Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens

Authors


Author for correspondence:
Hiroshi Ezura
Tel: +81 29 853 7734
Fax: +81 29 853 7734
Email: ezura@gene.tsukuba.ac.jp

Summary

  • • Ethylene evolution from plants inhibits Agrobacterium-mediated genetic transformation, but the mechanism is little understood. In this study, the possible role of ethylene in Agrobacterium-mediated genetic transformation was clarified.
  • • It was tested whether or not plant ethylene sensitivity affected genetic transformation; the sensitivity might regulate bacterial growth during co-cultivation and vir gene expression in Agrobacterium tumefaciens. For these experiments, melon (Cucumis melo) was used, in which ethylene sensitivity was controlled by chemicals, and Arabidopsis ethylene-insensitive mutants.
  • • Agrobacterium-mediated genetic transformation was inhibited in ethylene-sensing melon, whereas, in Arabidopsis ethylene-insensitive mutant, it was enhanced. However, the ethylene sensitivity did not affect bacterial growth. vir gene expression was inhibited by application of plant exudate from ethylene-sensitive plants. The inhibitory effect of the ethylene sensitivity on genetic transformation relieved the activation of vir gene expression in A. tumefaciens with vir gene inducer molecule (acetosyringone, AS) or A. tumefaciens mutant strain which has constitutive vir gene expression.
  • • These results indicate that ethylene evolution from a plant inoculated with A. tumefaciens inhibited vir gene expression in A. tumefaciens through the ethylene signal transduction in the plant, and, as a result, Agrobacterium-mediated genetic transformation was inhibited.

Introduction

Agrobacterium tumefaciens is an α-proteobacterium with the unique ability to transfer and integrate genes into the genome of a host plant (genetic transformation); thus, it is widely utilized in plant molecular genetics (Newell, 2000). The transformation process requires the tumour-inducing (Ti) plasmid of A. tumefaciens (van Larebeke et al., 1974, 1975; Watson et al., 1975; Currier & Nester, 1976). The Ti plasmid includes two regions (Hoekema et al., 1983): an oncogenic region (T-DNA) that encodes proteins involved in crown gall formation through auxin and cytokine synthesis after integration into the host plant genome (Akiyoshi et al., 1983); and the vir gene region, which encodes the proteins that actually transfer the T-DNA into plant cells and integrate it into the host genome (e.g. VirB, VirD and VirE function in type-IV secretory systems, transport and protection of the T-DNA from host DNases, respectively) (Zhu et al., 2000). The strain without vir regions has no ability for genetic transformation (Ooms et al., 1980). Therefore, activation of the vir genes is essential for genetic transformation.

Genetic transformation begins with the activation of the vir gene region, whereby A. tumefaciens is able to recognize the host plant. vir gene expression is triggered by phenols (Stachel et al., 1985, 1986;) and monosaccharides (Ankenbauer & Nester, 1990; Cangelosi et al., 1990; Shimoda et al., 1993) in the plant cell wall (i.e. signal compounds) under acidic conditions (pH 5.5). These compounds are sensed by a two-component regulatory system involving VirA (Lee et al., 1995, 1996). When VirA senses the signal compounds, it is autophosphorylated at His-474 (Jin et al., 1990b). VirA then phosphorylates the response regulator VirG, which directly regulates vir gene expression (Jin et al., 1990a). A mutant version of virG, virGN54D, in which the codon for asparagine at position 54 is substituted by an aspartate, causes constitutive activation of other vir genes, independent of virA (Pazour et al., 1992; Hansen et al., 1994).

The gaseous phytohormone ethylene is produced and perceived in response to a wide variety of environmental and developmental cues, including germination, flowering, drought and pathogen attack (Abeles et al., 1992). Recent studies have shown that ethylene also regulates Agrobacterium-mediated genetic transformation. The enhancement of ethylene production by application of 1-aminocyclopropane-1-carboxylic acid (ACC), which is the ethylene precursor, inhibits the genetic transformation of tomato and melon plants (Davis et al., 1992; Ezura et al., 2000). Furthermore, the endogenous ethylene also inhibits the genetic transformation of plant cells (Ezura et al., 2000; Han et al., 2005). In fact, the genetic transformation was enhanced by application of the ethylene biosynthetic inhibitor aminoethoxyvinylglycine (AVG) in co-cultivation medium in melon (Ezura et al., 2000) and bottle gourd (Han et al., 2005). The ethylene production in plants inhibits the genetic transformation, but the involvement of ethylene sensitivity to be clarified.

Although the inhibitory effect of ethylene on genetic transformation is clear, the mechanism remains to be clarified. One possible explanation involves decline of bacterial growth via defence response, because ethylene signal transduction induces the expression of genes related to defense such as chitinase, β-1,3-glucanase and PR1 (Deikman, 1997). For example, in tomato, increased ethylene sensitivity transgenic plant caused a decline in the bacterial population (Ciardi et al., 2001), and in Arabidopsis, ethylene-insensitive mutants enhanced the bacterial growth (Norman-Setterblad et al., 2000). Therefore, ethylene seems to inhibit Agrobacterium-mediated genetic transformation through decline of A. tumefaciens growth during co-cultivation.

To better understand the inhibitory effect of ethylene, we focused on the initial step in genetic transformation and bacterial growth. We measured vir gene expression in A. tumefaciens-exposed plant exudate from melon cotyledon-induced ethylene response with ACC to determine whether the initial step in genetic transformation is affected, and we evaluated the amount of bacterial growth during co-cultivation. Based on our results, we discuss the molecular mechanism underlying the inhibitory effect of ethylene on genetic transformation.

Materials and Methods

Plant materials

Seeds of the melon Cucumis melo L. var. cantaloupensis cv. Vedrantais and Arabidopsis thaliana (ecotype Columbia and ethylene-insensitive mutant etr1-1, ein2-5 and ein3-1) were grown under 16 : 8 h light : dark conditions at 25 and 22°C, respectively. For growth under sterile conditions, seeds were surface-sterilized (2 min in 70% ethanol, transferred to 2% (v/v) sodium hypochlorite, rinsed three times with sterile distilled water) and sown.

Ethylene response

Surface-sterilized melon seeds were sown on half-strength of Murashige and Skoog's medium (MS; Murashige & Skoog, 1962) and germinated at 25°C for 5 d under a 16 : 8 h light : dark photoperiod. The hypocotyl lengths of the germinated seedlings were then measured. The 1-aminocyclopropane- 1-carboxylic acid (ACC), a precursor of ethylene, and silver thiosulfate (STS), an inhibitor of ethylene response, was added to the germination medium as appropriate. Ten to 30 seedlings were used for each treatment.

Bacterial culture

Several bacterial strains were used (Table 1). These strains were cultured in liquid Luria–Bertani (LB) medium (1% triptone, 0.5% yeast extract, and 1% NaCl, pH 7.0) for 2 d at 28°C with shaking. The cells were then collected by centrifugation, washed and suspended in liquid MS without sucrose.

Table 1.  Bacterial strains and plasmids used in this study
Strain or plasmidRelevant featuresSource or reference
  1. Ap, ampiciline; Tc, tetracycline; Km, kanamycin; Gm, gentamicin.

Agrobacterium tumefaciens
 C58 Wild-type strainSciaky et al. (1978)
  A136 C58 chromosomal background; pTiC58 curedSciaky et al. (1978)
 C58C1RifRC58 chromosomal background; pTiC58 cured, harbouring pGV2260Deblaere et al. (1985)
Ti-plasmid
 pGV2260Nononcogenic Ti plasmid. The T-region is deleted and substituted by pBR322; Apr, TcrDeblaere et al. (1985)
Plasmids
 pIG121-HmBinary cloning vector for Agrobacteriu-mediated plant transformation; KmrHiei et al. (1994)
 pBBR1MCS-5Broad-host-range cloning vector. Compatible with IncP, IncQ and IncW group plasmid; GmrKovach et al. (1995)
 pBBR1MCS-5.virGN54DCarrying virGN54D in pBBR1MCS-5: virG mutant carrying an Asn-54 to Asp amino avid substution; Gmrvan der Fits et al. (2000)
 pBBRvirD2::uidAContaining virD2-uidA transrational fusion gene in pBBR1MCS-5, the expression of virD2-uidA is under control of virD promoterThis study

Constitutively vir gene expression in A. tumefaciens

The plasmid pBBR1MCS-5.virGN54D was kindly provided by Dr J. Memelink (Leiden University, the Netherlands). This plasmid carries the virGN54D version of virG, which constitutively confers vir gene activation in transformed A. tumefaciens and enhances gene transfer (Pazour et al., 1992; van der Fits et al., 2000). The plasmid was transformed into A. tumefaciens C58C1RifR cells by electroporation (Shen & Forde, 1989). A. tumefaciens C58C1RifR carrying the plasmid pBBR1MCS-5 (Kovach et al., 1995) was used as a control.

Construction of the virD2–uidA reporter system

The virC1-virD2 region involved in virD promoter was obtained by PCR from the genomic DNA of A. tumefaciens C58C1RifR with the primers of virD1-301F1 (5′-CCCTTTGAAAGAGCAAAACGTC-3′) and virD1 + 892R, 5′-TGACCACCGACATGTAAATGTGG-3′). The PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA), (pCRvirC1D2). The uidA gene was cloned from pBI221 (accession no. AF502128) by PCR with the primer uidAFw 5′-CTGCAGATGTTACGTCCTGTAGAAACC-3′ (PstI site underlined; start codon in bold) and uidARv 5′-GAGCTC TCATTGTTTGCCTCCCTGCTG-3′ (SacI site underlined; stop codon in bold). The fragment was inserted into the PstI site of the virC1D2 region (pCRvirD2::uidA) to form a translational fusion with the virD2 gene product, and the uidA gene expression will be under the control of virD promoter. Because the uidA gene is without an intron, it can produce active protein in bacterial cells (Reeve et al., 1999; Yuan et al., 2007). The virD2-uidA fragment was cloned with EcoRI, SacI into the pBBR1MCS-5 (pBBRvirD2::uidA). The pBBRvirD2::uidA was introduced into A. tumefaciens C58C1RifR by electroporation (Shen & Forde, 1989).

Bacterial inoculation

Surface-sterilized melon seeds were sown on half-strength of MS and germinated at 25°C for 5 d under a 16 : 8 h light : dark photoperiod. The ACC was added at the start of germination and at the time of co-cultivation. The STS was only added to the germination medium. To avoid the effect of silver ion on A. tumefaciens growth in the cotyledon segments during the co-cultivation, STS was supplied only to the germination medium, because silver ion and the related compounds are known as bacterial agents (Brady et al., 2003; Matsumura et al., 2003). Cotyledons from the germinated seedlings were hand-sectioned transversely into five pieces, and the three internal pieces were used for inoculation. The segments were soaked in an A. tumefaciens C58C1RifR (pIG121-Hm) cell suspension (108 cells ml−1) for 20 min at room temperature. The cell suspension was also diluted to 106 or 107 cells ml−1 if needed. The pIG121-Hm has a reporter gene (35S-uidA intron) in its T-DNA region (Hiei et al., 1994). Because the uidA gene possesses an intron, it can only produce active protein in plant cells, therefore making it a marker for genetic transfer to plant cells (Ohta et al., 1990). The inoculated segments were then placed on a co-cultivation medium (MS containing 1 mg l−1 6-benzylaminopurine, 2% glucose and 4% Gelrite (Wako Pure Chemicals, Osaka, Japan), pH 5.7). If necessary, ACC and acetosyringone (AS) were added to the co-cultivation medium at 200 and 100 µm, respectively. The inoculated segments were co-cultivated for 4 d at 25°C in darkness. After 4 d co-cultivation, the segments were crushed and subjected to β-glucuronidase (GUS) assay to estimate genetic transformation.

Tumour formation assay for A. thaliana

Surface-sterilized A. thaliana seeds were sown on MS and germinated at 22°C for 7 d under a 16 : 8 h light : dark photoperiod after a 4 d vernalization period (continuous darkness at 4°C). Intact A. thaliana plants were dipped into A. tumefaciens C58 or A136 suspension, blotted on sterilized filter paper to remove excess suspension, and co-cultivated for 7 d on MS with 0.4% Gelrite. The plants were rinsed in sterile distilled water, blotted, and then incubated on MS with 0.3% Gelrite containing 375 mg l−1 Augmentin. Four weeks after infection, numbers of plants that formed green tumour on the stems were counted.

Estimation of the A. tumefaciens population

A serial-dilution plate assay was used to estimate the A. tumefaciens population in melon cotyledon segments. Five randomly selected segments inoculated with the bacteria were aseptically crushed in sterile MS. Three replications with 15 cotyledon segments were performed for each treatment. The extracts were serially diluted to 10−1–10−6 with MS; then 20 µl of each suspension was spread on a plate containing LB without antibiotics and the plates were incubated at 28°C for 2 d. The number of colonies per plate was used to estimate the number of bacterial cells per segment.

Quantification of vir gene expression

For preparation of the exudate, 5-d-old melon seedlings were used. The growth was under sterile conditions, the cotyledons of the germinated seedlings were hand-sectioned transversely into five pieces, and the three internal pieces were used. A total of 100 segments were incubated in 100 ml of MS minus sucrose for 72 h (induction medium, IM). A. tumefaciens C58C1RifR harbouring the virD2-uidA reporter system was cultured in 100 ml of LB medium until the OD600nm = 0.5. The culture was then centrifuged and washed twice with MS without sucrose. The cells were then incubated with IM or MS lacking sucrose with AS for 20 h, after which they were collected and lysed with detergent. The lysate was subjected to a GUS assay to allow quantification of vir gene expression. GUS activity was assayed with X-Gluc (Jefferson et al., 1987) or fluorometric-β-glucuronidase assay.

Fluorometric-β-glucuronidase assay

A GUS assay was performed using the substrate 4-methylumbelliferyl-β-D-glucuronide (4MUG; Calbiochem, La Jolla, CA, USA) and the reaction product 4-methylumbelliferone (4MU; ICN Biomedicals, Aurora, OH, USA). Samples were crushed in extraction buffer (50 mm sodium phosphate, 10 mm EDTA, 0.1% Triton-X 100, 0.1% N-lauroyl sarcosine sodium salt and 10 mm 2-mercaptoethanol, pH 7.0) on ice. The crushed samples were centrifuged at 14 000 g for 5 min at 4°C, and the supernatants were collected. The protein concentrations of the extracts were measured by the Bradford method using a Bio-Rad Protein Assay kit (Bio-Rad, Tokyo, Japan). The protein extracts were subjected to a GUS assay in reaction buffer (10 ng of extracted protein µl−1, 0.5 mm 4MUG, 50 mm sodium phosphate, 10 mm EDTA, 0.1% Triton X-100, 0.1% N-lauroyl sarcosine sodium salt and 10 mm 2-mercaptoethanol, pH 7.0). The reactions were incubated at 37°C for 10 min, then stopped by the addition of 20 volumes of 200 mm Na2CO3 (pH 11.0) and subjected to fluorescent spectrophotometry using a Bio-Rad Versa Fluor Fluorometer with an excitation wavelength of 360 nm and an emission of 450 nm to detect the reaction product. The amount of 4MU in the solution was estimated from a standard curve based on a dilution series of 2–1000 nm of 4MU.

Results

Ethylene sensitivity in plants inhibits genetic transformation from A. tumefaciens to plant cells

Suppression of hypocotyl elongation is a typical ethylene response (Fig. 1a). The suppression was observed in melon seedlings germinated in the presence of ACC that enhances ethylene production (Wang et al., 2002). The typical ethylene response in hypocotyl was partially overcome by STS, which is known as an ethylene response inhibitor (Fig. 1a; Veen & Kwakkenbos, 1982; Veen, 1983). Modulations of ethylene response in the seedlings by 200 µm of ACC and 100 µm of STS were then used in the following studies on genetic transformation.

Figure 1.

Effect of the ethylene response on T-DNA transfer in melon (Cucumis melo) cotyledon segments and the ethylene response in Arabidopsis mutants. (a) Hypocotyl lengths of the melon seedlings. Seedlings grown under light on medium with (+) or without (–) 100 µm silver thiosulfate (STS) were measured. The open and closed columns represent the absence and presence of 1-aminocyclopropane-1-carboxylic acid (ACC) in the medium, respectively. Bars indicate SD (n = 30). (b) Occurrence of T-DNA transfer in segments of seedling cotyledons. The occurrence of T-DNA transfer was indicated by β-glucuronidase (GUS) activity in the segments. Bars indicate SD (n = 3). The open and closed columns represent the absence and presence of ACC, respectively. ACC was added to the germination and co-cultivation media. STS was applied only to the germination medium. Agrobacterium cells without (–) or carrying (+) the plasmid pIG121-Hm. Bacterial cell suspensions were prepared at 108 cells ml−1 for inoculation. Bars indicate SD (n = 3). The letters indicate statistical significance at the 5% confidence level based on Student's t-test. (c) Frequency of tumour formation in ethylene-insensitive Arabidopsis mutants. Each value is the average of three independent experiments. The letters represent statistically significant differences based on chi-square testing (P < 0.05).

Inoculation of A. tumefaciens C58C1RifR carrying pIG121-Hm resulted in higher GUS activity in melon cotyledon segment compared with no inoculation (Fig. 1b). This observation indicates the occurrence of genetic transformation in the melon cotyledon segment. The occurrence of genetic transformation was reduced by the presence of ACC in the germination and the co-cultivation medium. The reduction in occurrence of genetic transformation by ACC was partially overcome when the seedlings were germinated in the presence of STS.

To elucidate whether the ethylene sensitivity is involved in the stable transformation of plants, we inoculated A. thaliana (ecotype Colombia, Col) or the ethylene-insensitive mutants etr1-1, ein2-5 and ein3-1 with A. tumefaciens C58. Because stable transformation results in tumour formation (Chilton et al., 1977; Citovsky et al., 2007), we used the tumour formation to estimate the effect of the ethylene sensitivity. Intact A. thaliana plants inoculated with A. tumefaciens C58 formed green tumour on their stems but not in leaves. There were no tumours observed on the segments inoculated with A136, which lacks the Ti plasmid. The frequency of tumour formation was 8.1 ± 2.3% in Col inoculated with A. tumefaciens C58. For etr1-1, ein2-5 and ein3-1 inoculated with A. tumefaciens C58, the frequencies of tumour formation were significantly higher than those recorded for wild-type Col (22.6 ± 6.0, 36.4 ± 3.3 and 32.0 ± 12.7%, respectively; Fig. 1c). These results indicate that the ethylene sensitivity reduced the frequency of stable transformation in A. thaliana. Together, our results in melon and A. thaliana indicate that the ethylene sensitivity in plants affects Agrobacterium-mediated genetic transformation.

The ethylene response does not affect bacterial growth or population size

To show that the bacterial population present during co-cultivation is involved in gene transfer, the inoculated bacterial density was reduced from 108 to 106 cells ml−1. Four days after inoculation, the size of the A. tumefaciens C58C1RifR (pIG121-Hm) population was estimated from the number of colonies using the serial dilution method (Fig. 2a). Genetic transformation was defined as an increase in GUS activity in the inoculated segments (Fig. 2b). Uninoculated melon cotyledon segments were used as a control. The bacterial population reached 109 cells ml−1 during co-cultivation in melon segments that had been inoculated with 108 cells ml−1. The inoculated segments showed greater GUS activity than the uninoculated segments. Following infection with 107 cells ml−1, the bacterial population size also reached 109 cells ml−1 and the level of GUS activity was equal to that obtained following inoculation with 108 cells ml−1. In comparison, 108 cells ml−1 were present after co-cultivation in the samples inoculated with 106 cells ml−1. GUS activity in the cotyledons inoculated with 106 cells ml−1 was significantly lower than that in the cotyledons inoculated with 107 or 108 cells ml−1. This shows that a reduction in population size during co-cultivation affects genetic transformation.

Figure 2.

Effect of the Agrobacterium population size on T-DNA transfer in melon (Cucumis melo) cotyledon segments. (a) Estimation of the bacterial cell number in the segments 4 d after inoculation (DAI). Bars represent SD (n = 3). Letters indicate statistical significance at the 5% confidence level based on Student's t-test. A. tumefaciens C58C1RifR cell suspensions were prepared at 106–108 cells ml−1 just before inoculation. Uninoculated segments were used as controls (–). (b) Occurrence of T-DNA transfer in the segments depicted in (a). The occurrence of T-DNA transfer is indicated by the GUS activity in the segments. Bars indicate SD (n = 3). Letters denote statistical significance at the 5% confidence level based on Student's t-test. (c) Effects of 1-aminocyclopropane-1-carboxylic acid (ACC) on the Agrobacterium population in melon cotyledon segments. The number of Agrobacterium cells in each segment was estimated during co-cultivation. Bars represent SD (n = 3). Letters indicate statistical significance at the 5% confidence level based on Student's t-test. Black, grey and white columns refer to 0, 2 and 4 d after inoculation, respectively. ACC was added to the co-cultivation medium. A bacterial cell suspension was prepared at 108 cells ml−1 for inoculation.

From these results, we hypothesized that the plant ethylene response was involved in bacterial growth and could reduce the bacterial population size. To test this hypothesis, we estimated the bacterial population size in melon cotyledon segments that had not been exposed to ACC during germination and co-cultivation. Zero, 2 and 4 d after inoculation, the cotyledon segments were crushed and subjected to a serial-dilution plate assay. The original bacterial cell density was 107 cells ml−1, but the population size increased during co-cultivation, reaching 109 cells ml−1 4 d after inoculation. When applied with ACC in germination and co-cultivation, the final population size was the same (Fig. 2c), demonstrating that the plant ethylene response does not affect bacterial growth and population size.

Monitoring vir gene expression in A. tumefaciens C58C1RifR with the virD2-uidA reporter system

To observe vir gene expression, we constructed the virD2–uidA reporter plasmid pBBRvirD2::uidA (Fig. 3a), which was introduced into A. tumefaciens C58C1RifR by electroporation (Shen & Ford, 1989). A. tumefaciens C58C1RifR (pBBR1MCS-5) and A. tumefaciens C58C1RifR (pBBRvirD2::uidA) were each incubated with AS, which induces vir gene expression. After 20 h of incubation, the cells were collected and stained with X-Gluc, the substrate of β-glucuronidase (uidA, GUS). A. tumefaciens C58C1RifR (pBBRvirD2::uidA) treated with AS appeared blue, whereas the untreated strain appeared pale blue. X-Gluc did not stain A. tumefaciens C58C1RifR (pBBRMCS-5), regardless of the presence of AS (Fig. 3b). These results show that expression of virD2-uidA is controlled by the virD promoter. To determine the threshold AS concentration needed to induce vir gene expression, we adjusted the AS concentration from 0 to 100 µm. To measure the level of expression, GUS activity was assayed fluorometrically. After incubation, the bacteria were collected and lysed in detergent, and the lysate was tested for GUS activity. GUS activity was significantly higher in the cells inoculated with 100 µm AS; similarly, the activity in the cells was slightly greater following treatment with 10 µm AS than with 0 or 1 µm. Note that those cells treated with 1 µm AS showed the same level of GUS activity as those that were not exposed to AS. These results indicate that this system can detect vir gene expression in the presence of 10–100 µm AS, and that the system is able to monitor vir gene expression.

Figure 3.

Monitoring of vir gene expression. (a) Plasmid maps of pBBRvirD2::uidA. virD2::uidA translation is under the control of the virD promoter. (b) vir gene expression in Agrobacterium tumefaciens C58C1RifR monitored by β-glucuronidase (GUS) activity. Induction of vir gene expression was controlled by addition of 0.1 mm acetosyringone (AS). Agrobacterium tumefaciens C58C1RifR was incubated with X-Gluc for 1 h. MCS5 and virD2::uidA refer to C58C1RifR (pBBR1MCS-5) and C58C1RifR (pBBRvirD2::uidA), respectively. (c) Effect of AS concentration on vir gene expression. vir gene expression was detected by a quantitative GUS assay. The open and closed columns represent C58C1RifR (pBBR1MCS-5) and C58C1RifR (pBBRvirD2::uidA), respectively. The values represent means ± SD (n = 3). Letters indicate statistical significance at the 5% confidence level based on Student's t-test.

The ethylene response inhibits vir gene expression in A. tumefaciens

Expression of the vir genes in A. tumefaciens is essential for genetic transformation (Ooms et al., 1980); thus, we hypothesized that the plant ethylene response is involved in vir gene expression in A. tumefaciens (Fig. 4). To test this hypothesis, we used the reporter system described earlier and a melon exudate. The exudate was prepared from cotyledon segments as described in the Materials and Methods section, using seedlings in which the ethylene response was controlled with ACC in germination medium. The exudate was incubated with A. tumefaciens C58C1RifR (pBBRvirD2::uidA) for 20 h, and then the cells were collected by centrifugation and lysed with detergent. The lysate was then tested for GUS activity. A significant increase in GUS activity was observed when A. tumefaciens C58C1RifR (pBBRvirD2::uidA) was incubated with the melon exudate, whereas GUS activity decreased when the strain was incubated with exudate from ethylene-responsive melon plants (Fig. 4). Notably, the level of GUS activity observed using the exudate from ethylene-responsive plants was the same as that observed without melon exudate (Fig. 4). These data indicate that vir gene expression is not induced by exudates from ethylene-responsive plants. Thus, the plant ethylene response suppresses vir gene expression in A. tumefaciens C58C1RifR.

Figure 4.

The plant ethylene response affects vir gene expression in Agrobacterium tumefaciens C58C1RifR. The bacteria were incubated in leaf extract or MS for 20 h. vir gene expression was monitored by the virD2-uidA reporter system and the β-glucuronidase (GUS) activity in cell-free extract. Leaf exudates (−)•ACC (−), leaf exudates (+)•ACC(−) and leaf exudates (+)•ACC(+) show that A. tumefaciens C58C1RifR (pBBRvirD2::uidA) was incubated with MS medium, leaf exudates, and leaf exudates from ethylene-responding melon, respectively (ACC, 1-aminocyclopropane-1-carboxylic acid). Bars indicate SD (n = 3). Letters denote statistical significance at the 5% confidence level based on Student's t-test.

vir gene expression can reverse the inhibitory effect of ethylene on genetic transformation

The incubation of A. tumefaciens with exudate from ethylene-responsive plants did not induce vir gene expression (Fig. 4). This result suggests that the plant ethylene response might inhibit genetic transformation through the suppression of vir gene. To test this suggestion, we hypothesized that activation of vir gene expression would overcome the inhibitory effect of ethylene on genetic transformation. To enhance vir expression, we used two strategies. The first, the application of 100 µm of AS in co-cultivation medium, was able to activate vir gene expression in A. tumefaciens (Stachel et al., 1985; Yuan et al., 2007; Fig. 3). The second involves the inoculation with the A. tumefaciens strain C58C1RifR (pBBRvirGN54D, pIG121-Hm), which has constitutive vir gene expression. GUS activity indicating occurrence of genetic transformation was inhibited by the addition of ACC during germination and co-cultivation. However, in the presence of AS or inoculation of C58C1RifR (pBBRvirGN54D, pIG121-Hm), genetic transformation still occurred, even with the addition of ACC (Fig. 5). This result means that the vir gene activation overcame the inhibitory effect of the plant ethylene response on genetic transformation. Therefore, the suppression of vir gene by plant ethylene response leads to inhibition of Agrobacterium-mediated genetic transformation.

Figure 5.

Activation of the vir genes enhances T-DNA transfer in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC). The occurrence of T-DNA transfer was indicated by β-glucuronidase (GUS) activity. Open and closed columns, absence and presence of ACC during germination and co-cultivation, respectively; grey column, presence of ACC in the germination and co-cultivation medium, and the co-cultivation medium also contains acetosyringone (AS) at the same time. 121/MCS5 and 121/N54D, the inoculation of Agrobacterium tumefaciens C58C1RifR (pIG121-Hm, pBBR1MCS-5) and A. tumefaciens C58C1RifR (pIG121-Hm, pBBR1MCS-5. virGN54D), respectively; AS was included in the co-cultivation medium. Bars represent SD (n = 3). Letters indicate statistical significance at the 5% confidence level based on Student's t-test.

Discussion

Our results demonstrate that ethylene sensitivity is involved in Agrobacterium-mediated genetic transformation in melon cotyledon segments. The addition of ACC to the co-cultivation medium suppressed genetic transformation in the segments; however, this effect was overcome when STS was applied before germination (Fig. 1b). The ethylene-insensitive mutants of A. thaliana etr1-1, ein2-5 and ein3-1 showed a higher frequency of stable transformation than the wild-type Col (Fig. 1c). The etr1-1 has a mutation in the gene encoding the ethylene receptor protein (Schaller & Bleecker, 1995), whilst ein2-5 and ein3-1 are blocked at later steps in the ethylene signal transduction pathway (Roman et al., 1995). Therefore, ethylene itself does not affect the ability to carry out genetic transformation; rather, ethylene affects genetic transformation from A. tumefaciens through the ethylene-signalling pathway of the host plant. These observations indicate that the plant ethylene response and ethylene signalling suppress genetic transformation in plant cells.

vir gene expression was suppressed when A. tumefaciens was incubated with exudate from ethylene-responsive melon plants (Fig. 4). One possible explanation is that ethylene-responsive plants inhibit accumulation of inducers for vir gene expression of A. tumefaciens. Ethylene signal transduction elicits enzyme catalyses p-coumaric acid and the analog compounds, including a vir gene-inducer sinapic acid in the monolignol biosynthetic pathway of plants (Ashby et al., 1988; Lee et al., 1995; Raes et al., 2003). It is possible to think that ethylene decreases accumulation of inducers for vir gene expression. The production of inducers of vir gene expression has been reported in tobacco (Stachel et al., 1986) and wheat (Messens et al., 1990), but the effect of ethylene on accumulation of the inducers remains to be clarified. Another hypothesis is that the exudate from ethylene-responsive plants might include a molecular competitor of the activator, but this possibility might be low. This can be largely explained by the fact that the inhibition of genetic transformation by ethylene sensitivity was relieved not only by strain with constitutive vir gene expression but also with vir gene inducer molecules (Fig. 5). If ethylene-responsive plants produce competitive molecules, the application of acetosyringone should not relieve the inhibitory effect of ethylene response on genetic transformation. To demonstrate these predictions, further experiments will be required in future.

Recent studies showed that indole acetic acid and salicylic acid were involved in vir gene expression. When A. tumefaciens was exposed to these hormones directly, vir gene expression was inhibited (Liu & Nester, 2006; Yuan et al., 2007). However, the ethylene signal pathway seems to affect vir gene expression independently. Indole acetic acid and salicylic acid are competitors of vir gene inducer (Liu & Nester, 2006; Yuan et al., 2007). In contrast to these hormones, the ethylene signal pathway seems to decrease the accumulation of the inducer, because the inhibitory effect of ethylene on genetic transformation was relieved by the application of vir gene inducer (Fig. 5). This suggests that the pattern of ethylene might be different from IAA and salicylic acid. Therefore, ethylene might not suppress vir gene expression through these hormones. The relationship between ethylene response and these hormones on suppression of vir gene should to be clarified in future studies.

In Agrobacterium–host plant interaction, ethylene suppresses genetic transformation (Davis et al., 1992; Ezura et al., 2000; Han et al., 2005; Fig. 1b,c). The suppression mechanism involves activation of vir gene expression in Agrobacterium (Fig. 4). During the transformation process, the activation of vir gene results from the recognition of the susceptible plant cell through the phenolic compounds (Stachel et al., 1985). Therefore our results show that plant ethylene response affects the recognition step in the transformation process. In several legume–Rhizobium interactions, nodulation is sensitive to ethylene (Nukui et al., 2000, 2004; Okazaki et al., 2004). In addition, ethylene inhibits plant responses to the Rhizobium signal Nod in Medicago truncatula (Oldroyd et al., 2001). Nod allows legumes to recognize and associate with Rhizobium cells (Fisher & Long, 1992; Riely et al., 2004). In plant–microbe interactions, plant ethylene sensitivity should be considered to have a regulatory role in molecular recognition between the organisms.

Ethylene has been reported to be involved in the regulation of defence-related gene expression during plant–microbe interactions (Ecker & Davis 1987; Suzuki et al., 1998), and it is thought that plants might defend themselves against pathogenic disease by controlling bacterial growth. However, it was recently reported that in some plant–pathogen interactions, the ethylene-mediated defences could be uncoupled from pathogen growth (Lund et al., 1998; O'Donnell et al., 2001, 2003). For example, the ethylene-insensitive mutant etr1-1 and etr2-1 showed more severe symptoms than the wild-type, but the rates of Xanthomonas campestris pv. campestris growth were the same in the ethylene-insensitive mutants etr1-1 or etr2-1 and the wild-type (O'Donnell et al., 2003). In the Agrobacterium–plant interaction, disease symptoms accompany genetic transformation, and the plant ethylene response suppressed the appearance of disease symptoms without inhibiting bacterial growth. Our results seem to suggest that the ethylene response might control host recognition by pathogenic bacteria and not bacterial growth for the ethylene-mediated defences uncoupled from pathogen growth.

In this study, we demonstrated that the plant ethylene response, and not ethylene itself, suppresses genetic transformation (Fig. 1b,c); the plant ethylene response affects activation of the vir gene (Fig. 4); and the plant ethylene response does not affect bacterial growth (Fig. 2c). Activation of the vir gene is a recognition step in Agrobacterium-mediated genetic transformation. Therefore, this study indicated that the plant ethylene response suppresses the recognition step in the Agrobacterium-plant interaction.

Acknowledgements

We thank the members of the Ezura laboratory for helpful discussions. We are grateful to Dr S. Abdullah for his advice concerning the English of this article and Prof. H. Kamada (University of Tsukuba) for his helpful discussions on tumour formation assay in A. thaliana. This work was supported by the 21st Century Centers of Excellence Program and a grant-in aid for scientific research category B (no. 15380002) to HE from the Ministry of Education, Science, Sports, and Technology of Japan.

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