In their natural habitats, plants live in close contact with a myriad microorganisms. Plant-microbe associations can be mutually beneficial, such as the root nodule symbiosis with nitrogen-fixing bacteria or the more wide-spread association of plant roots with arbuscular mycorrhizal fungi (reviewed in Parniske, 2008; Markmann and Parniske, 2009). In contrast, pathogenic fungi or bacteria impair plant development and cause various disease symptoms in their hosts. The gram-negative Agrobacterium tumefaciens of the family Rhizobeaceae is a “special case.” It is a biotroph pathogen, which markedly alters the physiology and morphology of infected host plants. What makes Agrobacterium so special is its capability for interkingdom gene transfer. In nature, wild type A. tumefaciens (as well as A. rhizogenes and A. vitis) causes “crown gall disease,” characterized by the growth of tumor-like structures (calli) on host species. The genetic information for this anatomical reprogramming is encoded on the tumor-inducing (Ti) plasmid. The transfer DNA (T-DNA) derived from the Ti plasmid is imported into the host cell's cytoplasm and subsequently into the nucleus (Gelvin, 2003, 2005; Dafny-Yelin et al., 2008; Pitzschke and Hirt, 2010b). T-DNA transport is mediated by agrobacterial virulence factors, and—involuntarily—supported by proteins of the attacked host. Over the last decade, microbiologists and plant scientists have disclosed an impressive portfolio of agrobacterial infection strategies, some of which resemble those in other pathogen-host interactions. Plant defense mechanisms counteracting these strategies are equally diverse and impressive.

The principal steps and factors involved in Agrobacterium-mediated plant transformation are comparatively well-understood, and reviews can be found in e.g., (Gelvin, 2009, 2010a,b; Pitzschke and Hirt, 2010b). Briefly, agrobacteria sense phenolic substances that are secreted by wounded plant tissue. Reception of these signals drives the expression of bacterial virulence (vir) genes. Subsequently, Vir proteins are produced, and single-stranded T-DNA molecules are synthesized from the Ti plasmid. The T-complex, i.e., T-DNA associated with certain Vir proteins, is injected into the host cytoplasm. A sophisticated network of bacterial and plant factors mediates translocation of the T-DNA to its final destination, the host cell's nucleus.

Agrobacterium inserts substrates (T-DNA and virulence proteins including VirD2, VirE2, VirE3, VirD5, and VirF) into the host cell by a type IV secretion system (Cascales and Christie, 2003). This strategy is also employed for the delivery of microbial factors by other plant pathogens, including Xanthomonas campestris (Thieme et al., 2005) and Burkholderia (Engledow et al., 2004). Likewise, mammalian pathogens including Bordetella pertussis, Legionella pneumophila, Brucella spp., and Helicobacter pylori, use type IV machineries to export effector proteins to the extracellular milieu or the cell cytosol (Christie and Vogel, 2000). Remarkably, under laboratory conditions, agrobacteria can genetically transform virtually any type of eukaryote, ranging from yeast (Bundock et al., 1995) to human cells (Kunik et al., 2001) (reviewed in Michielse et al., 2005; Lacroix et al., 2006). The T-complex, consisting of T-DNA, bacterial virulence proteins (VirE2, VirD2) and the host factor VIP1 (VirE2-interacting protein 1) is imported into the nucleus. Subsequently, the proteinaceous components are stripped off, releasing the T-DNA from the T-complex. This step relies on degradation of VirE2, VirD2, and VIP1 by the plant SCF proteasomal machinery (see below). The bacterial F-box protein VirF, which is contained in and confers substrate specificity to the SCF complex, participates in this degradation. If the T-complex disintegrates before it is in contact with the host's chromatin, the delivered transgenes are expressed for only a few days. The loss of transgene activity at later stages likely results from the T-DNA being degraded by host nucleases (Gelvin, 2003). In contrast, if the T-DNA is shielded until the T-complex is in contact with chromatin, stable transformants can be obtained. Due to its affinity for histones, VIP1 most probably guides the T-DNA to its target destination, the chromatin (Lacroix et al., 2008).

Since the discovery of the gene transfer mechanism (Schell and Van Montagu, 1977; Holsters et al., 1978), Agrobacterium strains have been converted (“disarmed”) into efficient delivery systems for the genetic manipulation of plants. While transient expression approaches can provide rapid answers on e.g., subcellular localization, protein-protein interaction and promoter/effector relationships (Andrews and Curtis, 2005; Li et al., 2009; Pitzschke, 2013b), genetic engineering requires the transgene(s) to be stably integrated in the host genome.

The so-called disarmed/non-oncogenic A. tumefaciens strains employed are deprived of their Ti properties, and the T-DNA region is used as a vehicle for the introduction of tailor-made DNA sequences. Any DNA sequence placed between T-DNA “border sequences” (Ti-plasmid-derived 25-bp direct repeats) can be transferred (Gelvin, 2012). Disarmed strains, therefore, facilitate transformation, but do not provoke callus growth or other abnormalities caused by oncogenic strains. Consequently, phenotypic abnormalities that may be exhibited by transformed plants are primarily due to the particular transgene being expressed. Furthermore, by using armed and disarmed strains side-by-side, host responses that are independent of or dependent on Ti sequences can be distinguished.

The advent of full genome sequencing and microarray technologies has created the opportunity to draw a complete picture on Agrobacterium-induced changes at the transcript level. Gene expression profiling data have been generated for various plant species, and comprehensive databases (e.g., http://www.plexdb.org) and bioinformatics resources even allow comparison of transcriptional responses across multiple plant species (Dash et al., 2012). One major finding from diverse microarray studies was that agrobacteria largely modify host gene expression, particularly that of defense-related genes.

This fact had already been recognized in the “pre-microarray era.” cDNA-AFLP analysis of Ageratum conyzoides plant cell cultures enabled the identification of (non-oncogenic) Agrobacterium-induced transcripts, many of which encoded putative defense factors (Ditt et al., 2001). In a subsequent study the same research group observed an anti-correlation between Agrobacterium-mediated transformation efficiency and defense gene expression levels (Ditt et al., 2005). By the approach of suppression subtractive hybridization and DNA macroarrays, Veena Jiang et al. (2003) provided the first insight into the molecular kinetics of Agrobacterium -plant interactions. Transcriptional responses of tobacco BY-2 cell cultures to a subset of agrobacterial strains, impaired in T-DNA and/or Vir protein transfer, were monitored over a 36-h-period. All strains elicited a general defense response during early stages of infection. However, expression of defense-related genes was repressed at later stages—exclusively by the transfer-competent strains. More detailed expression profiling of selected genes furthermore disclosed the “unintentional” participation of the host cellular machinery in the transformation process (Veena Jiang et al., 2003).

Microbes attempting to invade their hosts betray themselves by the presence of so-called microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs). These molecules, which are recognized as “non-self” initiate the first line of defense, known as PAMP-triggered immunity (PTI) (Nurnberger et al., 2004; Sanabria et al., 2008; Boller and He, 2009) (see below). Pathogens, in turn, aim to overcome PTI activation by injecting certain effector proteins into the host cytoplasm. Perception of these pathogen-encoded effectors by cognate intracellular plant proteins raises the second line of defense, effector-triggered immunity (ETI) (Bonardi and Dangl, 2012; Gassmann and Bhattacharjee, 2012). This response is characterized by the induction of localized apoptosis (hypersensitive response, HR) and systemic defense signaling. Plants capable of activating ETI can thus not only restrict pathogen spread, but they can also fortify themselves against subsequent attacks (Shah and Zeier, 2013).

Many biological processes, including host-pathogen interactions, are controlled by SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. These complexes mediate the proteasomal degradation of specific target proteins. The F-box protein contained in SCF complexes confers substrate specificity (Lechner et al., 2006).

Although prokaryotes lack SCF complexes, F-box-encoding genes are found in some pathogenic bacteria. The translocation of F-box effectors appears to be a wide-spread “infection strategy.” Pathogens secrete F-box proteins into their hosts to abuse the SCF machinery, resulting in high infection rates. However, F-box effectors are intrinsically unstable proteins which are rapidly degraded by the host proteasome pathway (Magori and Citovsky, 2011b). The Citovsky laboratory uncovered yet another level of agrobacterial cleverness and callousness: Destabilization of the agrobacterial F-box protein VirF is counteracted by the bacterial effector, VirD5 (Magori and Citovsky, 2011a). As if this was not enough, agrobacteria also exploit additional host factors to maximize infection: Diverse pathogens, including Agrobacterium, induce expression of VBF (VIP1-binding factor), a host-encoded F-box protein. VBF can functionally replace the agrobacterial VirF in regulating VIP1 and VirE2 protein levels (Zaltsman et al., 2010b). Analogous to VirF, VBF destabilizes VirE2 and VIP1, most likely via SCF-mediated proteasomal degradation (Zaltsman et al., 2010a). A very recent study extends on this finding and highlights the importance of VBF at the final stage of T-DNA pre-integration (Zaltsman et al., 2013). As reported earlier, T-complexes can be reconstituted from ssDNA and VirE2 in vitro (Zupan et al., 1996). Its tight packaging by VirE2 molecules shields the ssDNA from the outside and makes it inaccessible to degradation by exogenously added DNAse. In the presence of extracts from wild type, but not from VBF antisense plants, this “shielding effect” was found to be rapidly lost. Thus, VBF-mediated uncoating of the T-complex indeed results in unmasking of the T-DNA (Zaltsman et al., 2013).

Micro-bombardment studies in N. benthamiana leaves have disclosed a cytoplasmic-nuclear distribution of VBF. In contrast, VBF/VIP1 complexes occur exclusively in the nucleus. Based on these observations, VBF may have additional functions in the cytoplasm, besides acting in T-complex disassembly in the nucleus, (Zaltsman et al., 2010b). Alternatively, VBF may re-locate upon pathogen attack (similar to VIP1). If this—currently hypothetic—scenario was true, a straight-forward question arises. Is VBF distribution phosphorylation-dependent; is it controlled by MAPKs? At least in silico, such scenario appears possible (Pitzschke, unpublished). MAPKs phosphorylate their targets at serine or threonine residues adjacent to a proline. A kinase interaction motif [KIM; R/K-x2-6-I/Lx(/L)], known to be recognized by mammalian MAPKs (Tanoue and Nishida, 2003), assists MAPK binding also in substrate proteins of plant MAPKs (Schweighofer et al., 2007). The VBF protein sequence contains one Ser-Pro dipeptide motif as well as one KIM (position 164-171) (Figure 1). Pathogen-activated MAPK(s), such as MPK3, may phosphorylate residue Ser17 and thereby initiate VBF nuclear translocation.

A plethora of developmental and stimulus-triggered responses are signaled via phytohormones. Auxin is involved in essentially all aspects of plant growth and development (Benjamins and Scheres, 2008; Ljung, 2013). Ethylene controls fruit ripening and plant senescence. It also mediates biotic stress and numerous other environmental responses (Merchante et al., 2013). Abscisic acid controls seed germination, stomatal movement and is tightly connected with diverse abiotic and biotic stress responses (Nakashima and Yamaguchi-Shinozaki, 2013). Salicylic acid (SA), jasmonate and ethylene primarily act in biotic stress protection. There is ample evidence for the existence of substantial crosstalk between plant hormone defense pathways (De Torres Zabala et al., 2009; Robert-Seilaniantz et al., 2011a; Boatwright and Pajerowska-Mukhtar, 2013). These reports highlighted the importance of the plant's need to dynamically balance absolute and relative levels of phytohormones. A complex and comprehensive review on plant hormones and pathogen response was published very recently (Denance et al., 2013).

Agrobacteria largely shift the “hormone balance” in their infected hosts. This effect on endogenous growth regulators will ultimately lead to agrobacterium-induced tumor formation. An elaborate study provided an insight into Agrobacterium-induced phytohormonal changes, and it allowed the researchers to separate tumor-dependent and-independent host responses. Lee et al. (2009) examined the physiological changes and adaptations during tumor development provoked by an oncogenic strain (C58) or a disarmed derivate (GV3101), which only lacks the T-DNA but not the Vir factors (VirD2, VirE2, VirE3, VirF) (Holsters et al., 1980). The oncogenic strain was found to cause much stronger host responses than the disarmed strain. The authors monitored the kinetics of Agrobacterium-induced concentration changes of plant hormones, including SA, ethylene, jasmonic acid and indole-3-acetic acid (IAA, the most important auxin). In parallel, they assessed transcriptional changes, with a focus on hormone biosynthesis genes. At the early stage of infection, IAA and ethylene started to accumulate, while later, after T-DNA integration, primarily SA levels increased.

In the subsequent sections particular attention is given to the roles of auxin and SA in the agrobacterium/plant interaction.

Plants exhibit an admirable perseverance in their battle against microbial manipulation. Even after unsuccessful attempts to escape Agrobacterium-induced genetic re-programming, the host cell does not surrender. Instead, transformed cells employ gene silencing mechanisms to limit the levels of T-DNA-derived transcripts. Evidence for the involvement of post-transcriptional gene silencing had been provided in a pioneering work by Dunoyer et al. (2006). Small interfering RNAs (siRNAs) directed against T-DNA oncogenes (tryptophan 2-monooxygenase and agropine synthase) were detected in Nicotiana benthamiana leaves 3 days after infiltration with virulent agrobacteria. Additional experiments in Arabidopsis further stressed the importance of gene silencing as a disease-limiting strategy. RNA interference-deficient mutant plants (rdr6, lacking a RNA-dependent RNA polymerase) were found to be hypersusceptible to agrobacterial infection, as evidenced by extensive tumor formation (Dunoyer et al., 2006). The researchers also conducted infection studies in leaves and stems of Nicotiana bethamiana carrying a post-transcriptionally-silenced reporter gene (green fluorescent protein, GFP). This approach enabled them to show that the siRNA protection strategy against T-DNA genes is efficient only at early stages of infection: Strong green fluorescence, high GFP mRNA concentrations and low siRNA concentrations were detected specifically in young tumors. Later in the infection process, the pathogen takes command. By specifically inhibiting siRNA synthesis, agrobacteria induce an anti-silencing state—thereby ensuring oncogene expression and tumor maturation (Dunoyer et al., 2006).

A more recent study furthermore documented that DNA methylation also plays a critical role in the regulation of T-DNA transcript levels (Gohlke et al., 2013). The authors compared the methylation pattern of mock- and Agrobacterium-inoculated Arabidopsis inflorescence stalks on a genome-wide level. Four-week-old tumors, arising from inoculation with the oncogenic A. tumefaciens strain C58 contained a globally hypermethylated genome. Intriguingly, a specifically low degree of methylation was observed in T-DNA-derived oncogenes (Ipt IaaH, IaaM). Data obtained from experiments with DNA methylation mutants lead to the conclusion that crown gall formation and oncogene expression correlate with the unmethylated state and, consequently that hypermethylation is a strategy to inhibit plant tumor growth.

Agrobacterium naturally has a wide host range in plants, primarily dicot species. Driven by the demand for higher yields and improved stress tolerance the accessibility to transformation has become a prime issue in crop science. Despite intensive research it is still poorly understood why some plant species can be transformed easily, while others are recalcitrant to Agrobacterium-mediated transformation. Transformation methods of model plants and important crop species are frequently updated, documenting the striving for simpler, more robust and more efficient protocols (reviewed in e.g., Pitzschke, 2013a). These protocols primarily focus on optimizing the conditions of Agrobacterium—explant co-incubation. Here, duration, light conditions and the concentration of supplemented acetosyringone and plant hormones are key parameters.

One central message emerges from enumerable transformation studies. The outcome of co-cultivation is primarily determined by the timing and intensity at which host defense responses are activated. Understanding the molecular language of the plant—Agrobacterium dialogue is therefore of substantial interest both to basic research and agricultural science.

Studies that compare different cultivars of the same species are particularly informative, and one such study shall be mentioned here. Transformation efficiencies between rice cultivars differ greatly. The indica variety lags far behind the japonica cultivars. A comparative study of the two cultivars in transient and stable transformation assays revealed that the lower transformation efficiency in indica rice was mainly due to less-efficient T-DNA integration into the host genome (Tie et al., 2012). Microarray analyses (1, 6, 12, and 24 h post-infection) revealed major differences in the Agrobacterium-induced changes in transcriptome profiles of the two cultivars. These differences were most pronounced at the early stages of infection (within the first 6 h). The authors observed an overall stronger response in the indica cultivar (Zs), with several genes being repressed, and they postulated that some of these genes may be required for the transformation process. From this study, one may conclude that (1) although T-DNA integration represents a late step in the transformation process, the “decision” that leads to failure or success is made early. This decision is made in a narrow time window, since many Zs-specific transcripts are repressed only transiently (at the 1 OR 6 h time-point only). (2) Agrobacteria manage to actively prevent repression of integration-assisting genes in the susceptible cultivar. Among others, gene ontology (GO) annotations “stress-responsive” and “lipid transport” are overrepresented in the group of indica-specific transcripts. The lower T-DNA integration efficiency in the indica cultivar may also be attributable to the specific repression of genes related to DNA damage repair. This assumption is in good agreement with the importance of the host DNA repair machinery in T-DNA integration reported earlier (Li et al., 2005b; Citovsky et al., 2007).

Because strong and prolonged host defense responses generally correlate with reduced transformation success (Figure 2), external attenuation of these responses may be a means to improve transformation efficiencies. The experimental approaches that can be taken to manipulate host defenses are as manifold as the defense strategies themselves. The problem can be tackled from different sides: (1) by using modified agrobacterial strains that elicit a weaker defense, as e.g., shown in a study on potato (Vences-Guzman et al., 2013); (2) by modifying the composition of plant media and/or growth conditions to keep defense levels low, e.g., Zhang et al. (2013a); (3) by transient and targeted manipulation of the plants non-self-recognition machinery (see below); (4) by counteracting the effect of antimicrobial substances. This strategy proved successful in tea, where L-glutamine was found to overcome the bactericidity of polyphenols (Sandal et al., 2007).

In an innovative study Tsuda and colleagues demonstrated how detailed knowledge on plant-microbe interactions can be employed for successful transformation. AvrPto encodes an effector protein from the bacterial plant pathogen Pseudomonas syringae. The protein suppresses plant immunity by interfering with plant immune receptors. The AvrPto gene was placed under the control of a dexamethasone-inducible promoter. In transgenic Arabidopsis plants carrying the inducible construct, dexamethasone pre-treatment largely improved transformation in agro-infiltrated leaves (Tsuda et al., 2012).

An entirely different “pre-treatment strategy” proved successful in perennial ryegrass (Lolium perenne L.) (Zhang et al., 2013a). Stable transformants were obtained at an impressively high rate (84%), and 60% of the transgenic calli were regenerated into green plantlets. This was achieved by combining two strategies, while either treatment alone had little effect (10–20% transformation efficiency): (1) Myo-inositol, a component of many standard media, was removed from the callus culture medium. (2) A cold shock pre-treatment was applied prior to agrobacterial infection.

Myo-inositol levels in plants are primarily controlled by a specific oxygenase, which catalyses the first step in the conversion of this sugar into plant cell wall polysaccharides (Endres and Tenhaken, 2009). The basis of the effect observed by Zhang and colleagues is still largely elusive. It appears that myo-inositol acts in different ways and at multiple levels: omission of myo-inositol promoted Agrobacterium binding to the cell surface. It also repressed H₂O₂ production in infected tissue. One indirect consequence of ROS production, callus browning, could furthermore be suppressed when including the cold pre-treatment (Zhang et al., 2013a). Worthwhile questions are: Does growth of cold-pre-treated calli on myo-inositol-free medium alter cell wall composition to support agrobacterial attraction, invasion and/or survival in L. perenne cells? If so, what is the critical difference? Can such favorable cell wall characteristics be imitated to facilitate agrobacterial transformation of other recalcitrant species?

The molecular battle between agrobacteria and plants is impressive, instructive and challenging (Figure 2). Impressive, because the arms race takes so many forms. Instructive, because discoveries from Agrobacterium-plant interaction studies may drive progress in other fields of microbe-host association research. Challenging, because the external conditions that permit or prohibit transformation including transgene expression are diverse, and the balance needs to be determined empirically. The current state of research provides substantial breeding ground for plant scientists to search for this balance in their favorite species in a more targeted manner.