Lycopersicum esculentum cv. Heinz 1370) and squash (Cucurbita maxima) were surface sterilized and transferred to sterile Erlenmeyer flasks containing 50 ml of deionized water. The flasks were incubated with mild shaking at room temperature for 15 days. Every 3 days, 10 ml of samples of the culture supernatants were withdrawn, acidified by addition of 100% phosphoric acid to a final concentration of 1.5% v/v, and extracted with an equal volume of ethyl acetate. Ethyl acetate was evaporated using nitrogen gas, and pellets were resuspended in 5 ml of AB minimal medium (pH 5.8), filter sterilized, and inoculated with strain A136(pCH114) (pCH116) (Chang et al., 1996). Cells were cultured overnight at 27∞C for 16 h and assayed for b-galactosidase activity (Miller, 1972). GUS specific activity of ooxA–gus fusion of strain KYC16 was measured and represented in units of bglucuronidase specific activity as described previously (Gallagher, 1992). © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Agrobacterium colonization of unwounded plants 1529 Seed germination and seedling inoculation Acknowledgements Seeds of N. tabacum cv Samsun NN were surface-sterilized by soaking in 50% bleach and 0.1% SDS for 10 min, followed by extensive washing. Seeds were then transferred to sterile water in Petri dishes and incubated for 48 h at 4∞C in the dark, after which the seedlings were maintained at room temperature with exposure to natural and artificial lighting. Unless indicated otherwise, before inoculation, bacteria were cultured overnight in Luria–Bertani medium with antibiotics. Cells were then washed with sterile water, and suspended to OD600 = 0.5 in 5% sucrose and 0.005% TRITON-X100. Approximately 100 hydroponically grown seedlings were dipped into the bacterial suspensions for 5 min, washed gently in 5% sucrose, and transferred to solidified MS medium lacking any carbon source (Murashige and Skoog, 1962). At the time of inoculation, seedlings were 1– 2 cm long and approximately 2 weeks old. After inoculation, seedlings were incubated at room temperature on a bench top with exposure to ambient fluorescent and natural lighting. We would like to acknowledge Stephen S. Chung, and Drs Samina Jafri and Reiko Akakura for constructing some of the strains used in the study. We thank Dr Rod Getchell for help with flow cytometry, Dr Anatol Eberhard for help with GC/MS, and Ms. Nahid Shaikh for help with plant inoculations and microscopic observations. We also would like to thank Dr Larry Walker for help with fluorescence spectrophotometry. Microscopy Individual seedlings were transferred to a glass slide, overlaid with a cover slip, and viewed on an Olympus BX61 microscope using 20¥ and 40¥ phase-contrast objectives. For detection of GFP, samples were viewed by epifluorescence microscopy using a Chroma 41017 (bandpass emission) filter cube [HQ 470/40, Q 495LP, HQ 525/50] or a Chroma 41012 (longpass emission) wide blue filter cube [HQ 480/40, Q 505LP, HQ 510LP]. Images were acquired using a Cooke SensiCam with a Sony Interline chip. Image acquisition was performed using SlideBook Software package (Intelligent Imaging) and figures were assembled using Photo-Paint (Corel, Salinas, CA). Recovery of bacteria from seedlings For each sample, six seedlings were transferred to liquid MS medium in a culture tube, which was then vortexed vigorously to remove the bound bacteria. In experiments involving the ooxA–gus fusion, six seedlings were transferred to liquid MS medium in an Eppendorf tube, and macerated. The liquid was separated from plant fragments by pipetting and centrifugation. Pellets were resuspended in 200 ml of 20% glycerol and stored at -80∞C until analysis. Analysis of vir gene induction by flow cytometry Strain AB601 was cultured at 27∞C in AB minimal medium (pH 5.8) in the presence of various concentrations of the vir gene inducer AS. Culture samples were collected at different time points and frozen in 25% glycerol at -80∞C. On the day of analysis, samples were thawed, diluted approximately 25fold in AB buffer, and analysed in a FACSCalibur flow cytometer (Beckton Dickinson) equipped with an argon laser emitting at 488 nm. Fluorescence data were collected using logarithmic amplifiers for 10 000 cells per sample (as determined by light side-scattering), and analysed using CellQuest software. © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 References Andrews, J.H., and Harris, R.F. (2000) The ecology and biogeography of microorganisms on plant surfaces. Annu Rev Phytopathol 38: 145–180. Atmakuri, K., Ding, Z., and Christie, P.J. (2003) VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol Microbiol 49: 1699–1713. Binns, A.N., and Thomashow, M.F. (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42: 575–606. Binns, A.N., and Constantino, P. (1998) The Agrobacterium oncogenes. In The Rhizobiaceae. Spaink, H.P., Kondorosi, A., and Hooykaas, P.J.J. (eds). Dordrecht: Kluwer Academic Publishers, pp. 251–266. Chang, C.H., Zhu, J., and Winans, S.C. (1996) Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein. J Bacteriol 178: 4710–4716. Chateau, S., Sangwan, R.S., and Sangwan-Norreel, B.S. (2000) Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J Exp Bot 51: 1961–1968. Cho, K., Fuqua, C., Martin, B.S., and Winans, S.C. (1996) Identification of Agrobacterium tumefaciens genes that direct the complete catabolism of octopine. J Bacteriol 178: 1872–1880. Cormack, B.P., Valdivia, R.H., and Falkow, S. (1996) FACSoptimized mutants of the green fluorescent protein (GFP). Gene 173: 33–38. Davies, P.J. (1990) Plant Hormones and Their Role in Plant Growth and Development. Dordrecht: Kluwer Academic Publishers. Dixon, R.A., and Paiva, N.L. (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085–1097. Escudero, J., and Hohn, B. (1997) Transfer and integration of T-DNA without cell injury in the host plant. Plant Cell 9: 2135–2142. Ewald, P.W. (1983) Host-parasite relations, vectors, and the evolution of disease severity. Annu Rev Ecol Syst 14: 465– 485. Fullner, K.J., Lara, J.C., and Nester, E.W. (1996) Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273: 1107–1109. Gallagher, S.R. (1992) GUS Protocols. San Diego, CA: Academic Press. Gelvin, S.B. (2003) Agrobacterium-mediated plant transformation: the biology behind the ‘gene-jockeying’ tool. Microbiol Mol Biol Rev 67: 16–37. Ghorbel, R., Dominguez, A., Navarro, L., and Penna, L. 1530 A. Brencic, E. R. Angert and S. C. Winans (2000) High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus. Tree Physiol 20: 1183–1189. Haseloff, J. (1999) GFP variants for multispectral imaging of living cells. Methods Cell Biol 58: 139–151. Haseloff, J., Siemering, K.R., Prasher, D.C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94: 2122–2127. Heath, J.D., Charles, T.C., and Nester, E.W. (1995) Ti plasmid and chromosomally encoded two-component systems important in plant cell transformation by Agrobacterium species. In Two-Component Signal Transduction. Hoch, J.A., and Silhavy, T.J. (eds). Washington, DC: American Society for Microbiology Press, pp. 367–385. Hirano, S.S., and Upper, C.D. (2000) Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae: a pathogen, ice nucleus, and epiphyte. Microbiol Mol Biol Rev 64: 624–653. Kahl, G. (1982) Molecular biology of wound healing: the conditioning phenomenon. In Molecular Biology of Plant Tumors. Kahl, G., and Schell, J.S. (eds). New York: Academic press, pp. 211–267. Kalogeraki, V.S., and Winans, S.C. (1997) Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188: 69–75. McAdams, H.H., and Arkin, A. (1997) Stochastic mechanisms in gene expression. Proc Natl Acad Sci USA 94: 814–819. McAdams, H.H., and Arkin, A. (1999) It’s a noisy business! Genetic regulation at the nanomolar scale. Trends Genet 15: 65–69. Matthysse, A.G., and Kijne, J.W. (1998) Attachment of Rhizobiaceae to plant cells. In The Rhizobiaceae. Spaink, H.P., Kondorosi, A., and Hooykaas, P.J.J. (eds). Dordrecht: Kluwer Academic Publishers, pp. 235–249. Matthysse, A.G., and McMahan, S. (1998) Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants. Appl Environ Microbiol 64: 2341– 2345. Miller, J. (1972) Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Monack, D.M., Mueller, A., and Falkow, S. (2004) Persistent bacterial infections: the interface of the pathogen and the host immune system. Nat Rev Microbiol 2: 747–765. Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497. Parrish, N.M., Dick, J.D., and Bishai, W.R. (1998) Mechanisms of latency in Mycobacterium tuberculosis. Trends Microbiol 6: 107–112. Rashkova, S., Zhou, X.R., Chen, J., and Christie, P.J. (2000) Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase. J Bacteriol 182: 4137–4145. Sangwan, R.S., Bourgeois, Y., and Sangwan-Norreel, B.S. (1991) Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol Gen Genet 230: 475–485. Sangwan, R.S., Bourgeois, Y., Brown, S., Vasseur, G., and Sangwan-Norreel, B. (1992) Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 188: 439– 456. Sciaky, D., Montoya, A.L., and Chilton, M.D. (1978) Fingerprints of Agrobacterium Ti plasmids. Plasmid 1: 238–253. Shen, W.H., Escudero, J., Schlappi, M., Ramos, C., Hohn, B., and Koukolikova-Nicola, Z. (1993) T-DNA transfer to maize cells: histochemical investigation of beta-glucuronidase activity in maize tissues. Proc Natl Acad Sci USA 90: 1488–1492. Smith, E.F., and Townsend, C.O. (1907) A plant tumour of bacterial origin. Science 25: 671–673. Stachel, S.E., and Zambryski, P.C. (1986) VirA and VirG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell 46: 325–333. Stachel, S.E., Messens, E., Vanmontagu, M., and Zambryski, P. (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318: 624–629. Stachel, S.E., Nester, E.W., and Zambryski, P.C. (1986) A plant-cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci USA 83: 379–383. Stewart, G.R., Robertson, B.D., and Young, D.B. (2003) Tuberculosis: a problem with persistence. Nat Rev Microbiol 1: 97–105. Stover, E., Swartz, H., and Burr, T. (1997) Crown gall formation in a diverse collection of vitis genotypes inoculated with Agrobacterium vitis. Am J Enol Vitic 48: 26–32. Swart, S., Logman, T.J.J., Smit, G., Lugtenberg, B.J.J., and Kijne, J.W. (1994) Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiaceae. Plant Mol Biol 24: 171–183. Taiz, L., and Zeiger, E. (2002) Plant Physiology. Sunderland, MA: Sinauer Associates. Tzfira, T., Rhee, Y., Chen, M.H., Kunik, T., and Citovsky, V. (2000) Nucleic acid transport in plant–microbe interactions: the molecules that walk through the walls. Annu Rev Microbiol 54: 187–219. Tzfira, T., Li, J., Lacroix, B., and Citovsky, V. (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20: 375–383. Wagner, V.T., and Matthysse, A.G. (1992) Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells. J Bacteriol 174: 5999–6003. Wang, L., and Winans, S.C. (1995) The 60 Nucleotide occR operator contains a subsite essential and sufficient for OccR binding and a 2nd subsite required for ligandresponsive DNA bending. J Mol Biol 253: 691–702. Zhu, J., Oger, P.M., Schrammeijer, B., Hooykaas, P.J.J., Farrand, S.K., and Winans, S.C. (2000) The bases of crown gall tumorigenesis. J Bacteriol 182: 3885–3895. Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.Y., and Cao, H. (2003) Identification of Arabidopsis rat mutants. Plant Physiol 132: 494–505. © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Agrobacterium colonization of unwounded plants 1531 Zupan, J., Muth, T.R., Draper, O., and Zambryski, P. (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23: 11–28. Supplementary material The following supplementary material is available for this article online: © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 57, 1522–1531 Fig. S1. Gas chromatograms of tobacco (A) and squash (C) seedling exudates, and mass spectra (B, D and E) of virinducing phenolic compounds detected in the exudates. Table S1. Expression of virB–lacZ of strain A136(pCH114) (pCH116) (Chang et al., 1996) in response to different concentrations of AS, and in response to exudates of seedlings of tobacco (Nicotiana tabacum cv. Samsun NN), tomato (Lycopersicum esculentum cv. Heinz 1370) and squash (Cucurbita maxima).