Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer.

Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant-Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.


INTRODUCTION
Agrobacterium tumefaciens is a soil-borne bacterium known to cause crown gall disease in plants. A. tumefaciens strains that induce crown gall have a large plasmid (tumor-inducing plasmid: Ti plasmid), which is essential for the establishment of crown gall disease (van Larebeke et al., , 1975Zaenen et al., 1974;Watson et al., 1975;Currier and Nester, 1976). Chilton et al. (1977) detected part of the Ti plasmid in the crown gall genome, showing that plant cell conversion resulted in genetic transformation. This work clearly showed that A. tumefaciens has the ability to transfer T-DNA into plant genomes (Chilton et al., 1977).
Since this discovery in 1977, many investigators have attempted to generate A. tumefaciens suitable for plant genetic engineering. In early techniques, the oncogenic T-DNA region of the Ti plasmid was replaced with genes of interest by single or double homologous recombination (Zambryski et al., 1983). Although the recombination steps are easy, they are limited by the potential recombination of repetitive sequences during or after recombination in the Ti plasmid replicon in A. tumefaciens. Hoekema et al. (1983) showed that A. tumefaciens is able to deliver the T-DNA even if the Ti plasmid is divided into two plasmids (the T-DNA region and the vir region). This finding made it possible to extract only the T-DNA region from A. tumefaciens, and the modification was easier without the sequence limitation. After this finding, a binary plant vector strategy became the standard method in plant genetic engineering (Hoekema et al., 1983;Bevan, 1984). Increasing the virulence of A. tumefaciens is a useful strategy for higher transformation efficiency. The identification and application of vir gene inducers, such as plant phenolic compounds (Stachel et al., 1985(Stachel et al., , 1986 and sugars (Cangelosi et al., 1990;He et al., 2009;Hu et al., 2013), enabled this technique to be adapted for a wide range of plant species, especially monocot crops. The selection of hyper-virulent strains also spread the adaptation throughout the host range. Superbinary vectors, which have shown higher vir gene expression, have provided critical improvements in transformation efficiency (Komari et al., 2006). Agrobacterium-mediated gene transfer has been well established through many modifications, and it is a basic technique in plant science, but the gene transfer efficiencies are not sufficient for many plant species, especially crops and biomass plants. Therefore, improvements in these techniques are required.
Some plant metabolites and phytohormones inhibit Agrobacterium-mediated transformation. In particular, ethylene showed a negative effect on Agrobacterium-mediated T-DNA transformation in many plant species. In this review, we focus on the effect of ethylene on Agrobacterium-mediated gene transfer into plant cells and introduce an engineering strategy by which to increase the transformation efficiency of A. tumefaciens via ethylene removal. The established A. tumefaciens strain is designated as super-Agrobacterium.

ETHYLENE SUPPRESSES Agrobacterium -MEDIATED T-DNA TRANSFER
The gaseous phytohormone ethylene regulates multiple physiological and developmental processes in plants, such as seedling emergence, leaf and flower senescence, ripening, organ abscission, growth-phase transitions, rhizobium-legume interaction, and plant-pathogen interaction (Beyer, 1981;Yang and Hoffman, 1984;Yang, 1985;Abeles, 1992;Ogawara et al., 2003). Ethylene synthesis is stimulated by biotic or abiotic stress. Ethylene also modulates A. tumefaciens-plant interactions. Ethylene is a crucial determinant of crown gall development. Plants treated with inhibitors of ethylene synthesis or perception, such as aminoethoxyvinylglycine (AVG), and ethyleneinsensitive Never-ripe mutants (tomato) suppress crown gall growth (Aloni et al., 1998). Because vascularization is essential for efficient assimilate import from the host plant into the tumor cells, if the vascularization is suppressed, nutrient supply stops (Malsy et al., 1992;Pradel et al., 1996Pradel et al., , 1999, resulting in the inhibition of crown gall development. Ethylene stimulates crown gall development by inducing vascular development (Wächter et al., 2003), and it induces crown gall development but inhibits Agrobacterium-mediated genetic transformation of plant cells. The enhancement of ethylene production by supplying its immediate precursor, 1aminocyclopropane-1-carboxylic acid (ACC), suppresses gene transfer in tomatoes and melons (Davis et al., 1992;Ezura et al., 2000). Ethylene is increased by wounding and A. tumefaciens infection stress during co-cultivation (Ezura et al., 2000). Reducing ethylene production during co-cultivation using ethylene biosynthesis inhibitors such as AVG or suppressing plant ethylene perception by adding silver ions to the tissue culture medium has improved the transformation efficiency in melons (Ezura et al., 2000), cauliflowers (Chakrabarty et al., 2002), apricots (Burgos and Alburquerque, 2003), apple trees (Petri et al., 2005;Seong et al., 2005), and bottle gourds (Han et al., 2005). The stable transformation frequency was also increased in the Arabidopsis thaliana ethylene-insensitive mutants etr1-1 and etr1-2 (Nonaka et al., 2008a).
These results indicate that ethylene inhibits gene transfer in plants. One possible explanation for this phenomenon might involve the plant defense response via ethylene signaling. Previous studies have found that ethylene regulates several genes that are involved in the defense response, including those that encode the PR proteins chitinase, β-1, 3-glucanase, and PR1 (Deikman, 1997) in addition to phytoalexin synthetic enzymes (Ecker and Davis, 1987), defensins (Penninckx et al., 1996), and hydroxy-Pro-rich glycoproteins (Toppan et al., 1982). These compounds suppress bacterial growth because of their antibacterial activity. Indeed, in tomatoes, the reduced expression of LeETR4, which encodes a tomato ethylene receptor, resulted in increased sensitivity to ethylene, an enhanced hypersensitive response, and the reduced growth of Xanthomonas campestris pv. vesicatoria compared to wild-type (WT) plants (Ciardi et al., 2000). Ethylene insensitivity results in reduced resistance to the potato soft rot agent Erwinia carotovora subsp. carotovora in A. thaliana etr1-1 and ein2-1 mutants (Norman-Setterblad et al., 2000). In general, ethylene levels increase in plant defense responses; however, the inhibitory effect of ethylene on Agrobacteriummediated gene transfer does not occur through plant defense. The model scheme is described in Figure 1. Microarray and differential display analysis showed that A. tumefaciens infection induces the plant genes necessary for transformation while simultaneously repressing host defense response genes (Veena et al., 2003). Ethylene evolution was induced by A. tumefaciens inoculation in the early stage of the infection (Nonaka et al., 2008b;Lee et al., 2009). Although A. tumefaciens infection increased ethylene levels, the plant genes that encode ethylene receptors and their downstream signaling components, including defense response genes, are not induced (Lee et al., 2009). These results support the hypothesis that the inhibitory effect of ethylene on Agrobacterium-mediated gene transfer is independent of the plant defense response. Indeed, the plant ethylene response did not affect A. tumefaciens growth during co-cultivation (Nonaka et al., 2008a). The plant ethylene response inhibits T-DNA transfer through the suppression of vir gene expression (Nonaka et al., 2008a). The application of exudate from plants showing an ethylene response to A. tumefaciens reduced vir gene expression, which decreased T-DNA transfer. Such an inhibitory effect was partially overcome by the application of acetosyringone, a vir gene inducer, and in an A. tumefaciens strain constitutively expressing the vir gene. From this result, two possibilities were considered: in plants showing an ethylene response, the amount of the vir gene inducer would be reduced or the antagonist of the vir gene inducer would be produced. This deficient recovery indicates that vir gene suppression is one of the reasons for a reduction in T-DNA transfer via the ethylene response. The partial restoration upon the application of the vir gene inducer suggests that there is another inhibitory mechanism of ethylene. Therefore, the suppression of the vir gene is not sufficient to explain the negative effect of ethylene on Agrobacterium-mediated gene transfer. Because the inhibitory mechanism of ethylene on Agrobacterium-mediated gene transfer still needs to be clarified, to improve the transformation frequency, the introduction of the ability to reduce ethylene in A. tumefaciens would be effective.

STRATEGY TO REDUCE ETHYLENE PRODUCTION IN PLANT CELLS INOCULATED WITH Agrobacterium
Ethylene is generated through the ethylene biosynthetic pathway, which was elucidated largely by the pioneering work of Yang and co-workers in the 1970s and 1980s (Wang et al., 2002). Ethylene is synthesized from the amino acid methionine, which is converted to S-adenosyl-L-methionine (SAM) by SAM synthase (ADS). SAM is the major methyl donor in plants and is involved in the methylation of lipids, proteins, and nucleic acids. SAM is converted by the enzyme ACC synthase (ACS) to 5 -methylthioadenosine (MTA), which is converted back to methionine via the Yang Cycle and to ACC, the precursor of ethylene. ACC is finally oxidized by ACC oxidase (ACO) to form ethylene, cyanide, and carbon dioxide. The conversion of SAM to ACC is considered to be the rate-limiting step in ethylene biosynthesis and consequently has been studied intensively. Therefore, the degradation of ACC, the immediate precursor of ethylene, effectively reduces ethylene production in plants. The reduction of ethylene via the degradation of ACC is found in some soil bacteria.
The pyridoxal 5-phosphate-dependent enzyme ACC deaminase catalyzes the decomposition of ACC to α-ketobutyrate and ammonia (Honma and Shimomura, 1978;Minami et al., 1998;Hontzeas et al., 2004;McDonnell et al., 2009). Although ACC deaminase genes have been isolated in a wide range of organisms, the ACC deaminase gene was isolated from many plant growthpromoting rhizobacteria (PGPR; Table 1). As shown in. PGPR with ACC deaminase activity can lower the ethylene level in plant cells through the degradation of ACC.
In response to tryptophan and other small molecules present in root exudates, IAA is synthesized and secreted by a PGPR that is bound to the surface of the root (Whipps, 1990;Hong et al., 1991;Fallik et al., 1994;Dimkpa et al., 2012). Some of the newly synthesized IAA is taken up by the plant and can stimulate ACS to convert SAM to ACC (Kende, 1993). The uptake and subsequent cleavage of ACC by the PGPR decrease the amount of ACC outside the plant (Penrose and Glick, 2001).
The K m of ACC deaminase is lower than ACC oxidase. The various plant ACC oxidase for ACC ranges from approximately 8 µM (for ripening apples) to 120 µM (for etiolated beans; Smith et al., 1992). The K m of ACC deaminase for ACC ranges from approximately 1.5 to 3.4 mM (Honma and Shimomura, 1978;Hontzeas et al., 2004). This means that ACC oxidase has about a 100-fold greater affinity for ACC than does ACC deaminase. Despite the fact that ACC oxidase has a much higher affinity for ACC, the kinetic calculations indicate that ACC deaminase can be more effective in lowering ACC than ACC oxidase when the amount of ACC deaminase is much greater than the amount of

Source organisms Reference
Bacteria (Gram-negative)

Enterobacter cloacae
Holguin and Glick (2001 ACC oxidase (Glick et al., 1998). These results indicate that the ACC metabolite reaction in PGPR was more effective than that in plant cells and that the ACC level are lower in inner plant cells than in external cells. Therefore, to maintain equilibrium between internal and external ACC levels, the plant increases the level of ACC exudate. PGPRs have the ability to utilize ACC as a sole source of nitrogen in plant roots (Glick et al., 1994;Jacobson et al., 1994) and to proliferate under conditions in which other soil bacteria cannot grow. The reduction of the inner ACC level caused by the utilization of ACC in bacteria causes the plant to synthesize more ACC and to effectively exude ACC from the plant. A significant correlation was found between in vitro bacterial ACC deaminase activity and the growth-promoting activity of these bacteria under pot and field trial conditions (Shaharoona et al., 2006a,b). In fact, many types of PGPR containing the ACC deaminase gene reduced ethylene production, resulting in physiological changes in many types of plants. The rhizobacterium www.frontiersin.org December 2014 | Volume 5 | Article 681 | 3 Variovorax paradoxus 5C-2, which possesses ACC deaminase, promotes the growth (leaf area and shoot biomass) and development of WT A. thaliana and the ethylene-overproducing mutant eto1-1 but does not have these effects in ethylene-insensitive mutants (etr1-1 and ein2-1; Chen et al., 2013). Furthermore, V. paradoxus 5C-2 decreased the ACC concentrations in the rosette leaves of WT plants by 59% and foliar ethylene emission in both WT plants and eto1-1 mutants by 42 and 37%, respectively, (Chen et al., 2013). Rhizobacteria possessing ACC deaminase induced maximum waterlogging tolerance in Ocimum sanctum, as treated waterlogged plants exhibited the maximum growth and biomass yield with minimal ethylene levels (Barnawal et al., 2012). Bacterial endophytes expressing ACC deaminase delay flower senescence (Ali et al., 2012). Mesorhizobium ciceri LMS-1 expressing an exogenous ACC deaminase increases nodulation abilities and chickpea plant resistance to soil constraints (Nascimento et al., 2012).

SUPER-Agrobacterium WITH ACC DEAMINASE ACTIVITY INCREASES T-DNA TRANSFER EFFICIENCY VIA A. tumefaciens
The introduction of ACC deaminase activity into A. tumefaciens may reduce ethylene levels during co-cultivation and increase A. tumefaciens-mediated transformation efficiency. Whole-genome sequence analysis revealed that the A. tumefaciens strain C58 does not have an ACC deaminase gene (Wood et al., 2001); therefore, this strategy seems to be effective in this species. The ACC deaminase gene was amplified from Pseudomonas sp. via PCR. This amplified fragment was inserted into pBBR1MCS-5 (pBBRacdS). The pBBR1MCS-5 plasmid is compatible with IncP, IncQ, and IncW plasmids, and it has a different origin of the RK2 vector, which is used as a binary vector (Kovach et al., 1994). Because their origins are different, pBBRacdS and the binary vector are able to coexist in A. tumefaciens. The expression of ACC deaminase was controlled by the lacZ promoter, which constitutively and strongly expresses genes in A. tumefaciens. The A. tumefaciens strain harboring ACC deaminase genes showed ACC deaminase activity and effectively inhibited ethylene production in melons (Figure 2A), resulting in increased transient T-DNA transformation in melons ( Figure 2B; Nonaka et al., 2008b). For the further improvement of Agrobacterium-mediated transformation, we attempted to increase the expression level of ACC deaminase. Klüsener et al. (2010) showed that virB1, virD1, and virE1 of A. tumefaciens were among the most highly expressed genes in acidic medium (AB medium at pH 5.5) containing 100 mol/L acetosyringone (Klüsener et al., 2010). We compared the activities of these three promoters to select the highest one, and the virD1 promoter showed the strongest transcription activity. The virD1 promoter conferred fourfold increased transcriptional activity compared with the lacZ promoter. The time course analysis (0-72 h) showed that ACC deaminase expression was induced at 6 h by adding acetosyringone to MS medium, and the high expression level was maintained until 72 h. By contrast, the expression of the ACC deaminase gene driven by the lacZ promoter started to decrease at 6 h. GUS-stained tomato cotyledons were categorized based on the stained area: less than 1, 1-3, 3-5, 5-10, 10-20, and more than 20%. (D) The frequency of each GUS staining category in tomato explants. Bacterial strains with significant differences (Student's t-test and Kruskal-Wallis test; P < 0.01) are indicated by different letters. cont: A. tumefaciens GV2260 (pBBR1MCS-5, pIG121); acdS: A. tumefaciens GV2260 (pBBRacdS, pIG121-Hm) acdS gene driven by lac promoter; virD1acdS: A. tumefaciens GV2260 (pvirD1acdS, pIG121-Hm) acdS gene driven by the virD1 promoter. Panels (A,B) are cited from Nonaka et al. (2008b). Panels (C,D) are cited from Someya et al. (2013).
Compared with the lacZ promoter, the virD1 promoter maintained ACC deaminase gene expression at a higher level for a long time, increasing the ACC deaminase activity in A. tumefaciens. Generally, the co-cultivation period is 72 to 96 h, so the virD1 promoter seems to be suitable as the ACC deaminase gene activator. This newly developed A. tumefaciens strain showed higher T-DNA transformation efficiency in tomatoes (Figures 2C,D) and higher biomass production than Erianthus ravennae, which shows very low transformation frequency. Therefore, A. tumefaciens with higher ACC deaminase activity is a powerful tool for the Agrobacterium-mediated genetic engineering of plants (Someya et al., 2013). Introducing ACC deaminase into A. tumefaciens is effective at increasing the stable transformation frequency. A. tumefaciens with ACC deaminase succeeded in increasing the stable transformation frequency in Egusi melons (Ntui et al., 2009)

CONCLUSION
Agrobacterium-mediated transformation is an important tool for plant genetic engineering. Although a sophisticated protocol has been established for model plants, such as A. thaliana, tobacco, and rice, the transformation efficiency was not sufficiently high for commercially important crops such as maize, sorghum, soybean, barley, and E. ravennae. There has therefore been a need for the improvement of this methodology. Among the various negative factors contributing to low Agrobacteriummediated transformation rates, ethylene has been well known as an inhibitor of transformation efficiency for a long time.
There are chemicals to reduce ethylene production or ethylene perception. However, they are hard to use because they are expensive, in a gaseous form or toxic to bacteria; they are thus not suitable for improving Agrobacterium-mediated transformation. Therefore, we attempted to reduce ethylene in A. tumefaciens. Some PGPRs have an enzyme that degrades the ethylene precursor ACC. This strategy is not toxic for bacteria, and it is very easy to perform. We introduced the enzyme ACC deaminase into A. tumefaciens. The strain has been designated as super-Agrobacterium. It showed increased transient gene delivery into melon cotyledons (Nonaka et al., 2008b), tomatoes, and E. ravennae (Someya et al., 2013). In addition, the super-Agrobacterium also increased stable transformation in Egusi melon and three canola species (Ntui et al., 2009;Hao et al., 2010). To improve the "super-Agrobacterium," a thorough analysis will be required in the future. One of the negative effects of ethylene is the suppression of vir gene expression in A. tumefaciens. However, the constitutive vir gene expression strain or the addition of the vir gene expression inducer could only partially overcome the inhibitory effect of ethylene on vir gene expression. This result showed the possibility of a different inhibitory mechanism that is caused by ethylene; therefore, a thorough analysis will be required in the future. Our study showed that there are two points that should be considered when improving the efficiency of Agrobacterium-mediated gene transfer: first, the transient transformation frequency is important for increasing the stable transformation efficiency. Second, the regulation of plant-derived signals is an effective method for increasing the A. tumefaciens-mediated gene transfer frequency. For a more sophisticated strategy by which to increase transformation, it is effective to analyze the plant-derived signals in Agrobacterium-plant interactions. The removal of these signals would further increase the A. tumefaciens-mediated transformation frequency in recalcitrant plants. chlororaphis O6 is inhibited by ZnO nanoparticles but enhanced by CuO