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20 October 2017 Agrobacterium-Mediated Plant Transformation: Biology and Applications
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Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.


Agrobacterium tumefaciens is a soil phytopathogen that naturally infects plant wound sites and causes crown gall disease via delivery of transferred (T)-DNA from bacterial cells into host plant cells through a bacterial type IV secretion system (T4SS). Through the advancement and innovation of molecular biology technology during the past few decades, various important bacterial and plant genes involved in tumorigenesis were identified. With the help of more comprehensive knowledge of how A. tumefaciens interacts with host cells, A. tumefaciens has become the most popular plant transformation tool to date. Any gene of interest can now easily be used to replace the oncogenes in the T-DNA region of various types of binary vectors to perform plant genetic transformation with A. tumefaciens. Arabidopsis, the most-studied model plant with powerful genetic and genomic resources, is readily transformable by A. tumefaciens for stable and transient transformation in several ecotypes tested, although variable transformation efficiencies in different accessions were observed.

The Agrobacterium-plant interaction is a complex process that can be divided into several functional steps, which have been reviewed extensively (Bhattacharya et al., 2010; Gelvin, 2010b, 2012; Lacroix and Citovsky, 2013; Pitzschke, 2013; Christie et al., 2014). In this article, we summarize the major achievements in the field in two main areas: first, the key genes and molecular events of Agrobacterium-plant interactions; and second, current status and methods of Agrobacterium-mediated stable and transient transformation methods. The aim of this article is to provide an overview for scientists with microbiology or plant background to have a better understanding for the other side, which could help them pursue their research interests in a more comprehensive way.


The initial research interest in the genus Agrobacterium resulted from the search for the causative agent of plant diseases including crown gall, cane gall, and hairy root. Agrobacterium tumefaciens harboring the tumor-inducing (Ti) plasmid induces galls on roots and crowns of numerous dicot angiosperm species and some gymnosperms; whereas Agrobacterium rhizogenes harboring the root-inducing (Ri) plasmid induces abnormal root production on host plants. The neoplastic tumor-like cell growth is induced by expression of the oncogenes residing in the transferred-DNA (T-DNA) transported from these bacteria into the plant nucleus and integrated into the plant genome.

The A. tumefaciens-mediated plant genetic transformation process requires the presence of two genetic components located on the bacterial Ti-plasmid. The first essential component is the T-DNA, defined by conserved 25-base pair imperfect repeats at the ends of the T-region called border sequences. The second is the virulence (vir) region, which is composed of at least seven major loci (virA, virB, virC, virD, virE, virF, and virG) encoding components of the bacterial protein machinery mediating T-DNA processing and transfer. The VirA and VirG proteins are two-component regulators that activate the expression of other vir genes on the Ti-plasmid. The VirB, VirC, VirD, VirE and perhaps VirF are involved in the processing, transfer, and integration of the T-DNA from A. tumefaciens into a plant cell. Figure 1 shows the major steps of the Agrobacterium-mediated plant transformation process. The current knowledge and recent findings regarding the key events of Agrobacterium-mediated plant transformation process are reviewed in the following sections. Table 1 summarizes the major transformation steps and known plant factors participating in these steps.

Figure 1.

Major steps of the Agrobacterium tumefaciens-mediated plant transformation process.

(1) Attachment of A. tumefaciens to the plant cells. (2) Sensing plant signals by A. tumefaciens and regulation of virulence genes in bacteria following transduction of the sensed signals. (3) Generation and transport of T-DNA and virulence proteins from the bacterial cells into plant cells. (4) Nuclear import of T-DNA and effector proteins in the plant cells. (5) T-DNA integration and expression in the plant genome.


Attachment of A. tumefaciens to plant cells

Bacterial attachment to host cells is a crucial early step in disease development for many plant and animal pathogens. The isolation of A. tumefaciens mutants unable to bind to plant cells led to the identification of several chromosomal virulence (chv) genes, chvA, chvB, and pscA, required for the attachment processes. chvA, chvB, and pscA are involved in the synthesis, processing, and export of cyclic ß-1,2-glucan and other sugars (Thomashow et al., 1987; Zorreguieta et al., 1988; Cangelosi et al., 1989; O'Connell and Handelsman, 1989). These mutants are either avirulent or highly attenuated in virulence under many inoculation conditions (Douglas et al., 1982; Douglas et al., 1985; Matthysse, 1987). During attachment, the A. tumefaciens cells synthesize cellulose fibrils that entrap large numbers of bacteria at the wounded sites (Matthysse et al., 1981 ; Matthysse, 1983). The cellulose deficient A. tumefaciens mutant is still able to attach to the carrot cell surface but cannot form aggregates (Matthysse, 1983). These mutants are virulent but are more susceptible to being washed off the plant cells (Matthysse et al., 1981 ; Matthysse, 1983). Thus, the major role of cellulose fibrils may be in aiding attachment of A. tumefaciens to the plant cell.

Table 1.

Plant factors involved in Agrobacterium-mediated plant transformation process




Aside from the cellulose and cyclic ß-1,2-glucan, A. tumefaciens produces an additional exopolysaccharide, unipolar polysaccharide (UPP), that participates in bacterial attachment (Tomlinson and Fuqua, 2009; Li et al., 2011; Xu et al., 2012; Heindl et al., 2014). The unipolar polysaccharide helps A. tumefaciens cells attach to the plant surfaces in a polar fashion because the UPP is mainly produced at the cell pole. The UPP is rarely observed in A. tumefaciens planktonic cells or in colonies on solid media, suggesting that the production of UPP is a signature of A. tumefaciens permanent attachment (Li et al., 2011; Xu et al., 2012; Xu et al., 2013). The UPP consists of at least two types of sugars, N-acetylglucosamine and N-acetylgalactosamine (Tomlinson and Fuqua, 2009; Xu et al., 2012). The A. tumefaciens UPP biosynthetic mutant uppE is defective in both bacterial attachment and biofilm formation, supporting the idea that the UPP plays an important role in bacterial attachment (Xu et al., 2012; Heindl et al., 2014). Interestingly, while A. tumefaciens attachment to plant tissues often leads to biofilm formation and is required for virulence, A. tumefaciens strains lacking UPP or impaired in biofilm formation remain efficient in T-DNA transfer. Thus, biofilm formation and UPP-mediated bacterial attachment may not play a role during Agrobacterium T-DNA translocation process, or their importance may not be discernible using the current infection assays under laboratory conditions.

Plant factors are also required for bacterial attachment to the plant cell surface. One such surface factor may belong to a vitronectin protein family. In animal cells, vitronectin, a component of the extracellular matrix, is utilized as a specific receptor by several pathogenic bacterial strains (Paulsson, and Wadstrom, 1990). Human vitronectin and anti-vitronectin antibodies both inhibited the binding of A. tumefaciens to cultured plant cells (Wagner and Matthysse, 1992). Further, A. tumefaciens chvB, pscA, and att mutants that are unable to bind to plant cells exhibit reduced binding to vitronectin (Wagner and Matthysse, 1992). Plant proteins immunologically related to the animal substrate adhesion molecule vitronectin, named PVN1 (plant vitronectin-like protein 1), have been identified in tobacco (Zhu et al., 1993; Zhu et al., 1994). PVNI is localized in the cell wall and may mediate cell adhesion (Zhu et al., 1994). However, whether PVN1 is a receptor for Agrobacterium binding remains to be investigated.

Nam et al. (1997) identified several Arabidopsis ecotypes that are deficient in binding A. tumefaciens and are resistant to A. tumefaciens root mediated transformation. Two of these ecotypes, BI-1 and Petergof, showed lower transient transformation efficiency than a more susceptible ecotype, suggesting that the transformation process was blocked at step(s) prior to integration. Further, microscopic analysis revealed that fewer bacteria bound to the root surface of B1-1 and Petergof than to a highly transformable ecotype such as Aa-0. In addition, Nam et al. (1999) identified three Arabidopsis T-DNA insertion mutants in the ecotype Ws with resistance to Agrobacterium transformation (rat mutants). The root explants of rat1, rat3, and rat4 mutants are deficient in binding A. tumefaciens. RAT1 encodes an arabinogalactan protein (AtAGP17, At2g23120) that is found in the extracellular matrix associated with the plant cell wall (Gaspar et al., 2004). Interestingly, although the Arabidopsis rat1 and rat3 mutants and the BI-1 and Petergof ecotypes are recalcitrant to A. tumefaciens root-mediated transformation, they are transformed as well as are the corresponding wild-type plants and highly transformable ecotypes using a flower vacuum infiltration method (Mysore et al., 2000a). These results suggested that plant proteins needed for transformation of somatic tissues may not be required for the transformation of the female gametophyte. In addition, a genetic screen to identify Arabidopsis hyper-susceptible to A. tumefaciens transformation (hat) mutants uncovered the heterozygous mutant mtf1-1, in which the mRNA transcript of the MTF1 (myb family transcription factor, At2g40970) gene exhibited more than a 12-fold reduction as compared to wild-type plants (Sardesai et al., 2013). The increased A. tumefaciens attachment to roots and improved stable and transient transformation efficiencies in mtf1-1 suggested that the MTF1 played a negative role in mediating the A. tumefaciens transformation process. Interestingly, the presence of A. tumefaciens-secreted cytokinin caused decreased MTF1 gene expression via the cytokinin response regulator ARR3-mediated signaling pathway. Because gene expression of AT14A (At3g28290), encoding a putative transmembrane receptor for a cell adhesion molecule, was increased in the mtf1 mutant and the at14a mutant showed decreased transformation rates and A. tumefaciens attachment to plant roots, AT14A might be an anchor point for A. tumefaciens binding (Sardesai et al., 2013).

Sensing and regulation of virulence genes of A. tumefaciens in response to plant signals

In nature, wounded plant tissues secrete a wide range of chemical compounds that can function as chemotactic agents to attract A. tumefaciens to the wounded sites for infection in plants. Wounded plants secrete sap with a characteristic acidic pH (5.0 to 5.8) and high content of different phenolic compounds, including lignin and flavonoid precursors. While these compounds can be used to ward off microbial attachment, these conditions stimulate A. tumefaciens virulence gene expression. The best studied and most effective vir gene inducers are monocyclic phenolics such as 3,5-dimethoxy acetophenone (acetosyringone, AS), as shown by induction of vir::lacZ fusions (Stachel et al., 1985). Several other environmental conditions are also important for optimal vir gene induction, including acidity, low phosphate, temperature, and sugars. However, phenolics are the only signal that is absolutely required (Brencic et al., 2003; Gao and Lynn, 2005; McCullen and Binns, 2006; Lin et al., 2008; Subramoni et al., 2014).

A. tumefaciens uses the VirA/VirG two-component regulatory system to regulate vir gene induction (Stachel et al., 1986; Jin et al., 1990a, 1990b, 1990c). To initiate the signaling pathway, plant phenolics interact either directly or indirectly with the transmembrane sensory protein VirA. Although two chromosomally encoded proteins, p10 and p21, but not VirA, are able to bind the phenolic signal in vitro (Lee et al., 1992), genetic evidence strongly suggests that VirA directly senses the phenolic compounds required for vir gene activation (Lee et al., 1995; Lee et al., 1996). ChvE, a chromosomally encoded glucose/galactose binding protein, interacts with VirA and enhances vir gene activation by binding to sugars (Shimoda et al., 1993; Doty et al., 1996). A. tumefaciens also uses the ChvE protein to recognize and bind various plant sugars that may help the bacterium to establish its host range (Hu et al., 2012). In addition, the affinity of the ChvE protein for sugar acids is increased at low pH, suggesting a link between the sugar and acidity response in A. tumefaciens (Hu et al., 2012). A. tumefaciens utilizes the ChvG/ChvI two-component system to activate the transcription of virG and induces expression of several other bacterial genes (Mantis and Winans, 1992; Chang and Winans, 1996), including genes involved in Chemotaxis and motility, several chromosome-encoded virulence genes, succinoglycan biosynthesis genes, and the gene cluster encoding the type VI secretion system (T6SS) (Yuan et al., 2007a; Wu et al., 2008; Wu et al., 2012). Recent studies further identified ExoR as a periplasmic negative regulator acting upstream of the ChvG sensor kinase to repress acid-inducible gene expression (Wu et al., 2012; Heckel et al., 2014) and uncovered a role for the T6SS in interbacterial competition activity during the plant colonization process (Ma et al., 2014). These data strongly suggested that these acid-inducible genes may play a role in A. tumefaciens survival and competitive fitness during Agrobacterium-plant interactions.

Because phenolic molecules are bacteriostatic at high concentration, A. tumefaciens also harbors cytochrome P450-like enzymes such as VirH2 to detoxify a phenolic vir gene inducer, ferulic acid (Kalogeraki et al., 1999; Brencic et al., 2003), suggesting that virH may be involved in the detoxification of harmful phenolics secreted by the wounded plants (Kanemoto et al., 1989). On the other hand, plant factors can also act as inhibitors of the A. tumefaciens sensory machinery. A major organic exudate of maize seedling roots, 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (HDMBOA), has been demonstrated to provide innate immunity and hinder successful A. tumefaciens infection of maize plants (Zhang et al., 2000); an o-imidoquinone decomposition intermediate, (3Z)-2,2-dihydroxy-N-(4-methoxy-6-oxocyclohexa-2,4-die-nylidene) acetamide, is a specific inhibitor of signal perception in A. tumefaciens (Maresh et al., 2006; Lin et al., 2008). These inhibitors may account for the recalcitrance of some plants to Agrobacterium-mediated transformation.

Several studies revealed that two plant hormones, auxin and cytokinin, not only contribute to tumor growth, but also affect agrobacterial physiology and gene expression (Liu and Nester, 2006; Hwang et al., 2010; Hwang et al., 2013b). It has been shown that IAA, the plant hormone auxin overproduced upon T-DNA integration, affects bacterial growth and inhibits vir gene induction by competing with phenolic inducers for interaction with the VirA protein. It has been proposed that relatively low auxin levels may promote transformation during the initial stages of A. tumefaciens infection, whereas the higher level of auxin produced in the crown gall tissues may prevent further transformation by inhibiting bacterial growth and virulence. In addition to auxin, A. tumefaciens-secreted cytokinin also affects bacterial virulence by regulating vir promoter activity and bacterial growth (Hwang et al., 2010, 2013b). Other studies also indicate that the plant defense molecule salicylic acid (SA) influences the A. tumefaciens infection process by inhibiting the expression of vir genes, bacterial growth, bacterial attachment to plant cells, and virulence (Anand et al., 2008; Yuan et al., 2007a, 2007b). The plant defense roles of SA against bacterial infections are further supported by genetic studies showing that mutant plants with SA overproduction are resistant to A. tumefaciens infection, whereas mutant plants with lower SA accumulation have a higher percentage of tumor formation (Anand et al., 2008; Yuan et al. 2007b; Lee et al., 2009). Another plant hormone, ethylene, can repress vir gene expressions in A. tumefaciens but shows no significant inhibitory effects on bacterial growth and population size (Nonaka et al., 2008). In an Arabidopsis ethylene-insensitive mutant, the A. tumefaciens-mediated transformation efficiency increased (Nonaka et al., 2008). In summary, intricate temporal and spatial regulation and a delicate balance of various plant hormones likely play important roles in modulating A. tumefaciens infection efficiency (Gohlke and Deeken, 2014; Subramoni et al., 2014).

Transport of T-DNA and virulence proteins via type IV secretion system (T4SS)

Virulence gene expression leads to the production of a single-stranded T-DNA, termed the T-strand, which is then transported into the host cell. VirD1 and VirD2 proteins function together as a site- and strand-specific endonuclease which binds to the supercoiled Ti plasmid at the T-DNA borders, relaxes it and nicks the bottom T-DNA strand between the third and fourth bases of the T-DNA borders (Stachel et al., 1986; Yanofsky and Nester, 1986; Albright et al., 1987; Jayaswal et al., 1987; Stachel et al., 1987; Wang et al., 1987; Filichkin and Gelvin, 1993). This process results in the generation of single-strand T-DNA molecules (T-strand) covalently attached to VirD2 at the 5′ end of the T-strand at the right border nick (Herrera-Estrella et al., 1988; Ward and Barnes, 1988; Young and Nester, 1988; Howard et al., 1989). Recently, three VirD2-binding proteins (VBP) from A. tumefaciens were identified by pull-down assays using AS-treated A. tumefaciens crude extract (Guo et al., 2007a). These proteins are functionally redundant, but removal of all three vbp genes in A. tumefaciens affects bacterial virulence on Kalanchoe plants (Guo et al., 2007a). The VBP1 protein not only interacts with the VirD2-bound T-strand, but also interacts with three components of the Type IV secretion system (T4SS), VirD4, VirB4, and VirB11, via two independently binding domains (Guo et al., 2007b). The VBP may form a dimer in the A. tumefaciens cytoplasm and recruit the VirD2-bound T-strand to the T4SS apparatus through interactions with the VirD4 proteins (Guo et al., 2007b; Padavannil et al., 2014).

T-DNA export requires the vir-encoded T4SS for delivery across the bacterial envelope and the plasma membrane of the host plant cell (Zechner et al., 2012; Bhatty et al., 2013; Chandran, 2013; Christie et al., 2014). This A. tumefaciens T4SS consists of 12 proteins (VirB1-B11 and VirD4), which form two functional components: a filamentous T-pilus and a membrane associated transporter complex that is responsible for translocating substrates across both bacterial cell membranes (Alvarez-Martinez and Christie, 2009; Fronzes et al., 2009a; Waksman and Franzes, 2010; Wallden et al., 2010). This transport complex can be further subdivided into four functional subassemblies: the VirD4 coupling protein as substrate receptor, an inner-membrane translocase (VirB3-B4-B6-B8-B11), an outer-membrane core complex (VirB7-B9-B10), and an extracellular T-pilus (VirB2-B5) (Christie et al., 2014). While it is clear that each of the VirB2-11 and VirD4 proteins is essential for T-DNA and effector transport, it remains unknown whether the extracellular T-pilus is part of the translocation channel.

The VirD4 protein is an integral inner membrane protein with ATPase activity functioning as a coupling protein responsible for presentation of DNA and protein substrates to the VirB transporter. Studies using a transfer DNA immunoprecipitation (TrIP) assay revealed that the transferring T-DNA substrates make close contacts with the VirD4 protein, further supporting the function of VirD4 as the substrate receptor (Cascales and Christie, 2004a). Genetic studies of the virD4 mutants also revealed that DNA substrate binding of VirD4 protein and delivery to VirB11 both activate the ATP hydrolysis activities of VirD4 and VirB11. These signals may cause a conformational change of VirB10 which plays an important role in regulating DNA substrate transfer through the T4SS apparatus opening and assembly (Atmakuri et al., 2004; Cascales and Christie, 2004b; Cascales et al., 2013).

VirB4 is an ATPase and contains a Walker A nucleotide triphosphate binding motif essential for virulence (Christie et al., 1989; Fullner et al., 1994). The VirB4 ATPase also directly participates in T-pilus biogenesis. VirB4 can interact with the VirB2 Tpilin and induce changes in the VirB2 membrane topological state via an ATP-dependent mechanism, suggesting that the VirB4 ATPase may function as a dislocase to mediate membrane extraction of pilin monomers during pili biogenesis (Kerr and Christie, 2010; Wallden et al., 2010). In addition, VirB11 may function with VirB4 to coordinate a structural change in VirB2 pilin protein and mediate VirB2 binding to other T4SS components, which in turn affects pilus polymerization (Kerr and Christie, 2010). Accumulating genetic studies and recent structural studies support a model in which VirB4 dimers or homomultimers contribute both structural information for the assembly of a trans-envelope channel competent for DNA transfer and an ATP-dependent activity for configuring this channel as a dedicated export machine (Fronzes et al., 2009a; Durand et al.,2010, 2011; Kerr and Christie, 2010; Li et al., 2012; Pena et al., 2012; Wallden et al., 2012). In addition, the TrIP assay results showed that translocating DNA substrates first interact with the VirD4 receptor, followed by VirB11 ATPase, suggesting VirB11 may function together with VirB4 and deliver the T-DNA substrate to the transfer channel of the T4SS (Cascales and Christie, 2004a; Bhatty et al., 2013; Chandran, 2013; Christie et al., 2014).

Recent electron microscopy imaging has revealed the structure of purified VirB3-10 complex encoded by R388 T4SS, in which VirB3, VirB4, and likely VirB5, VirB6, and VirB8 are part of the inner membrane complex (Low et al., 2014). This result is consistent with a predicted inner-membrane translocase complex consisting the VirB3, VirB4, VirB6, VirBS, and VirB11 based on subcellular localization, protein-protein interaction, and biochemical studies (Christie et al., 2014). This study also revealed the outer membrane complex consisting of VirB7-B9-B10, which is in agreement with previous cryoelectron microscopy and crystallography of purified VirB7, VirB9, and VirB10 proteins encoded from pKM101 (Chandran et al., 2009; Fronzes et al., 2009b). This outer-membrane core complex contains 14 copies each of VirB7, VirB9, and VirB10; and is inserted in both the inner and outer membranes (Fronzes et al., 2009a, 2009b). The structure of the core complex is cylindrical and contains two layers, the inner (I) and outer (O) layers (Fronzes et al., 2009b; Wallden et al., 2012; Bhatty et al., 2013). Upon sensing ATP energy utilization by the VirD4 and VirB11 ATPases, the VirB10 protein conformation changes and may help regulate the O layer hole opening to allow substrate transfer through the T4SS channel (Cascales and Christie, 2004b). While the majority of T4SS components can be assembled into subcomplexes when expressed in a heterologous E. coli system, an alpha-crystallin-type small heat shock protein, HspL was shown to be important for stability of several VirB proteins and efficient T-DNA transfer and tumorigenesis (Tsai et al., 2009). HspL expression is induced by AS in response to VirB protein expression and functions as a VirB8 chaperone (Tsai et al., 2009, 2010). Moreover, overexpression of HspL in A. tumefaciens enhanced transient transformation efficiencies of both susceptible and recalcitrant Arabidopsis ecotypes. A. tumefaciens virulence during heat shock was also considerably improved by over-expression of the HspL, revealing the importance of the VirBS chaperone HspL during A. tumefaciens infections (Hwang et al., 2015).

The extracellular T-pilus consists of major subunit VirB2 and minor subunit VirB5, which is located at the pilus tip (Lai and Kado, 1998; Schmidt-Eisenlohr et al., 1999; Aly and Baron, 2007). Genetic and environmental studies show that each virB gene is required for T-pilus biogenesis; however, only the virB2 gene is required for VirB2 pro-pilin processing and peptide cyclization (Eisenbrandt et al., 1999; Lai et al., 2000, 2002). Non-polar virB mutants abolish T-pilin transport and T-pilus biogenesis, and support a model in which the VirB-specific transporter is not only used for translocating the T-complex, but also for exporting cyclic T-pilin subunits to the bacterial cell surface, perhaps as a scaffold to facilitate efficient assembly of the T-pilus (Lai and Kado, 2000; Christie et al., 2014). However, several amino acid substitutions in the T4SS components VirB6, VirB9, VirB10, and VirB11 have been demonstrated to block the polymerization of VirB2 pilin to form the extracellular T-pilus but do not significantly affect substrate transfer. These “uncoupling” mutations still require intracellular VirB2 for substrate transfer (Zhou and Christie, 1997; Sagulenko et al., 2001 ; Jakubowski et al., 2003, 2005, 2009; Banta et al., 2011; Garza and Christie, 2013). Wu et al. (Wu et al., 2014a) further generated several uncoupling virB2 mutants with single amino acid substitutions in the VirB2 protein that retain wild-type levels of tumor formation on tomato stems but show reductions in transient transformation efficiencies of Arabidopsis seedlings. Taken together, these results suggest the auxiliary role of the filamentous T-pilus in optimizing the A. tumefaciens transformation efficiency of certain plant species or infection conditions.

Biochemical studies show that VirB5 co-fractionates as a minor component in pilus preparations and contributes to T-pilus assembly (Schmidt-Eisenlohr et al., 1999). Additionally, immunogold staining demonstrated that VirB5 is located at the tips of the cell-bound T-pili and the ends of detached T-pili, suggesting the VirB5 might be involved in host-bacteria contact (Aly and Baron, 2007). This notion is supported by two lines of evidence. One is that the VirB5 homolog CagL is localized on the T4SS pilus tip of Helicobacter pylori and functions in binding host human cells, likely by interacting with the integrin receptor (Kwok et al., 2007; Backert et al., 2008). Evidence that the CagL protein, the VirB5 homologue in the H. pylori, functions as a specialized adhesin binding to the human integrin ß1 and may activate the downstream host cell response, suggested a putative host cell receptor for the bacterial T4SS protein (Kwok et al., 2007; Backert et al., 2008). However, whether A. tumefaciens VirB5 also serves as an adhesin remains to be determined. Secondly, addition of exogenous VirB5 or expression of secreted VirB5 in the tobacco transgenic plant was able to enhance T-DNA expression (Lacroix and Citovsky, 2011). Interestingly, the VirB5 protein interacts with the cytokinin biosynthetic enzyme Tzs (trans-zeatin synthesizing) protein in yeast and in vitro (Aly et al., 2008). The tzs gene in the nopaline strains of A. tumefaciens shares significant homology with the ipt gene, is located outside the T-DNA region and is not incorporated into the host plant genome during infection (Akiyoshi et al., 1985, 1987; Beaty et al., 1986; Powell et al., 1988). The loss of Tzs protein expression and trans-zeatin secretion by the tzs mutants correlates with reduced stable and transient transformation efficiencies on Arabidopsis roots, suggesting the Tzs, by synthesizing trans-zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens (Hwang et al., 2010; Hwang et al., 2013b). Because Tzs was detected in the A. tumefaciens membrane fraction (Lai et al., 2006; Aly et al., 2008) and a functional T4SS is required for Tzs localization on the cell surface (Aly et al., 2008), Tzs may function extracellularly, likely at the interface of the Agrobacterium-plant interaction. Since AT14A has partial sequence similarity to the animal integrin receptor, has been shown to mediate the cell wallplasma membrane-cytoskeleton continuum in A. thaliana cells (Lu et al., 2012), and is critical for A. tumefaciens attachment to plant roots (Sardesai et al., 2013), it is plausible to hypothesize that T-pilus tip protein VirB5 may interact with AT14A for A. tumefaciens binding to the plant cell surface.

VirB1 may have two functions in the VirB transporter. The amino-terminal region of VirB1 contains a signal peptide and a motif homologous to that of lytic transglycosylases and lysozymes (Mushegian et al., 1996; Baron et al., 1997), suggesting that VirB1 may function to prepare sites in the bacterial envelope for transporter assembly by localized lysis of the peptidoglycan layer. The virB1 non-polar mutant is highly attenuated in virulence and cannot assemble T-pili (Berger and Christie, 1993; Lai and Kado, 2000), suggesting that pilus assembly across an intact peptidoglycan layer is inefficient or unstable. VirB1 is processed to a 73-residue, carboxy-terminal fragment termed VirB1*. The secreted VirB1* may play a direct role in T-pilus assembly, either by stabilizing VirB5 or by mediating VirB2-VirB5 interaction (Llosa et al., 2000; Župan et al., 2007). VirB5 may directly interact with VirB2 or confer its effect by means of other proteins, e.g., VirB7. Energy for T-pilus assembly may be provided by VirB 11, and it may be transduced to the pilus assembly complex in the outer membrane via VirB6 and VirB7 (Krall et al., 2002; Christie et al., 2005). This current view of the T4SS structure is in accordance with the T-DNA transfer route in the T4SS that was proposed based on the results of TrIP assays (Cascales and Christie, 2004a). The T-DNA substrate contacts six of the 12 components of the T4SS machine, including the VirD4 receptor, followed by the VirB 11 ATPase, VirB6/irB8, and VirB2 pilin/VirB9 proteins (Cascales and Christie, 2004a). However, whether the VirB2 protein in contact with T-DNA is part of T4SS translocation channel or filamentous pilus has not been determined.

A. tumefaciens cells attach to plant cells at the bacterial poles and several VirB proteins, as well as VirD4, were observed at cell poles, suggesting that the VirB/D4 transfer apparatus localizes at the poles (Matthysse, 1987; Kumar and Das, 2001 ; Kumar et al., 2002; Jakubowski et al., 2004; Judd et al., 2004, 2005). However, recent studies using very high resolution deconvolution microscopy suggested that the A. tumefaciens VirB/D4 transfer apparatus forms helically arranged foci around the bacteria cells to help make intimate lateral contact with plant cells, which might maximize substrate transfer through the T4SS (Aguilar et al., 2010, 2011 ; Cameron et al., 2012). Several possible functions could be assigned to the T-pilus. First, the T-pilus could serve as a conduit for several components needed for virulence, including the T-pilin subunit, VirE2 proteins and single-stranded T-DNA that is piloted by the covalently linked VirD2 protein (Cascales and Christie, 2004a; Cascales et al., 2013). Second, the T-pilus could serve as a bridge to bring the bacterium and the host cell into close proximity while T-DNA is transferred into the host cell through T4SS translocation channel. Kelly and Kado (2002) described a presumably coiled thread-like interconnection with an average width of approximately 30 nm between A. tumefaciens and Streptomyces lividans cells during A. tumefaciens transformation. This interconnecting structure is dependent on virB genes and appears only under the same conditions as those required for T-pilus formation. One possible model derived from this observation is that the T-pilus may retract and subsequently draw the bacterial cell into sufficiently close contact with the target host cells for T-DNA transfer to occur.

So far, limited knowledge exists about what kind of plant proteins might be involved in the initial contact of A. tumefaciens T4SS components with host cell surfaces. Two kinds of plant proteins that interact with VirB2 protein were identified. The first class is composed of three related proteins with unknown function, RTNLB1 (reticulon-like protein B-1, At4g23630), RTNLB2 (At4g11220), and RTNLB4 (At5g41600) proteins; the second class is an AtRAB8B GTPase (At3g53610). The level of RTNLB1 protein is transiently increased immediately after A. tumefaciens infection. Overexpression of RTNLB1 in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacteriummediated transformation while transgenic RTNLB and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by A. tumefaciens than are wild-type plants. These data suggested that the three RTNLB proteins and AtRAB8 are involved in the initial interaction of A. tumefaciens with plant cells (Hwang and Gelvin, 2004).

Cytoplasmic trafficking and nuclear import of T-DNA and effector proteins in plant cells

The VirD2 protein attaches to the 5′ end of the T-strand and serves as a pilot protein to guide the T-DNA from the bacteria into the plant cell. In addition to T-strands, several virulence effector proteins can be exported from the bacterial cells independently of each other and of the T-DNA. In addition to VirD2, VirE2, VirD5, VirE3, VirF, MobA, and Atu6154 in A. tumefaciens, and the GALLS protein in A. rhizogenes, have been shown to be transferred from bacteria into plant cells via the A. tumefaciens transfer apparatus, using the Cre recombinase reporter assay for translocation (CRAfT) (Vergunst et al., 2000, 2003, 2005). C-terminal clusters of positively-charged amino acids (specifically an Arg-x-Arg motif) of T4SS effectors function as the secretion signal for the A. tumefaciens transfer apparatus. In addition to genetic evidence, split-GFP studies directly visualized VirE2 protein translocation in plant and yeast cells (Li et al., 2014b; Sakails et al., 2014). However, active movement of linear and filament VirE2 and transgene expression only occur in tobacco cells but not in yeast cells, suggesting that active VirE2 trafficking is important for efficient T-DNA expression. Because translocated VirE2 forms the filamentous structure in the absence of T-DNA, the associations and assistance of host plant cell proteins may be required for VirE2 movement (Li et al., 2014b; Sakalis et al., 2014). Indeed, using split-GFP technology to visualize VirE2 movement in agroinfiltrated N. benthamiana leaf epidermal cells, recent studies showed that VirE2 is transported into plant cells via clathrin-mediated endocytosis (Li and Pan, 2017) and trafficked via an ER/actin network that is powered by myosin XI-K (Yang et al., 2017).

Both VirD2 and VirE2 proteins contain plant-active nuclear localization signal (NLS) sequences that are believed to be involved in T-DNA import into the nucleus. By the yeast interaction trap (two-hybrid) approach, several plant proteins that interact with either VirD2 or VirE2 proteins were identified (Balias and Citovsky, 1997; Deng et al., 1998; Bako et al., 2003; Tao et al., 2004; Bhattacharjee et al., 2008). Two VirD2-interacting proteins, ROC1 (peptidyl-prolyl cis-trans isomerase CYP1, At4g38740) and the CyP A proteins of Arabidopsis, belong to a large cyclophilin family of peptidyl-prolyl cis-trans isomerases, which are highly conserved in plants, animals, and prokaryotes (Deng et al., 1998). The VirD2 domain interacting with the cyclophilins is in the central region of VirD2 protein, which is distinct from the nuclear localization signal domains (Deng et al., 1998). Although the biological role of cyclophilins in A. tumefaciens infection is unclear, they were proposed to maintain the proper conformation of VirD2 within the host cell cytoplasm and/or nucleus during T-DNA nuclear import and/or integration (Deng et al., 1998; Bako et al., 2003). Another cellular factor encoded by a tomato protein phosphatase 2C (PP2C) interacted with the VirD2 NLS region (Tao et al., 2004). An Arabidopsis abi1 (At4g26080) mutant showed higher sensitivity to A. tumefaciens transformation than did the wild-type plant, whereas over-expression of PP2C in tobacco protoplasts inhibited nuclear import of a ß-glucuronidase-VirD2 NLS fusion protein (Tao et al., 2004). These data suggested that phosphorylation of the VirD2 protein near the NLS region may influence the import of the T-complex.

The VirD2 protein was also found to interact with an Arabidopsis protein, AtKAPa/IMPA-1 (At3g06720), which belongs to a large karyopherin family known to mediate nuclear import of NLS-containing proteins (Ballas and Citovsky, 1997). AtKAPa-VirD2 interactions, which occur both in the yeast two-hybrid system and in vitro, require the presence of the VirD2 carboxyl-terminal NLS (Ballas and Citovsky, 1997). There are nine importin a members in Arabidopsis. The I MPA-2 (At4g16143), I MPA-3 (At4g02150), and I MPA-4 (At1 g09270) show interactions with VirD2 and VirE2 with different strengths in yeast, in vitro, and in plants (Bhattacharjee et al., 2008; Lee et al., 2008). In addition, IMPA-1 interacts with the VirE2 protein in the plant cytoplasm and I MPA-4 interacts with VirE2 predominantly in the plant nucleus (Lee et al., 2008). Stable and transient transformation assay results showed that an IMPA1, IMPA2, or IMPA3 T-DNA insertion mutant remains susceptible to Agrobacterium-mediated transformation, whereas the IMPA4 T-DNA insertion mutant is resistant to Agrobacterium-mediated root transformation (Bhattacharjee et al., 2008). The resistant phenotype of the impa4 mutant could be complemented by the IMPA4 gene but could not be complemented by other importin a genes under the control of their native promoters, which might be due to the different promoter expression profiles of importin a genes. Additionally, an Arabidopsis T-DNA insertion mutant of the importin ß-3 exhibits lower Agrobacterium-mediated transformation efficiencies, suggesting that importin ß-like transportins might be also involved in Agrobacterium-mediated transformation (Zhu et al., 2003b). Collectively, these data indicate that after the TDNA and Vir proteins enter the plant cells, the host cell nuclear import machinery may be hijacked to mediate the nuclear targeting of T-DNA (Gelvin, 2010b, 2012; Lacroix and Citovsky, 2013).

Yeast two-hybrid assays identified two VirE2-interacting plant proteins, VIP1 (At1g43700) and VIP2 (At5g59710) (Tzfira et al., 2001, 2002; Anand et al., 2007). The VIP1 protein shows homology to a class of basic leucine zipper (bZIP) proteins, and is known to localize to the cell nucleus (Tzfira et al., 2001). VIP1 anti-sense transgenic tobacco plants exhibit significantly reduced nuclear import of GUS-VirE2 but not of GUS-VirD2, suggesting a role for VIP1 in VirE2 nuclear import (Tzfira et al., 2001). The early stage of T-DNA expression is blocked in VIP1 anti-sense tobacco transgenic plants, whereas the over-expression of VIP1 in transgenic tobacco plants enhances the early steps of the A. tumefaciens infection process (Tzfira et al., 2001, 2002). Further, the evidence of VIP1 interacting with VirE2-ssDNA complexes in vitro (Tzfira et al., 2001) with VirE2 and importin α to form a ternary complex (Citovsky et al., 2004) suggested that VIP1 might function as an adaptor between VirE2 and the conventional nuclear import machinery of plant cells. Nuclear targeting of the VIP1-VirE2 complex could be triggered by the phosphorylation of VIP1 on serine-79 by the mitogen-activated protein kinase 3 (MPK3, At3g45640) (Djamei et al., 2007). Substitution of the serine-79 to alanine, mimicking the non-phosphorylated form of VIP1, resulted in the cytoplasmic localization of VIP1 (Djamei et al., 2007). These data led to the proposed ‘Trojan horse’ model, which suggested that A. tumefaciens infection activates MPK3-mediated phosphorylation of VIP1 in plant cells, and thereby promotes VIP1-VirE2-ssDNA complex trafficking to the plant nucleus (Djamei et al., 2007). However, a recent study by Shi et al. (2014) indicated that transformation efficiencies of the Arabidopsis vip1 mutant and the VIP1 overexpression transgenic plants were not altered when infected by numerous tested A. tumefaciens strains. Furthermore, overexpression of several forms of VIP1 did not alter the subcellular localization of VirE2 (Shi et al., 2014). The conflicting results from these VIP1 studies might be due to different plant systems, experiment setups, and/or analysis methods (Tzfira et al., 2001; Li et al., 2005a; Shi et al., 2014). Thus, a modified model of VirE2-VIP1 interactions in A. tumefaciens infection process has been proposed, in which the nuclear import of T-DNA is largely affected by the VirD2, and VirE2 may play an accessory structural role to facilitate T-DNA targeting to the plant nucleus. VirE2 proteins may interact with VIP1 in the cytoplasm regions and prevent some endogenous VIP1 from activating plant defense genes, thus promoting A. tumefaciens infection (Pitzschke, 2013; Shi et al., 2014).

As described above, A. tumefaciens may utilize the host cell nuclear import system to help T-DNA and effector proteins to enter plant nucleus. The plant cytoskeleton may also play a role in T-complex cytoplasmic trafficking. Salman et al. (2005) utilized single-particle-tracking methods to follow the movement of fluorescently labeled VirE2-ssDNA complexes in a cell-free system. Chemical disruptions of microtubule networks and dynein motors blocked VirE2 complex movement, suggesting the possible involvement of microtubules in A. tumefaciens T-complex nuclear targeting (Salman et al., 2005; Tzfira, 2006). However, cytoskeleton inhibitor treatment together with real time VirE2 trafficking imaging experiments in N. benthamiana demonstrated that cytoplasmic movement of VirE2 does not require microtubules but does rely on actin powered by myosin (Yang et al., 2017). The involvement of actin in VirE2 trafficking is also supported by the findings that Arabidopsis mutant plants impaired in actin genes (ACT2, At3g18780 and ACT7, At5g09810) expressed in the roots were resistant to stable and transient transformation, suggesting the involvement of the actin cytoskeleton in Agrobacterium-mediated transformation (Zhu et al., 2003b). In addition, both VirD2 and VirE2 proteins interact with filamentous actin in an in vitro cosedimentation assay. Inhibitors of the actin cytoskeleton lowered the A. tumefaciens transformation efficiency of tobacco BY-2 cells (Gelvin, 2012).

Integration into the plant genome and expression of the T-DNA

T-DNA integration into the plant cell genome is the final step of the Agrobacterium-mediated transformation process. Unlike other mobile DNA elements such as transposons and retroviruses, the T-DNA does not encode enzymatic activities required for integration. Thus, T-DNA insertion into the plant DNA must be mediated by proteins transported from A. tumefaciens itself, namely VirD2 and VirE2, and/or host cell factors.

VirD2 might play a dual role in the T-DNA integration process, ensuring both its fidelity and efficiency. The integration of the 5′ end of the T-strand into plant DNA is generally precise. This may result from the linkage of VirD2 to the 5′ end of the T-strand, which provides protection from exonucleases inside the plant cells (Durrenberger et al., 1989; Tinland et al., 1995; Rossi et al., 1996). A region in the C-terminal portion of VirD2 called the ω domain is implicated in the integration process (Shurvinton et al., 1992; Tinland et al., 1995; Mysore et al., 1998). However, it is not clear how exactly the ω domain of VirD2 is involved in T-DNA integration because the pattern of integration of the T-DNA is not affected by deletion of the ω sequence (Shurvinton et al., 1992; Tinland et al., 1995; Bravo-Angel et al., 1998; Mysore et al., 1998). Involvement of VirD2 in protecting the integrity of the right border was also demonstrated in a fungal transformation system (Michielse et al., 2004); this study further showed that VirE2 similarly protects the left end of the T-DNA, while VirC2 helps ensure correct processing. Importantly, VirC2 is also required for single-copy integration in this system (Michielse et al. 2004).

A report by Bake et al. (2003) demonstrated that VirD2 interacts with, and is phosphorylated both in vitro and in alfalfa cells by, the nuclear CAK2Ms kinase, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. VirD2 is also found in tight association with the TATA box-binding protein (TBP) in vitro and in Agrobacterium-transformed Arabidopsis cells (Bako et al., 2003). The TBP protein is implicated in the control of transcription-coupled repair (TCR), which ensures preferential and effective removal of DNA lesions from transcribed genes (Bake et al., 2003). CAK2Ms isolated by immunoprecipitation phosphorylate cyclin-dependent kinase (CDK2) and the C-terminal domain (CTD)of the RNA polymerase II largest subunit, which may serve as a TBP-binding platform (Bako et al., 2003). The functional conservation of TBP and CDK-activating protein kinase (CAK2) orthologs in eukaryotes indicates that these nuclear VirD2-binding factors may provide a link between T-DNA integration and transcription-coupled repair.

Ziemienowicz et al (2000) demonstrated that VirD2 protein does not possess general ligase activity using an in vitro ligation assay. Enzyme(s) present in plant extracts, likely DNA ligase, and E. coli T4 DNA ligase were able to ligate the 5′ end of T-DNA from an artificial T-DNA-VirD2 complex to a partly double-stranded oligonucleotide (Ziemienowicz et al., 2000). An Arabidopsis orthologue of the yeast and mammalian DNA ligase IV gene termed AtLIG4 (At5g57160) is required for the repair of DNA damage because seedlings of the Atlig4 mutant are hyper-sensitive to the DNA-damaging agents methyl methanesulfonate and X-rays (Friesner and Britt, 2003; van Attikum et al., 2003). However, using tumorigenesis and germline transformation assays, the Arabidopsis Atlig4 mutant is not impaired in T-DNA integration. Thus, DNA ligase IV may not be required for T-DNA integration in certain plant tissues. Similarly, the ku80 Arabidopsis mutant plant is hyper-sensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out non-homologous end joining recombination (Gallego et al., 2003). ku80 (At1g48050) mutant plants show no significant defect in the efficiency of floral dip transformation of plants with A. tumefaciens (Friesner and Britt, 2003; Gallego et al., 2003). However, in other studies, contradictory data indicated that Arabidopsis ku80 or ku70 (At1g16970) mutant plants and rice plant lines down-regulated in ku70, ku80, or Iig4 showed different transformation responses (van Attikum et al., 2001 ; Friesner and Britt, 2003; Gallego et al., 2003; Li et al., 2005b; Jia et al., 2012; Nishizawa-Yokoi et al., 2012; Vaghchhipawala et al., 2012; Mestiri et al., 2014; Park et al., 2015). These discrepancies might be the result of different techniques and different plant tissues used to examine transformation efficiency, or they may reveal more complex and redundant pathways for T-DNA integration mechanisms during A. tumefaciens infections (Tzfira et al., 2004a; Citovsky et al., 2007; Gelvin, 2010a, 2010b; Magori and Citovsky, 2012; Lacroix and Citovsky, 2013).

The contradictory conclusions for the involvement of NHEJ for T-DNA integration implied that an alternative DNA repair pathway might be responsible for T-DNA integration. A recent break-through showed that T-DNA integration in A. thaliana requires the polymerase theta (Pol 6, At4g32700) (van Kregten et al., 2016). This discovery was inspired by the similar signature of double-strand breaks (DSBs) of T-DNA integration with other systems. A kind of genome scar ‘filler DNA’ is usually found in the site of T-DNA integration as well as the Pol θ-mediated error-prone DSB repair in mammalian cells (Mateos-Gomez et al., 2015). Pol 6 mutant plants tebichi/polq showed resistant to T-DNA integration but were susceptible to Agrobacterium-mediated transient transformation demonstrated via both floral dip and root transformation. Based on the knowledge of molecular action of Pol θ, it has been proposed that T-DNA could target the random DSBs in the genome and the microhomology between the ends of T-DNA and the DSBs is required for the integration of T-DNA. Enhanced understanding of the mechanism of T-DNA integration may allow researchers to improve transgene targeting by introducing artificial breaks in the plant genome, which ideally will lead to specific integration of DNA fragments in a designated location (van Kregten et al., 2016).

Identification of additional cellular factors involved in the TDNA interaction comes from genetic approaches. Nam et al. (1999) identified several Arabidopsis rat mutants deficient in TDNA integration. One mutant, rat5, which harbors a mutation in the H2A histone gene (HTA1, At5g54640), is deficient in stable but not transient T-DNA expression, suggesting the involvement of HTA1 in T-DNA integration (Mysore et al., 2000b). The rat5 mutant plant is highly recalcitrant to stable transformation by root inoculation. However, the mutant plant is efficiently transformed by flower vacuum infiltration (Mysore et al., 2000a). The highest levels of HTA1 gene expression are detected in the elongation zone of the root, a region that is most susceptible to transformation (Yi et al., 2002). The HTA1 promoter activity is induced by wounding or by A. tumefaciens infections (Yi et al., 2006). The HTA1 cDNA not only complements the rat5 mutant, but was also shown to increase transient and stable transformation rates of wild-type plants, indicating that histone H2A-1 may play an role in transformation (Mysore et al., 2000b; Yi et al., 2006; Tenea et al., 2009). Tenea et al. (2009) demonstrated that overexpression of Arabidopsis cDNAs encoding seven tested histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11, At5g65350) genes increased both Agrobacterium-mediated transformation and transgene expression; whereas none of the seven tested histone H2B (HTB) cDNAs, nor other tested histone H3 (HTR) cDNAs, increased transformation. Overexpression of HTA, HFO, or HTR11 cDNA increased expression of incoming single-stranded or double-stranded forms of a gusA gene in plant cells. These results suggested that several histone proteins may enhance transgene expressions within the plant cells and therefore may increase A. tumefaciens infection efficiency (Tenea et al., 2009; Gelvin, 2010b).

The VirE2-interacting protein, VIP1, may also participate the T-DNA integration step, possibly by creating a link to the host cell chromatin component histone protein. VIP1 shows interaction with plant histone H2A-1 in vivo, and with purified Xenopus core histones H2A, H2B, H3 and H4 in vitro (Li et al., 2005a; Loyter et al., 2005). In addition, the VIP1 protein associates with purified plant nucleosomes in vitro (Lacroix et al., 2008). VIP1 is also able to provide links between plant nucleosomes with VirE2 and with a synthetic minimal T-complex in vitro by forming quaternary nucleosome-VIP1 -VirE2-ssDNA complexes (Lacroix et al., 2008). Another VirE2-interacting protein, VIP2, is a basic domain/leucine zipper motif-containing protein. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and the Arabidopsis vip2 mutant are both defective in T-DNA integration but not in transient T-DNA expression. The transcripto me analyses of Arabidopsis vip2 mutant revealed that expression of several histone genes was decreased, suggesting VIP2 may function as a transcriptional regulator to affect histone gene expression, thereby participating in the T-DNA integration step indirectly (Anand et al., 2007). When the T-DNA is inserted into the host chromosomal DNA wrapped in chromatin, the chromatin structure may need to be remodeled or removed to accommodate external T-DNA insertions. Several genetic studies indicated that T-DNA integration may be influenced by several chromatin proteins, including histone deacetylase (HDA1IHDT19, At4g38130; HDA2/HDT1, At3g44750; HD2B/ HDT2, At5g22650), and two subunits of the chromatin assembly factor 1 (FAS1, At1g65470; FAS2, At5g64630) (Zhu et al., 2003b; Endo et al., 2006; Crane and Gelvin, 2007; Gelvin and Kim, 2007).

After the T-complex enters the plant nucleus, the associated bacterial virulence proteins and host proteins may need to be removed from the T-DNA to allow efficient T-DNA integration. The disassembly of the T-complex is likely mediated by targeted proteolysis, which is accomplished by the VirF protein and the plant ubiquitin proteasome complex (Tzfira et al., 2004b; Zaltsman et al., 2010a, 2010b). virF mutant bacterial strains show weak virulence on some plant species, including Nicotiana glauca and tomato, but can efficiently transform other plant species, suggesting the VirF protein may function as a host-range factor (Melchers et al., 1990; Regensburg-Tuïnk and Hooykaas, 1993). The virF mutant can be complemented by expressing VirF in transgenic plants, showing that the VirF protein functions in plant cells (Regensburg-Tuïnk and Hooykaas, 1993). VirF is an F-box protein and interacts with three host Skp-1 related proteins, ASK1 (At1g75950), ASK2 (At5g42190), and ASK10 (At3g21860) proteins (Schrammeijer et al., 2001). Both F-box proteins and Skp1 proteins are core components of the SCF (Skp1-Cullin-F-box protein) family of E3 ubiquitin ligases and serve to mediate targeted protein destabilization by the 26S proteasome. The VirF protein can interact with the VIP1 protein and mediate VirE2 protein degradation in the presence of VIP1 in yeast and plant cells, suggesting the VirF-mediating protein degradation is important for efficient transformation (Tzfira et al., 2004b). The involvement of members of the host SCF-E3 ligase complex were further deciphered by gene silencing and gene expression studies (Anand et al., 2012). The SCF-E3 ligase is composed of four different proteins: an F-box protein that recruits substrates; SKP1 that functions as an adaptor protein; Cullin (CUL1); and a RING-finger protein (RBX1) that interacts with both Gullin and E2 conjugating enzymes (Anand et al., 2012; Magori and Citovsky, 2012). Microarray analysis results showed that only ASK1, ASK2, and ASK20, among 21 Arabidopsis SKP1-LIKE (ASK) genes, are induced after A. tumefaciens infections. The Arabidopsis ask1 and ask2 mutants showed lower A. tumefaciens infection efficiencies, suggesting the SKP1 proteins may be positively involved in A. tumefaciens infection (Anand et al., 2012; Magori and Citovsky, 2012). However, the transformation rates of both RBX1 and CUL1c gene silenced N. benthamiana were not significantly affected (Anand et al., 2012). The Arabidopsis SGT1a (suppressor of the G2 allele of SKP1, At4g23570) and SGT1b (At4g11260) genes that encode accessory proteins interacting with the SCF-complex were induced following A. tumefaciens infection (Anand et al., 2012). Only the sgt1b mutant showed lower tumor formation efficiencies on roots when infected with A. tumefaciens, suggesting the potential role of SGT1 in the A. tumefaciens-plant interaction (Anand et al., 2012). Another plant SCF-E3 ligase complex member, the VIP1-binding F-box protein (VBF, At1g56250), is able to substitute for VirF function in plants and its expression is also induced by A. tumefaciens infection (Ditt et al., 2006; Zaltsman et al., 2010b). The VBF protein interacts with VIP1 and targets VIP1 and VIP1-VirE2 complexes for proteasomal destabilization via the SCFVBF complex (Zaltsman et al., 2010b). Additionally, the involvement of the VBF protein in uncoating a synthetic single-stranded DNA packaged by VirE2 was demonstrated in a cell-free proteasomal degradation system (Zaltsman et al., 2013). This proteasomal uncoating of synthetic minimal T-complexes is dependent on the presence of functional VBF and VIP1 proteins; and is inhibited by a known selective inhibitor of proteasomal activity, MG132 (Lacroix and Citovsky, 2013; Zaltsman et al., 2013).

The VirF protein may have multiple functions during A. tumefaciens infection, in addition to mediating the host cell ubiquitin-proteasome system (UPS) to unmask the T-complex and help T-DNA become integrated into the host genome (Gelvin, 2010a; Magori and Citovsky, 2012; Lacroix and Citovsky, 2013; Pitzschke, 2013). As noted above, upon A. tumefaciens infection, the VIP1 protein is phosphorylated by the mitogen-activated protein kinase 3 (MPK3) and functions as a transcription factor that induces expression of several stress-responsive genes (Djamei et al., 2007; Pitzschke, 2013). A. tumefaciens may utilize VirF to repress the VIP1-mediated host defense responses via VIP1 protein degradation. At the same time, premature or excessive degradation of VirE2-VIP1 protein complexes by VirF might also hinder T-DNA nuclear import and integration. In order to work against host cell UPS-mediated VirF degradation, A. tumefaciens exports another effector protein, VirD5, into plant cells; this protein interacts with VirF and protects it from rapid degradation by the defensive action of the host UPS (Magori and Citovsky, 2011). Similarly, VirD5 interacts with the Arabidopsis VIP1 protein and is able to form a ternary complex of VirD5-VIP1-VirE2 (Wang et al., 2014). VirD5 can compete with VBF for VIP1 binding and help stabilize VIP1 and VirE2 proteins, supporting the idea that the VirD5 protein may target T-complex to prevent its degradation in the plant cell nucleus (Wang et al., 2014). Finally, the VirE3 protein can be transferred into plant cells (Vergunst et al., 2000, 2003, 2005; Schrammeijer et al., 2003). Interestingly, VirE3 may interact with VirE2 and expression of VirE3 in VIP1 antisense tobacco plants can complement both nuclear import of VirE2 and transformation susceptibility (Lacroix et al., 2005). Thus, the A. tumefaciens may use the VirE3 effector protein as a substitute for VIP1 in plants to facilitate VirE2 nuclear targeting during infection. Collectively, these examples illustrate how the A. tumefaciens-mediated plant transformation process represents an impressive arms race, in which the plant cells try to fight off pathogen infections while the pathogen utilizes bacterial effectors to evade or outweigh plant defense responses.


Stable transformation

A. tumefaciens can genetically transform various host cells including numerous dicot and some monocot angiosperm species and gymnosperms (De Cleene and De Ley, 1976). In addition, A. tumefaciens can transform non-plant organisms, including fungi, algae, sea urchin embryos, human cells, and the gram positive bacterium Streptomyces lividans (Bundock et al., 1995, 1999; Piers et al., 1996; de Groot et al., 1998; Kunik et al., 2001 ; Kelly and Kado, 2002; Hooykaas, 2004; Kumar et al., 2004; Pelczar et al., 2004; Michielse et al., 2005; Bulgakov et al., 2006; Lacroix et al., 2006). In order to perform effective and large-scale functional genomic studies, highly efficient and simple genetic transformation methods for the studied organisms are crucial. The A. tumefaciens-mediated transformation method has several advantages over other transformation methods; it is easy to use, relatively inexpensive, and generally results in low copy number and well-defined DNA insertions into the host cell chromosome (Koncz et al., 1994; Pawlowski and Somers, 1996; Hansen et al., 1997; Enríquez-Obregón et al., 1998; Shou et al., 2004; Travella et al., 2005; Zhang et al., 2005; Gao et al., 2008). The traditional transformation methods such as protoplast transformation by electroporation, microinjection, or polyethylene glycol fusion are not as well-suited for production of transgenic plants, because the regeneration of plants from protoplasts is time-consuming and low efficiency (Fromm et al., 1985, 1986; Uchimiya et al., 1986; de la Peña et al., 1987; Newell, 2000; van den Eede et al., 2004; Banta and Montenegro, 2008). Microprojectile bombardment, also called biolistics, is the most important alternative to Ti plasmid DNA delivery systems for plants (Sanford, 2000). Spherical gold or tungsten particles are coated with DNA, accelerated to high speed with a particle gun, such that they penetrate into plant tissues (Kikkert et al., 2004). Microprojectile bombardment can be used to introduce foreign DNA into various plant tissues of a wide range of plant species. The gene of interest needs not to be cloned into a specialized transformation vector in order to be transformed into plant cells (Herrera-Estrella et al., 2005). However, the bombardment process tends to cause multiple-copy DNA insertions and the loss of molecular integrity of the DNA, and there are limitations on the size of the DNA. The complex DNA integration patterns usually result in genetic instability and/ or epigenetic silencing of the transgene in the transgenic plants (Newell, 2000; Kikkert et al., 2004; Lorence and Verpoorte, 2004; van den Eede et al., 2004; Altpeter et al., 2005; Herrera-Estrella et al., 2005). Due to these limitations and drawbacks of physical and chemical transformation methods, the A. tumefaciens-mediated gene transfer method is still the most frequently used and most popular method for generation of transgenic plants and genetically modified crops (Table 2).

The transformation methods can be categorized into two types: transient transformation and stable transformation. For transient transformation, T-DNA integration into the host genome is not required, and T-DNA expression usually lasts for a few days (Wroblewski et al., 2005; Marion et al., 2008; Jones et al., 2009; Kim et al., 2009; Li et al., 2009; Tsuda et al., 2012; Wu et al., 2014b). It presents useful advantages, in that it allows results of experimental treatments to be observed in a short period of time, as discussed below. On the other hand, stable transformation is a long process that often requires established tissue culture techniques to promote whole plant growth from the transformed cells or tissues. For stable transformation, the T-DNA must integrate in the host cell genome, so that it is subsequently passed on to the next generation (Bent, 2006; Gelvin, 2006; Tague and Mantis, 2006; Rivero et al., 2014).

In order to utilize pathogenic A. tumefaciens to generate transgenic plants, several obstacles need to be overcome. First, the oncogenes from the wild-type T-DNA are removed to eliminate the pathogenicity of the bacterium, and the strain becomes nonpathogenic (disarmed) without affecting its ability to transfer T-DNA. Second, the gene of interest and selection markers for transgenic plants need to be introduced into the T-DNA, and molecular biology tools are needed for DNA cloning in vitro. The Ti plasmid size is large and usually low-copy number, which makes isolation and cloning of the Ti plasmid quite challenging. In order to overcome these difficulties, most research teams utilize a binary vector system, in which the T-DNA region is carried on a broad-host range replicon and the vir genes required for T-DNA transfer are located on the disarmed Ti-plasmid (Barton et al., 1983; de Framond et al., 1983; Hoekema et al., 1983; Zambryski et al., 1983; Bevan, 1984). This binary vector system has provided the plant research community enormous versatility and flexibility, and has enabled an upsurge in the production of transgenic plants.

Table 2.

Summary of different plant transformation methods


With advances in recombinant DNA technology, T-DNA binary vectors have evolved over the past years to be highly developed and more specialized for different purposes (Hellens et al., 2000; Chung et al., 2005; Komari et al., 2006; Lee and Gelvin, 2008; Mehrotra and Goyal, 2012; Anami et al., 2013). T-DNA-based binary vectors generally consist of several features, including (1) the T-DNA right and left border repeat sequences that define the T-DNA region; (2) selectable marker genes to function in bacteria (E. coli and A. tumefaciens) and in plants; (3) restriction endo-nuclease sites within the T-DNA to allow insertion of one or more gene(s) of interest; (4) origin(s) of replication to allow plasmids to replicate in bacteria. In order to identify the transformed cells or plants, it is essential to be able to detect the foreign DNA that has been integrated into plant chromosome. Various antibiotic or herbicide resistance genes have been inserted into the T-DNA region of the binary vectors and used for transgenic plant selection. The neomycin phosphotransferase (NPTII) gene for kanamycin resistance and hygromycin phosphotransferase (HPT), conferring resistance to hygromycin, are the most widely used selectable marker gene systems in plants. Several other frequently used selection systems utilize other antibiotics, including chloramphenicol, gentamicin, streptomycin, bleomycin, blasticidin; herbicides, including phosphinothricin, gluphosinate, glyphosate, bromoxynil; or metabolic markers, including acetolactate synthase, threonine dehydratase, phosphomannose isomerase (Gheysen et al., 1998; Newell, 2000; Banta and Montenegro, 2008; Lee et al., 2008; Lee and Gelvin, 2008; Anami et al., 2013). Furthermore, in plant gene functional studies and other quantification studies, the use of reporter genes allows transformed cells to be quantified. The frequently used reporter gene products, such as ß-D-glucuronidase (GUS), both firefly and bacterial luciferases (LUC), green fluorescent protein (GFP) and other fluorescent proteins, can be detected in transformed plant tissues. The GUS activity in the transformed plant tissues can be observed by the blue staining after hydrolysis of the 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid; or can be quantitatively assayed by a fluorometric analysis that requires hydrolysis of the 4-methylumbelliferyl ß-D-glucuronide. The GFP protein is a useful in vivo marker to examine gene expression or recombinant protein localization, because it can be excited with either ultraviolet or blue light without addition of any substrates or cofactors.

Based on the agrobacterial chromosomal background and the resident Ti plasmids, the commonly used disarmed A. tume-faciens strains originated from two representative wild isolates: C58 (Lin and Kado, 1977) and Ach5 (Kovacs and Pueppke, 1994). The A. tumefaciens strains AGL1, EHA101, EHA105, and GV3101 (pMP90), have the nopaline type C58 chromosomal background and the modified Ti plasmids from either the pTiBo542 or the pTiC58 plasmids (Hood et al., 1986; Koncz and Schell, 1986; Lazo et al., 1991). The A. tumefaciens strain LBA4404 has the octopine type Ach5 chromosomal background and the octopine-type Ti plasmid pAL4404 derived from the pTiAch5 plasmid (Ooms et al., 1982). Genetic background and modified Ti plasmid combinations of the disarmed A. tumefaciens strains noticeably affect the bacterial host range and the transformation efficiencies of various plant species (Hwang et al., 2010; Hwang et al., 2013a) (Table 3).

The host species A. thaliana is characterized by several traits, including small plant size, short generation time, large quantity of seeds, relatively small genome size, and ease of transformation by A. tumefaciens, which has led it to become the model system for plant biologists to address questions of basic and applied technology. Several Agrobacterium-mediated transfer methods for A. thaliana were developed using various A. thaliana ecotypes, plant tissue types, bacterial strains, binary vectors, and selection marker (Lloyd et al., 1986; Feldmann and Marks, 1987; Sheikholeslam and Weeks, 1987; Valvekens et al., 1988; Akama et al., 1992; Clarke et al., 1992; Huang and Mā, 1992; Sangwan et al., 1992; Chang et al., 1994). Here, two major stable transformation methods of A. thaliana are described as follows.

Table 3.

Frequently used disarmed Agrobacterium strains


Stable transformation using root explants

Root explant is one of the plant tissues that is frequently used to genetically transform A. thaliana (van den Elzen et al., 1985; Valvekens et al., 1988; Akama et al., 1992; Clarke et al., 1992; Kemper et al., 1992; Czako et al., 1993; DeNeve et al., 1997; De Buck et al., 1998, 2000, 2009). There are several advantages of root expiants for genetic transformation by A. tumefaciens. A. thaliana root cells are competent for regeneration, can be easily acquired in large quantities, and can be maintained in sustained cultures (Valvekens et al., 1988; Sangwan et al., 1992; Czako et al., 1993). Furthermore, a relatively low percentage of transformants show polyploidy when root explants are used for A. tumefaciens transformation, in comparison to leaf transformation or direct gene transfer methods, such as microprojectile bombardment (Altmann et al., 1994). Additionally, root transformations of Arabidopsis tend to result in higher percentage of single T-DNA insertions when compared with leaf-disc transformations (Grevelding et al., 1993). The root transformation assay also provides a reproducible and quantitative assay system for plant biologists to examine transformation efficiencies of somatic cells (Gelvin, 2006). Several studies have utilized quantitative root transformation assays to determine virulence of different A. tumefaciens strains and relative transformation efficiencies of different A. thaliana mutants or ecotypes. Based on the results obtained from these root transformation assays, bacterial and plant proteins that are involved in Agrobacterium-mediated plant transformation process can be identified and characterized; several of these were described above (Nam et al., 1997, 1998, 1999 Bundock et al., 1999; Mysore et al., 2000a; Zhu et al., 2003a, 2003b; Tsai et al., 2009; Gelvin, 2010a, 2010b; Hwang et al., 2010; Tsai et al., 2010; Gelvin, 2012; Hwang et al., 2013b, 2015).

It is known that different factors affect transformation efficiency, including plant growth temperature, plant-bacteria co-incubation time and temperature, light intensity and periods, addition of AS, pretreatment with phytohormones, different A. thaliana ecotypes, different A. tumefaciens helper strains, and other factors (Vergunst et al., 1998; Zambre et al., 2003; Gelvin, 2006). The optimum growth temperature for Arabidopsis is 25°C. It is recommended that night temperature can be 2 to 4°C lower than the day temperature. During co-cultivation of plant root expiants with A. tumefaciens, the transformation efficiencies and plant regeneration frequencies were relatively higher at 25°C in comparison to 21 °C (Vergunst et al., 1998). On the other hand, although A. tumefaciens grows well at 28°C, accumulation of some of the VirB proteins constituting the T4SS T-DNA delivery machinery, as well as virulence on the host species Kalanchoe, is strongly compromised at this temperature (Fullner and Nester 1996; Banta, et al. 1998; Baron et al., 2001); these data indicate that pre-induction with AS (see below) should be performed at 19–21°C. Furthermore, A. tumefaciens can lose virulence when grown at 37°C due to loss of the Ti plasmid in the bacteria (Hamilton and Fall, 1971; Kerr, 1971). Light conditions during Agrobacterium-mediated plant transformation also affect transformation efficiencies significantly. The transformation efficiency is higher under continuous light than under a 16 hour light/8 hour dark photoperiod (Vergunst et al., 1998; Zambre et al., 2003). Another important factor for obtaining high transformation frequency is addition of 50–200 µM AS, a natural wound response molecule, to the A. tumefaciens culture prior to or during the co-incubation period in order to fully induce virulence gene expression in bacteria (Sheikholeslam and Weeks, 1987; Vergunst et al., 1998). It has also been suggested that coincubation periods should not exceed two days in order to avoid overgrowth of bacteria (Vergunst et al., 1998; Gelvin, 2006). After bacteria and root expiant coincubation, different antibiotics, such as carbenicillin, cefotaxime, vancomycin HCI, or timentin, may be added into the selection media to eliminate A. tumefaciens. Timentin is an antibacterial combination consisting of the semisynthetic antibiotic ticarcillin disodium and the ß-lactamase inhibitor clavulanate potassium and has frequently been used in A. thaliana root transformation methods (Nam et al., 1997, 1998, 1999; Vergunst et al., 1998; Bundock et al., 1999; Mysore et al., 2000a; Zhu et al., 2003a).

Different opine-type A. tumefaciens strains show different degrees of virulence on different plant species, partly because the tzs and virF genes exist in only one type of Ti-plasmid. It has been suggested that nopaline-type A. tumefaciens strains are preferable for transformation of A. thaliana and other plants from the Brassicaceae family (Nam et al., 1997; Gelvin, 2006; Hwang et al., 2013a). Similarly, different wild-type A. thaliana ecotypes show different competence for A. tumefaciens transformation (Nam et al., 1997; Chateau, 2000; Mysore et al., 2000a; Hwang et al., 2013a); ecotypes Aua/Rhon (Aa-O), Bensheim/Bergstrasse (Be-O), Wassilewskija (Ws), Columbia (Col-O), and C-24 are more susceptible to A. tumefaciens root transformation (Nam et al., 1997). Furthermore, competence of several A. thaliana eco-types can be enhanced when the explants are pre-cultured on media containing the phytohormones auxin and cytokinin (Valvekens et al., 1988; Sangwan et al., 1991, 1992; Hwang et al., 2013b; Sardesai et al., 2013).

Stable transformation by floral dipping

The transformation and regeneration of plants is a vital tool in both basic and applied plant biology research fields. The typical plant transformation method includes delivery of target gene into plant cells by A. tumefaciens transformation, followed by selection and regeneration of transgenic plants from the transformed cells through tissue culture techniques. Established plant tissue culture systems and sterile conditions are essential to regenerate plants. Suitable plant growth regulators are added to help regeneration of transgenic plants. However, several difficulties exist in the typical molecular transformation method. First, some A. thaliana ecotypes, such as Antwerpen (An-1), Angleur (Ang-0), Bologna (BI-1), Blanes/Gerona (Bla-2), Calver (Cal-0), and Dijon-G, and mutant lines, are resistant to transformation and/ or regeneration (Nam et al., 1997; Chateau, 2000; Mysore et al., 2000a; Hwang et al., 2013a). In addition, regeneration of whole plants from transformed somatic cells sometimes leads to somatic mutations. The presence of phytohormones in the tissue culture during regeneration also increases the chance of somatic mutations (Bent, 2000; Bent, 2006; Tague and Mantis, 2006). In order to resolve these difficulties, several plant transformation methods have been established and improved (Feldmann and Marks, 1987; Bechtold et al., 1993; Chang et al., 1994; Katavic et al., 1994; Clough and Bent, 1998; Richardson et al., 1998; Martinez-Trujillo et al., 2004; Zhang et al., 2006). Feldmann and Marks (1987) first reported that imbibition of A. thaliana germinating seeds with an appropriate A. tumefaciens strain can generate transformed progeny in the next generation. This method was applied to provide the first insertion mutagenized lines of A. thaliana. Another whole plant transformation method was presented by Chang et al. (1994) and by Katavic et al. (1994) in which reproductive inflorescences were cut off and the wounded sites were inoculated with A. tumefaciens. The treated plants were grown to maturity, harvested, and the progeny screened for transformants under selection. These methods avoid tissue culture steps and the resulting somaclonal variation, and a relatively short time is needed to acquire transformed progeny. However, the frequency of obtaining transformants in the next generation is highly variable and sometimes inconsistent for routine use.

An improved in planta transformation method was later proposed by Bechtold et al. (1993) in which the uprooted A. thaliana plants were vacuum infiltrated with the appropriate A. tumefaciens culture, infiltrated plants were then immediately replanted, and harvested seeds were finally screened for successful transformants. This method was developed to preclude altogether the requirement for plant tissue culture or regeneration steps (Bechtold et al., 1993; Bechtold and Pelletier, 1998). However, the removal and replanting of plants constrain the usefulness of this method. It was later discovered that submergence of inflorescences in the early flowering stages in a A. tumefaciens suspension is sufficient to obtain successful transformants (Clough and Bent, 1998). Furthermore, the vacuum infiltration process can be eliminated; simple dipping of developing floral tissues into the bacterial suspension is enough to acquire transformants in the next generation (Clough and Bent, 1998; Richardson et al., 1998; Bent, 2000). A. tumefaciens strains are first cultured in rich media with appropriate antibiotic selection until stationary phase (OD600= 0.8–1.0). The A. tumefaciens cultures are then collected by centrifugation and the pellet is resuspended in a solution containing 5% sucrose and the surfactant Silwet L-77 (Clough and Bent, 1998; Bent, 2000). Both sucrose and surfactant 0.05% Silwet L-77 are important for the success of the floral dip method (Clough and Bent, 1998; Richardson et al., 1998; Bent, 2000; Bent, 2006). However, Silwet L-77 can be toxic to plants at high concentrations, and 0.02% L-77 or as low as 0.005% L-77 can be used in the floral dip method. Repeated applications of A. tumefaciens can enhance the transformation efficiency (Clough and Bent, 1998; Martinez-Trujillo et al., 2004). After bacterial inoculation, covering plants for 1 day to maintain humidity also increase the transformation rates (Bent, 2000; Bent, 2006). Spraying of the A. tumefaciens is a useful alternative to transform Arabidopsis, and this method may provide the possibility of in planta transformation of plant species that are too large for dipping or vacuum infiltration (Chung et al., 2000). With these floral dip or spray methods, 0.1– 3.0% of the seeds is typically found to be transgenic. This floral dip method is simple and can be easily performed by nonspecialists, which avoids extensive labor and the issues and equipment associated with regeneration. This method has been successfully used on a large number of A. thaliana ecotypes and mutants that might be resistant to transformation by other methods targeting somatic cells or tissues (Mysore et al., 2000a). The most striking contribution of this method is the high-throughput generation of A. thaliana T-DNA insertion mutant lines (Alonso et al., 2003; Alonso and Stepanova, 2003). Plant researchers can now easily obtain various T-DNA insertion mutants for most Arabidopsis genes from various stock centers (Table 4) (Scholl et al., 2000; Pan, 2003; Li et al., 2014a).

In the A. thaliana vacuum infiltration method, most of the primary transformants are hemizygous at any T-DNA insertion site, suggesting that A. tumefaciens transformation may occur late in the floral development, possibly after the divergence of male and female gametophyte cell lineages (Bechtold et al., 1993; Bent, 2000; Bent, 2006). It was later discovered that the majority of transgenic plants result from transformation of the female gametophyte; this conclusion is based on the following observations (Ye et al., 1999; Bechtold et al., 2000; Desfeux et al., 2000; Bechtold et al., 2003): First, no transformed progeny were obtained after inoculation of A. tumefaciens on pollen donor plants, while 0.48% of transformed progeny were produced after inoculation of A. tumefaciens on pollen recipient plants (Desfeux et al., 2000). Second, various promoters fused to a GUS marker gene were used to observe sites of delivery of T-DNA (Ye et al., 1999; Bechtold et al., 2000; Desfeux et al., 2000). GUS staining was mainly observed in the ovules of the flowers and only rarely in pollen, suggesting that ovules are the primary target for A. tumefaciens transformation (Ye et al., 1999; Desfeux et al., 2000). In A. thaliana flowers, the gynoecium is an open and vase-like structure, which can fuse to form closed locules almost three days prior to anthesis. Based on this observation, the timing of A. tumefaciens infection is important to allow A. tumefaciens to enter the interior of the gynoecium prior to locule closure and ensure successful transformation (Desfeux et al., 2000). Third, most of the transformants contain T-DNA that is derived from the maternal chromosome set, based on a genetic linkage study (Bechtold et al., 2000). The floral dip or infiltration transformation methods have been successfully applied to wheat, radish, Medicago truncatula, and some other members from the Brassicaceae, such as pakchoi (Brassica campestris), Shepherd's purse (Capsella bursa-pastoris), Lepidium campestre, and Arabidopsis lasiocarpa (Liu et al., 1998; Trieu et al., 2000; Curtis and Nam, 2001 ; Tague, 2001 ; Wang et al., 2003; Inan, 2004; Agarwal et al., 2008; Bartholmes et al., 2008; Zale et al., 2009; Lenser and Theißen, 2014). Other bacteria, including Ensifer adhaerens, Rhizobium sp. NGR234, Sinorhizobium meliloti, and Mesorhizobium loti, contained the foreign vir genes and T-DNA regions on separate plasmids and have been used for floral dip transformation of A. thaliana (Broothaerts et al., 2005; Wendt et al., 2012). Recently, the Rhizobium etli CFN42 genome analysis results showed that it has functional and high homologs of vir genes in its p42a plasmid. The R. etli can both stably and transiently transform tobacco plants when the T-DNA region is introduced into the R. etli on a binary vector (Lacroix and Citovsky, 2016). Although these bacteria are not naturally able to transform plants because they lack native T-DNA and/or vir genes, most of them belong to Rhizobiaceae family, specifically the Alpha-Proteobacteria class.

The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein (Cas) technology is a recent advance and powerful tool for genome editing in eukaryotic cells. The technique utilizes Streptococcus pyogenes Cas9 endonuclease and single chimeric guide RNA (sgRNA) to generate DNA double-stranded breaks in targeted genome sequences. It has been successfully used to edit genomes in animal systems (Cong et al., 2013; Hwang et al., 2013c; Wang et al., 2013) and was recently applied in plants as well. CRISPR can create genome-edited transgenic plants if a stable transformation approach (such as floral dip) is used (Zhang et al., 2015). In summary, the floral dip or infiltration transformation method has now become the most important and frequently used Agrobacteriummediated transformation method in Arabidopsis research.

Transient transformation

In general, months are required to generate a transgenic line, and even longer if a homozygous genotype is desirable. Agro-bacterium-mediated transient transformation is a rapid alternative to assay gene function, promoter behaviour and protein function when generation of a stable transgenic line is unnecessary. A binary vector carrying a reporter system (Le. promoter-of-interest fused with a GUS/Iuciferase/fluorescence protein gene) or a fusion protein driven by a constitutive promoter (e.g. for subcellular localization, protein activity or protein-protein interaction study) can be naturally processed to generate single stranded T-DNA and delivered into the nucleus of the host. In the host nucleus, the single stranded T-DNA can be synthesized into double-stranded T-DNA, which is readily transcribed and translated into the reporter protein/fusion protein for analysis without genome integration. Transient transformation can be also used for gene silencing by expressing small interfering RNAs (siRNAs) and microRNAs (miRNAs) in plant tissues (McHale et al., 2013). As integration is not the main purpose in the transient system, a plant selectable marker is usually not required in the construct to be delivered. The drawback however, is the highly variable transformation efficiency for individual Agrobacterium strains, target plant species (including different A. thaliana ecotypes) and specific tissues. Although A. thaliana is not the easiest species to transiently transform, due to its importance in plant biology, extensive optimizations have been carried out. Below, we have summarized the reported methods (an overview can be seen in Table 5) that facilitate the transient transformation in A. thaliana.

Transient transformation in roots

Root explants of various A. thaliana ecotypes respond differently to Agrobacterium-mediated transient transformation. Some ecotypes such as Bl-1, Petergof, Cal-0, and Dijon-G are poorly transformed (Nam et al., 1997). Attachment assays revealed that the wild type A. tumefaciens virulent strain C58 binds poorly to roots of the Bl-1 and Petergof ecotypes, in contrast to highly efficient binding to roots of susceptible ecotypes Aa-0 and WS. This indicates that the binding ability of agrobacteria to the root tissue could be directly related to transformation efficiency. The Arabidopsis rat4 mutant, with a deletion of the cellulose synthase-like gene CSLA9 (At5g03760), showed a reduction in transient transformation efficiency (Zhu et al., 2003a). It has been shown that cellulose is not essential but important for efficient binding of A. tumefaciens cells to host cells for subsequent infection (Zhu et al., 2003a; Matthysse et al., 2005). The CSLA9 promoter is specifically active in the elongation zone of roots, the root tissue that is most susceptible to Agrobacterium-mediated transient transformation (Zhu et al., 2003a). RTNLB1 (reticulon-like protein B-1, At4g23630), which was found to interact with the A. tumefaciens T-pilus protein VirB2, is important for stable and transient transformation efficiency of root expiants (Hwang and Gelvin, 2004).

Table 4.

Selected Arabidopsis stock and database resources.


Agrobacterium rhizogenes is a relative of A. tumefaciens which cause adventitious root development at the site of infection (also known as “hairy root disease”) and can be manipulated to co-transfer the T-DNA borne on a binary vector into root cells (Limpens et al., 2004). A. rhizogenes-mediated transformation appears to be a fast and effective tool to study the function of genes involved in root biology. It has been employed in RNA interference (RNAi) studies in roots (Limpens et al., 2004) and the disarmed strain can also be used for transient expression at the root epidermis without affecting the root morphology (Campanoni et al., 2007).

Table 5.

Summary of Agrobacterium-mediated transient transformation assays in plants


Transient transformation in adult leaves via agrofiltration

Due to the anatomical specificity, root tissues may not be suitable for experiments related to defense, chloroplasts, or for protein purification. Vegetative tissues like leaves are more abundant and sacrificing the plant is not necessary. For species Nicotiana benthamiana and lettuce, which can reach ~80% mesophyll cells, it is possible to achieve nearly 100% transient transformation efficiency in some regions of the leaf (Wroblewski et al., 2005). In contrast, successful transient transformation in A. thaliana leaves by the same method can be challenging and depends on ecotype and A. tumefaciens strain. As mentioned above, most laboratory A. tumefaciens strains are derivatives of two wild type virulent strains, C58 and Ach5. For instance, GV3101(pMP90) from C58 and LBA4404 from Ach5 are routinely used for transient transformation of various plant species (Kim et al., 2009). Different A. tumefaciens strains produce variable opines: nopaline from pTiC58, octopine from pTiAch5, agropine from A281 and succinamopine from EHA105 (Dessaux et al., 1992; Frandsen, 2011). Although different opine-producing strains seem to work better in certain plant species, it remains unclear whether the specificity of opine utilization contributes to different transformation efficiencies in specific plant species. Strain C58 cured of pTiC58, also known as C58C1, harbouring the octopine-type pTiB6S3DT-DNA and a pCH32 helper plasmid is regarded as the best; in a study of more than 10 A. tumefaciens wild type and disarmed strains, it achieved the highest transient transformation efficiency of A. thaliana, lettuce and tomato leaves (Wroblewski et al., 2005). Phenolics have been reported to influence transient transformation efficiency. As is true for stable transformation, it is now routine to add AS during transformation. Pre-treating A. tumefaciens with AS before agroinfiltration of in A. thaliana leaves can also increase the transient transformation efficiency (Mangano et al., 2014). Detergents/surfactants are also used to improve cuticular penetration of fluid on leaf surfaces. Silwet L-77 is one of the widely used surfactants in transient transformation while Triton X-100 was also shown to significantly enhance the efficiency (Kim et al., 2009).

Plant defense responses also play a key role in limiting transient transformation. Arabidopsis senses A. tumefaciens elongation factor EF-Tu through a transmembrane Leucine-rich-repeat (LRR) receptor kinase, EFR (At5g20480), to trigger downstream immune responses. A. thaliana efr mutants fail to elicit defence responses effectively against A. tumefaciens, and as a result the mutants are more susceptible to transient transformation in agroinfiltrated Arabidopsis leaves, as assessed using GUS as a reporter (Zipfel et al., 2006). Effector AvrPto from the plant bacterial pathogen Pseudomonas syringae weakens the immunity of Arabidopsis by targeting multiple pattern recognition receptors (PRRs) (Hauck et al., 2003; Xiang et al., 2008). The effector was engineered into A. thaliana, such that expression was driven by a dexamethasone (DEX)-inducible promoter. In the transgenic line, AvrPto was expressed only when DEX was applied. Pre-treating the plant with DEX before A. tumefaciens infiltration suppressed the plant immunity and hence the transient expression efficiency was significantly enhanced (Tsuda et al., 2012). Salicylic acid (SA) plays an important role in defense against bacteria, a recent report shows that use of nahG (a bacterial SA hydroxylase) expressing Arabidopsis plants can also enhance the transient transformation efficiency in Arabidopsis leaves. (Rosas-Díaz et al., 2017)

Leaf transient transformation has been employed for virusinduced gene silencing (VIGS). VIGS uses viral vectors to generate double-stranded RNA of a gene of interest to be silenced. While performing VIGS in Solanaceous plant species, including N. benthamiana, tomato, pepper, potato and petunia is straightforward, it is relatively difficult in A. thaliana due to the inconsistent transformation efficiency. Optimization of Tobacco rattle virus (TRV)-based VIGS in A. thaliana involved minimal modification of the N. benthamiana protocol, but transformation has to be done in seedlings at the two- to three-leaf stage (BurchSmith et al., 2006). Light intensity and temperature were shown to affect systemic movement of the silencing signal in transient agroinfiltration in N. benthamiana. High light intensities (≥ 450 µE/m2/s) and temperatures (≥ 30°C) localized the silencing signal in the leaf tissue that was infiltrated. In contrast, lower light intensities with a constant temperature of 25°C supported strong systemic movement of the silencing signal (Patii and Fauquet, 2015). On the other hand, the transient VIGS technique can be applied to improve overall stable transformation efficiency. A. thaliana AGO2 (At1g31280) and NRPD1A (At1g63020) are inhibitors of Agrobacterium-mediated transformation. Transient transformation with AGO2 and NRPD1a silencing VIGS vectors by agroinfiltration of leaves prior to transformation with a gene of interest by floral dip was shown to increase the number of transgenic seeds by 6.0- and 3.5-fold, respectively (Bilichak et al., 2014).

Transient transformation in seedlings

Transient transformation of seedlings has the advantage of allowing analysis in the whole-plant cellular context, in contrast to localized agroinfiltration in adult leaves. Most seedling optimization has aimed to provide fast and convenient protocols for a preliminary analysis of uncharacterised genes and constructs. Vacuum infiltration was introduced to facilitate whole A. thaliana seedling adsorption of agrobacterial cells (Marion et al., 2008), while the Fast Agrobacterium-mediated seedling transformation (FAST) protocol optimized the cell density (OD600=0.5) and concentration of Silwet L-77 (0.005%) without the need for vacuuming (Li et al., 2009). FAST was demonstrated to work for Catharanthus roseus seedling transformation. However, it is worth noting that the FAST method strongly induces host defence genes Zct1 and Orca3; this should be taken into account in experimental design (Weaver et al., 2014). Optimization of A. tumefaciens culture conditions can also improve seedling transformation efficiency. The AGROBEST (Agrobacterium-mediated enhanced seedling transformation) method achieves high transient transformation efficiency in whole seedlings, allowing gain-of-function studies, by using a pH-buffered medium supplemented with AB salts and glucose. It has been shown that transient expression of GUS or LUC of Col-O or efr-1 seedlings was significantly enhanced when AGROBEST was used during A. tumefaciens inoculation (Wu et al., 2014b). Similar to what was observed for agroinfiltration in adult leaves (Wroblewski et al., 2005), C58C1 (pTiB6S3δT-DNA, pCH32) achieves higher transient transformation efficiency than the wild type C58 virulent strain or the C58-derived disarmed strain GV3101(pMP90) in A. thaliana seedlings (Wu et al., 2014b). This study also showed an inverse association of root growth inhibition and transient expression efficiency, suggesting that C58C1 (pTiB6S3δT-DNA, pCH32) may circumvent a plant defense barrier to enable high transient expression levels in A. thaliana seedlings. However, the genetic factors and mechanisms underlying the ability of this chimeric disarmed A. tumefaciens strain to achieve the highest transient transformation efficiency await future investigations. By expressing a gene of interest separately with a GFP marker in one bicistronic vector, fluorescent GFP signals are used to identify and locate the cells that are successfully transformed by the vector; in the same cells, the signals also indicates expression of the gene of interest. It has been demonstrated that the bicistronic vector transiently expressing SYP121 (At3g11820) was able to rescue the syp121 mutant inward K+ channel current phenotype (Chen et al., 2011 ).


From the discovery of Agrobacterium tumefaciens to the marketing of genetically modified (GM) crops, tremendous knowledge in plant gene function, cell biology, plant-microbe interaction and biotechnology has been unveiled. In particular, the optimized protocols for A. thaliana transformation have provided an irreplaceable tool for studying plant genetics by generation of random T-DNA insertion mutants. Seed stocks with various genomic disruptions by T-DNA insertion are available to order and have been used by plant scientists around the world. This enormous resource has greatly transformed plant research by streamlining studies of genes of interest. The convenience of Agrobacterium-mediated transformation in A. thaliana is also an ideal pilot scheme to test the effects of gene constructs before applying them in other plant species such as crops. At the same time, due to its role in plant applications, the biology of A. tumefaciens has been extensively studied. The most notable area is the study of the mechanism of T4SS, which mediates gene transfer from A. tumefaciens to plants. By comprehensively reviewing the functions of key genes involved in Agrobacterium-plant interactions and the transformation process including the recent breakthrough discovery of Pol θ in T-DNA integration, we hope to draw the attention of plant community to the importance of A. tumefaciens research and to the many unresolved questions about T-DNA translocation and integration process that await future studies.

Although A. tumefaciens is an excellent tool for dicot transformation, the efficiency in monocots, including in some important crop plants, is lagging far behind. Among the reasons for the poor efficiency may be the unique defense mechanisms in monocots and the absence of essential (efficient) components in monocots for T-DNA transfer and integration. One future direction could be investigating the defence signalling pathways in monocots against A. tumefaciens and introducing essential components into monocots. Combining the efficient transformation by A. tumefaciens with the CRISPR technique is also a growing trend in genome editing; this enables the removal of unwanted gene fragments such as antibiotic markers and nonessential foreign genes in the genome. The unmarked genome should help reduce public concerns about the potential toxicity of the current GM crops because of whole T-DNA integration. Apart from T4SS, A. tumefaciens possesses the T6SS which carries antibacterial activity for interbacterial competition; its secretion activity was shown to be suppressed when virulence proteins including T4SS were massively expressed (Wu et al., 2012). Some bacterial T6SS effectors are able to manipulate host immunity in animal systems, but there is no solid evidence to suggest that A. tumefaciens or other plant pathogens can use their T6SS effectors to manipulate host immunity in plants. Future efforts to identifying bacterial T6SS effectors targeting plants could be beneficial to enhance transformation efficiency. Given the increasing demands for plant engineering in medicine, environmental protection and food security, it is expected that A. tumefaciens will continue to play an important role in plant sciences, and understanding the biology of agrobacteria as well as its interactions with plants will remain essential, contributing to scientific developments throughout the plant domain.


We thank the anonymous reviewers for careful reading and valuable suggestions of this review. We also thank the Hwang and Lai laboratory members for suggestions and Miss Shin-Fei Chi for help with the figure. This work is made possible by grants from Academia Sinica and Ministry of Science and Technology (MOST 104-2311-B-001-025-MY3), Taiwan, to E.-M.L. and from Ministry of Education and Ministry of Science and Technology (MOST 105-2313-B-005-008), Taiwan, to H.-H.H. M.Y has been awarded a postdoctoral fellowship from Academia Sinica.


  1. Agarwal, S., Loar, S., Steber, C., and Zale, J. ( 2008). Floral transformation of wheat. Methods Mol. Biol. 478, 105–113. Google Scholar
  2. Aguilar, J., Cameron, T.A., Zupan, J., and Zambryski, P. ( 2011). Membrane and core periplasmic Agrobacterium tumefaciens virulence type IV secretion system components localize to multiple sites around the bacterial perimeter during lateral attachment to plant cells. mBio 2, e00218–00211. Google Scholar
  3. Aguilar, J., Zupan, J., Cameron, T.A., and Zambryski, P.C. ( 2010). Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells. Proc. Natl. Acad. Sci. USA 107, 3758–3763. Google Scholar
  4. Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K., and Shimura, Y. ( 1992). Efficient transformation of Arabidopsis thaliana'. comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains. Plant Cell Reps. 12, 7–11. Google Scholar
  5. Akiyoshi, D.E., Regier, D.A., and Gordon, M.P. ( 1987). Cytokinin production by Agrobacterium and Pseudomonas spp. J. Bacteriol. 169, 4242–4248. Google Scholar
  6. Akiyoshi, D.E., Regier, D.A., Jen, G., and Gordon, M.P. ( 1985). Cloning and nucleotide sequence of thetzsgene from Agrobacterium tumefaciens strain T37. Nucleic Acids Res. 13, 2773–2788. Google Scholar
  7. Albright, L.M., Yanofsky, M.F., Leroux, B., Ma, D.Q., and Nester, E.W. ( 1987). Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J. Bacteriol. 169, 1046–1055. Google Scholar
  8. Alonso, J.M. and Stepanova, A.N. ( 2003). T-DNA mutagenesis in Arabidopsis. Methods Mol. Biol. 236, 177–188. Google Scholar
  9. Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C., and Ecker, J.R. ( 2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657. Google Scholar
  10. Altmann, T., Damm, B., Frommer, W., Martin, T., Morris, P., Schweizer, D., Willmitzer, L., and Schmidt, R. ( 1994). Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement. Plant Cell Reports 13, 652–656. Google Scholar
  11. Altpeter, F., Baisakh, N., Beachy, R., Bock, R., Capell, T., Christou, P., Daniell, H., Datta, K., Datta, S., Dix, P.J., Fauquet, C., Huang, N., Kohli, A., Mooibroek, H., Nicholson, L., Nguyen, T.T., Nugent, G., Raemakers, K., Romano, A., Somers, D.A., Stoger, E., Taylor, N., and Visser, R. ( 2005). Particle bombardment and the genetic enhancement of crops: myths and realities. Mol. Breed. 15, 305–327. Google Scholar
  12. Alvarez-Martinez, C.E., and Christie, P.J. ( 2009). Biological diversity of prokaryotic type IVsecretion systems. Microbiol. Mol. Biol. Rev. 73, 775–808. Google Scholar
  13. Aly, K.A., and Baron, C. ( 2007). The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens. Microbiology 153, 3766–3775. Google Scholar
  14. Aly, K.A., Krall, L., Lottspeich, F., and Baron, C. ( 2008). The type IV secretion system component VirB5 binds to the trans-zeatin biosynthetic enzyme Tzs and enables its translocation to the cell surface of Agrobacterium tumefaciens. J. Bacteriol. 190, 1595–1604. Google Scholar
  15. Anami, S., Njuguna, E., Coussens, G., Aesaert, S., and Van Lijsebettens, M. ( 2013). Higher plant transformation: principles and molecular tools. Int. J. Dev. Biol. 57, 483–494. Google Scholar
  16. Anand, A., Krichevsky, A., Schornack, S., Lahaye, T., Tzfira, T., Tang, Y., Citovsky, V., and Mysore, K.S. ( 2007). Arabidopsis VIRE2 INTER-ACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants. Plant Cell 19, 1695–1708. Google Scholar
  17. Anand, A., Uppalapati, S.R., Ryu, C.M., Allen, S.N., Kang, L., Tang, Y., and Mysore, K.S. ( 2008) Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens. Plant Physiol. 146, 703–715. Google Scholar
  18. Anand, A., Rojas, C.M., Tang, Y., and Mysore, K.S. ( 2012). Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation. New Phytol. 195, 203–216. Google Scholar
  19. Ankenbauer, R.G., and Nester, E.W. ( 1990). Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides. J. Bacteriol. 172, 6442–6446. Google Scholar
  20. Atmakuri, K., Cascales, E., and Christie, P.J. ( 2004). Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol. Microbiol. 54, 1199–1211. Google Scholar
  21. Backert, S., Fronzes, R., and Waksman, G. ( 2008). VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems? Trends Microbiol. 16, 409–413. Google Scholar
  22. Bako, L., Umeda, M., Tiburcio, A.F., Schell, J., and Koncz, C. ( 2003). The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants. Proc. Natl. Acad. Sci. USA 100, 10108–10113. Google Scholar
  23. Ballas, N., and Citovsky, V. ( 1997). Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc. Natl. Acad. Sci. USA 94, 10723–10728. Google Scholar
  24. Banta, L.M., and Montenegro, M. ( 2008). Agrobacterium and plant biotechnology. In Agrobacterium: From Biology to Biotechnology, T. Tzfira, and V. Citovsky, eds, (Springer Science+Business Media, Berlin), pp. 73–147. Google Scholar
  25. Banta, L.M., Bohne, J., Lovejoy, S.D., and Dostal, K. ( 1998). Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption. J. Bacteriol. 180, 6597–6606. Google Scholar
  26. Banta, L.M., Kerr, J.E., Cascales, E., Giuliano, M.E., Bailey, M.E., McKay, C., Chandran, V., Waksman, G., and Christie, P.J. ( 2011). An Agrobacterium VirB10 mutation conferring a Type IV secretion system gating defect. J. Bacteriol. 193, 2566–2574. Google Scholar
  27. Baron, C., Domke, N., Beinhofer, M., and Hapfelmeier, S. ( 2001). Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains. J. Bacteriol. 183, 6852–6861. Google Scholar
  28. Baron, C., Llosa, M., Zhou, S., and Zambryski, P.C. ( 1997). VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1. J. Bacteriol. 179, 1203–1210. Google Scholar
  29. Bartholmes, C., Nutt, P., and Theiβen, G. ( 2008). Germline transformation of Shepherd's purse (Capsella bursa-pastoris) by the ‘floral dip’ method as a tool for evolutionary and developmental biology. Gene 409, 11–19. Google Scholar
  30. Barton, K.A., Binns, A.N., Matzke, A.J.M., and Chilton, M.-D. ( 1983). Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell 32, 1033–1043. Google Scholar
  31. Beale, M., Dupree, P., Lilley, K., Beynon, J., Trick, M., Clarke, J., Bevan, M., Bancroft, I., Jones, J., May, S., van de Sande, K., and Leyser, O. ( 2002). GARNet, the genomic Arabidopsis resource network. Trends Plant Sci. 7, 145–147. Google Scholar
  32. Beaty, J.S., Powell, G.K., Lica, L., Regier, D.A., MacDonald, E.M.S., Hommes, N.G., and Morris, R.O. ( 1986). Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis. Mol. Gen. Genet. 203, 274–280. Google Scholar
  33. Bechtold, N., and Pelletier, G. ( 1998). In planta Agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266. Google Scholar
  34. Bechtold, N., Ellis, J., and Pelletier, G. ( 1993). In-planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sci. 316, 1194–1199. Google Scholar
  35. Bechtold, N., Jaudeau, B., Jolivet, S., Maba, B., Vezon, D., Voisin, R., and Pelletier, G. ( 2000). The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana. Genetics 155, 1875–1887. Google Scholar
  36. Bechtold, N., Jolivet, S., Voisin, R., and Pelletier, G. ( 2003). The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens. Transgenic Res. 12, 509–517. Google Scholar
  37. Bent, A.F. ( 2000). Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiol. 124, 1540–1547. Google Scholar
  38. Bent, A.F. ( 2006). Arabidopsis thaliana floral dip transformation method. Methods Mol. Biol. 343, 87–104. Google Scholar
  39. Berger, B.R., and Christie, P.J. ( 1993). The Agrobacterium tumefaciens VirB4 gene-product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J. Bacteriol. 175, 1723–1734. Google Scholar
  40. Bevan, M. ( 1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711–8721. Google Scholar
  41. Bhattacharjee, S., Lee, L.Y., Oltmanns, H., Cao, H., Veena, Cuperus, J., and Gelvin, S.B. ( 2008). Atlmpa-4, an Arabidopsis importin α isoform, is preferentially involved in Agrobacterium-mediated plant transformation. Plant Cell 20, 2661–2680. Google Scholar
  42. Bhattacharya, A., Sood, P., and Cltovsky, V. ( 2010). The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection. Mol. Plant Pathol. 11,705–719. Google Scholar
  43. Bhatty, M., Laverde Gomez, J.A., and Christie, P.J. ( 2013). The expanding bacterial type IV secretion lexicon. Res. Microbiol. 164, 620–639. Google Scholar
  44. Bilichak, A., Yao, Y.L., and Kovalchuk, I. ( 2014). Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis. Plant Biotechnol. J. 12, 590–600. Google Scholar
  45. Bravo-Angel, A.M., Hohn, B., and Tinland, B. ( 1998). The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens. Mol. Plant Microbe Interact. 11, 57–63. Google Scholar
  46. Brencic, A., Eberhard, A., and Winans, S.C. ( 2003). Signal quenching, detoxification and mineralization of vir gene-inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens. Mol. Microbiol. 51, 1103– 1115. Google Scholar
  47. Broothaerts, W., Mitchell, H.J., Weir, B., Karnes, S., Smith, L.M.A., Yang, W., Mayer, J.E., Roa-Rodríguez, C., and Jefferson, R.A. ( 2005). Gene transfer to plants by diverse species of bacteria. Nature 433, 629–633. Google Scholar
  48. Bulgakov, V.P., Kiselev, K.V., Yakovlev, K.V., Zhuravlev, Y.N., Gontcharov, A.A., and Odintsova, N.A. ( 2006). Agrobacterium-mediated transformation of sea urchin embryos. Biotechnol. J. 1, 454–461. Google Scholar
  49. Bundock, P., den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P.J.J. ( 1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14, 3206–3214. Google Scholar
  50. Bundock, P., Mroczek, K., Winkler, A.A., Steensma, H.Y., and Hooykaas, P.J.J. ( 1999). T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis. Mol. Gen. Genet. 261, 115–121. Google Scholar
  51. Burch-Smith, T.M., Schiff, M., Liu, Y., and Dinesh-Kumar, S.P. ( 2006). Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142, 21–27. Google Scholar
  52. Cameron, T.A., Roper, M., and Zambryski, P.C. ( 2012). Quantitative image analysis and modeling indicate the Agrobacterium tumefaciens type IV secretion system is organized in a periodic pattern of foci. PLoS ONE 7, e42219. Google Scholar
  53. Campanoni, P., Sutter, J.U., Davis, C.S., Littlejohn, G.R., and Blatt, M.R. ( 2007). A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs. Plant J. 51, 322–330. Google Scholar
  54. Cangelosi, G.A., Martinetti, G., Leigh, J.A., Lee, C.C., Theines, C., and Nester, E.W. ( 1989). Role for Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan. J. Bacteriol. 171, 1609–1615. Google Scholar
  55. Cascales, E., and Christie, P.J. ( 2004a). Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304, 1170–1173. Google Scholar
  56. Cascales, E., and Christie, P.J. ( 2004b). Agrobacterium VirB10, an ATP energy sensor required for type IV secretion. Proc. Natl. Acad. Sci. USA 101, 17228–17233. Google Scholar
  57. Cascales, E., Atmakuri, K., Sarkar, M.K., and Christie, P.J. ( 2013). DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system. J. Bacteriol. 195, 2691–2704. Google Scholar
  58. Chandran, V. ( 2013). Type IV secretion machinery: molecular architecture and function. Biochem. Soc. Trans. 41, 17–28. Google Scholar
  59. Chandran, V., Fronzes, R., Duquerroy, S., Cronin, N., Navaza, J., and Waksman, G. ( 2009). Structure of the outer membrane complex of a type IV secretion system. Nature 462, 1011–1015. Google Scholar
  60. Chang, C.H., and Winans, S.C. ( 1996). Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene. J. Bacteriol. 178, 4717–4720. Google Scholar
  61. Chang, S.S., Park, S.K., Kim, B.C., Kang, B.J., Kim, D.U., and Nam, H.G. ( 1994). Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta. Plant J. 5, 551–558. Google Scholar
  62. Chateau, S. ( 2000). Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J. Exp. Bot. 51, 1961–1968. Google Scholar
  63. Chen, Z.H., Grefen, C., Donald, N., Hills, A., and Blatt, M.R. ( 2011). A bicistronic, Ubiquitin-10 promoter-based vector cassette for transient transformation and functional analysis of membrane transport demonstrates the utility of quantitative voltage clamp studies on intact Arabidopsis root epidermis. Plant Cell Environ. 34, 554–564. Google Scholar
  64. Christie, P.J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., and Cascales, E. ( 2005). Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol. 59, 451–485. Google Scholar
  65. Christie, P.J., Ward, J.E., Gordon, M.P., and Nester, E.W. ( 1989). A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc. Natl. Acad. Sci. USA 86, 9677–9681. Google Scholar
  66. Christie, P.J., Whitaker, N., and Gonzalez-Rivera, C. ( 2014). Mechanism and structure of the bacterial type IV secretion systems. Biochim. Biophys. Acta 1843, 1578–1591. Google Scholar
  67. Chung, M.H., Chen, M.K., and Pan, S.M. ( 2000). Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res. 9, 471–476. Google Scholar
  68. Chung, S.-M., Frankman, E.L., and Tzfira, T. ( 2005). A versatile vector system for multiple gene expression in plants. Trends Plant Sci. 10, 357–361. Google Scholar
  69. Citovsky, V., Kapelnikov, A., Oliel, S., Zakai, N., Rojas, M.R., Gilbertson, R.L., Tzfira, T., and Loyter, A. ( 2004). Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro. J. Biol. Chem. 279, 29528–29533. Google Scholar
  70. Citovsky, V., Kozlovsky, S.V., Lacroix, B., Zaltsman, A., Dafny-Yelin, M., Vyas, S., Tovkach, A., and Tzfira, T. ( 2007). Biological systems of the host cell involved in Agrobacterium infection. Cell Microbiol. 9, 9–20. Google Scholar
  71. Clarke, M.C., Wei, W., and Lindsey, K. ( 1992). High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciens. Plant Mol. Biol. Reporter 10, 178–189. Google Scholar
  72. Clough, S.J., and Bent, A.F. ( 1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. Google Scholar
  73. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., and Zhang, F. ( 2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819– 823. Google Scholar
  74. Crane, Y.M., and Gelvin, S.B. ( 2007). RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation. Proc. Natl. Acad. Sci. USA 104, 15156–15161. Google Scholar
  75. Curtis, I.S., and Nam, H.G. ( 2001). Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method - plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res. 10, 363–371. Google Scholar
  76. Czako, M.I., Wilson, J., Yu, X., and Morton, L.s. ( 1993). Sustained root culture for generation and vegetative propagation of transgenic Arabidopsis thaliana. Plant Cell Rep. 12, 603–606. Google Scholar
  77. De Buck, S., De Wilde, C., Van Montagu, M., and Depicker, A. ( 2000). Determination of the T-DNAtransfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root expiants. Mol. Plant Microbe Interact. 13, 658–665. Google Scholar
  78. De Buck, S., Jacobs, A., Van Montagu, M., and Depicker, A. ( 1998). Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root expiants and tobacco protoplasts. Mol. Plant Microbe Interact. 11, 449–457. Google Scholar
  79. De Buck, S., Podevin, N., Nolf, J., Jacobs, A., and Depicker, A. ( 2009). The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Plant J. 60, 134–145. Google Scholar
  80. De Cleene, M., and De Ley, J. ( 1976). The host range of crown gall. Bot. Rev. 42, 389–466. Google Scholar
  81. de Framond, A.J., Barton, K.A., and Chilton, M.-D. ( 1983). Mini—Ti: A new vector strategy for plant genetic engineering. Nat. Biotechnol. 1, 262–269. Google Scholar
  82. de Groot, M.J.A., Bundock, P., Hooykaas, P.J.J., and Beijersbergen, A.G.M. ( 1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 16, 839–842. Google Scholar
  83. de la Peña, A., Lörz, H., and Schell, J. ( 1987). Transgenic rye plants obtained by injecting DNA into young floral tillers. Nature 325, 274–276. Google Scholar
  84. Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., and Leemans, J. ( 1985). Efficient octopine Ti plasmidderived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 13, 4777–4788. Google Scholar
  85. DeNeve, M., DeBuck, S., Jacobs, A., VanMontagu, M., and Depicker, A. ( 1997). T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J. 11, 15–29. Google Scholar
  86. Deng, W., Chen, L., Wood, D.W., Metcalfe, T., Liang, X., Gordon, M.P., Comai, L., and Nester, E.W. ( 1998). Agrobacterium VirD2 protein interacts with plant host cyclophilins. Proc. Natl. Acad. Sci. USA 95, 7040–7045. Google Scholar
  87. Desfeux, C., Clough, S.J., and Bent, A.F. ( 2000). Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123, 895–904. Google Scholar
  88. Dessaux, Y., Petit, A., and Tempe, J. ( 1992). Opines in Agrobacterium biology. In Molecular Signals in Plant-Microbe Communications. (Boca Raton: CRC Press), pp. 109–136. Google Scholar
  89. Ditt, R.F., Kerr, K.F., de Figueiredo, P., Delrow, J., Comai, L., and Nester, E.W. ( 2006). The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens. Mol. Plant Microbe Interact. 19, 665–681. Google Scholar
  90. Djamei, A., Pitzschke, A., Nakagami, H., Rajh, I., and Hirt, H. ( 2007). Trojan horse strategy in Agrobacterium transformation: Abusing MARK defense signaling. Science 318, 453–456. Google Scholar
  91. Doty, S.L., Yu, M.C., Lundin, J.I., Heath, J.D., and Nester, E.W. ( 1996). Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens. J. Bacteriol. 178, 961–970. Google Scholar
  92. Douglas, C., Halperin, W., Gordon, M., and Nester, E. ( 1985). Specific attachment of Agrobacterium tumefaciens to bamboo cells in suspension cultures. J. Bacteriol. 161, 764–766. Google Scholar
  93. Douglas, C.J., Halperin, W., and Nester, E.W. ( 1982). Agrobacterium tumefaciens mutants affected in attachment to plant cells. J. Bacteriol. 152, 1265–1275. Google Scholar
  94. Durand, E., Oomen, C., and Waksman, G. ( 2010). Biochemical dissection of the ATPase TraB, the VirB4 homologue of the Escherichia coli pKM101 conjugation machinery. J. Bacteriol. 192, 2315–2323. Google Scholar
  95. Durand, E., Waksman, G., and Receveur-Brechot, V. ( 2011). Structural insights into the membrane-extracted dimeric form of the ATPase TraB from the Escherichia coli pKM101 conjugation system. BMC Struct. Biol. 11, 4. Google Scholar
  96. Durrenberger, F., Crameri, A., Hohn, B., and Koukolikova-Nicola, Z. ( 1989). Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc. Natl. Acad. Sci. USA 86, 9154–9158. Google Scholar
  97. Eisenbrandt, R., Kalkum, M., Lai, E.M., Lurz, R., Kado, C.I., and Lanka, E. ( 1999). Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. J. Biol. Chem. 274, 22548–22555. Google Scholar
  98. Endo, M., Ishikawa, Y., Osakabe, K., Nakayama, S., Kaya, H., Araki, T., Shibahara, K.-i., Abe, K., Ichikawa, H., Valentine, L., Hohn, B., and Toki, S. ( 2006). Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants. EMBO J. 25, 5579–5590. Google Scholar
  99. Enríquez-Obregón, G.A., Vázquez-Padrón, R.I., Prieto-Samsonov, D.L., De la Riva, G.A., and Selman-Housein, G. ( 1998). Herbicide-resistant sugarcane (Saccharum officinarum L.) plants by Agrobacteriummediated transformation. Planta 206, 20–27. Google Scholar
  100. Feldmann, K.A., and Marks, M.D. ( 1987). Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A nontissue culture approach. Mol. Gen. Genet. 208, 1–9. Google Scholar
  101. Filichkin, S.A., and Gelvin, S.B. ( 1993). Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease. Mol. Microbiol. 8, 915–926. Google Scholar
  102. Frandsen, R.J. ( 2011). A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciensmediated transformation. J. Microbiol. Methods 87, 247–262. Google Scholar
  103. Friesner, J., and Britt, A.B. ( 2003). Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J. 34, 427–440. Google Scholar
  104. Fromm, M., Taylor, L.P., and Walbot, V. ( 1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82, 5824–5828. Google Scholar
  105. Fromm, M., Taylor, L.P., and Walbot, V. ( 1986). Stable transformation of maize after gene transfer by electroporation. Nature 319, 791–793. Google Scholar
  106. Fronzes, R., Christie, P.J., and Waksman, G. ( 2009a). The structural biology of type IV secretion systems. Nat. Rev. Microbiol. 7, 703–714. Google Scholar
  107. Fronzes, R., Schafer, E., Wang, L., Saibil, H.R., Orlova, E.V., and Waksman, G. ( 2009b). Structure of a type IV secretion system core complex. Science 323, 266–268. Google Scholar
  108. Fullner, K.J., and Nester, E.W. ( 1996). Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J. Bacteriol. 178, 1498–1504. Google Scholar
  109. Fullner, K.J., Stephens, K.M., and Nester, E.W. ( 1994). An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. Mol. Gen. Genet. 245, 704–715. Google Scholar
  110. Gallego, M.E., Bleuyard, J.Y., Daoudal-Cotterell, S., Jallut, N., and White, C.l. ( 2003). Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis. Plant J. 35, 557–565. Google Scholar
  111. Gao, C., Long, D., Lenk, I., and Nielsen, K.K. ( 2008). Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment. Plant Cell Rep. 27, 1601–1609. Google Scholar
  112. Gao, R., and Lynn, D.G. ( 2005). Environmental pH sensing: resolving the VirA/VirG two-component system inputs for Agrobacterium pathogenesis. J. Bacteriol. 187, 2182–2189. Google Scholar
  113. Garcia-Hernandez, M., Berardini, T.Z., Chen, G., Crist, D., Doyle, A., Huala, E., Knee, E., Lambrecht, M., Miller, N., Mueller, L.A., Mundodi, S., Reiser, L., Rhee, S.Y., Scholl, R., Tacklind, J., Weems, D.C., Wu, Y., Xu, I., Yoo, D., Yoon, J., and Zhang, P. ( 2002). TAIR: a resource for integrated Arabidopsis data. Funct. Integr. Genomics 2, 239–253. Google Scholar
  114. Garza, I., and Christie, P.J. ( 2013). A putative transmembrane leucine zipper of Agrobacterium VirB10 is essential for T-Pilus biogenesis but not type IV secretion. J. Bacteriol. 195, 3022–3034. Google Scholar
  115. Gaspar, Y.M., Nam, J., Schultz, C.J., Lee, L.Y., Gilson, PR., and Gelvin, S.B. ( 2004). Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium Transformation. Plant Physiol. 135, 2162– 2171. Google Scholar
  116. Gelvin, S.B. ( 2006). Agrobacterium transformation of Arabidopsis thaliana roots. Methods Mol. Biol. 343, 105–114. Google Scholar
  117. Gelvin, S.B. ( 2010a). Finding a way to the nucleus. Curr. Opin. Microbiol. 13, 53–58. Google Scholar
  118. Gelvin, S.B. ( 2010b). Plant proteins involved in Agrobacterium-mediated genetic transformation. Annu. Rev. Phytopathol. 48, 45–68. Google Scholar
  119. Gelvin, S.B. (2012). Traversing the Cell: Agrobacterium T-DNA's journey to the host genome. Front. Plant Sci. 3, 52. Google Scholar
  120. Gelvin, S.B., and Kim, S.l. ( 2007). Effect of chromatin upon Agrobacterium T-DNA integration and transgene expression. Biochim. Biophys. Acta 1769, 410'421. Google Scholar
  121. Gheysen, G., Angenon, G., and Van Montagu, M. ( 1998). Agrobacterium-mediated plant transformation: a scientifically intriguing story with significant applications. In Transgenic Plant Research, K., Lindsey, ed, (Harwood Academic Publishers), pp. 1'33. Google Scholar
  122. Gohlke, J., and Deeken, R. (2014). Plant responses to Agrobacterium tumefaciens and crown gall development. Front. Plant Sei. 5, 155. Google Scholar
  123. Grevelding, C., Fantes, V., Kemper, E., Schell, J., and Masterson, R. ( 1993). Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics, whereas multiple insertions are found in leaf discs. Plant Mol. Biol. 23, 847–860. Google Scholar
  124. Guo, M., Hou, Q., Hew, C.L., and Pan, S.Q. ( 2007a). Agrobacterium VirD2-binding protein is involved in tumorigenesis and redundantly encoded in conjugative transfer gene clusters. Mol. Plant Microbe Interact. 20, 1201–1212. Google Scholar
  125. Guo, M., Jin, S., Sun, D., Hew, C.L., and Pan, S.Q. ( 2007b). Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus. Proc. Natl. Acad. Sci. USA 104, 20019–20024. Google Scholar
  126. Hamilton, R.H., and Fall, M.Z. ( 1971). The loss of tumor-initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Experientia 27, 229–230. Google Scholar
  127. Hansen, G., Shillito, R.D., and Chilton, M.D. ( 1997). T-strand integration in maize protoplasts after codelivery of a T-DNAsubstrate and virulence genes. Proc. Natl. Acad. Sci. USA 94, 11726–11730. Google Scholar
  128. Hauck, P., Thilmony, R., and He, S.Y. ( 2003). A Pseudomonas syringae type III effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants. Proc. Natl. Acad. Sci. USA 100, 8577– 8582. Google Scholar
  129. Heckel, B.C., Tomlinson, A.D., Morton, E.R., Choi, J.H., and Fuqua, C. ( 2014). Agrobacterium tumefaciens ExoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression. J. Bacteriol. 196, 3221–3233. Google Scholar
  130. Heindl, J.E., Wang, Y., Heckel, B.C., Mohari, B., Feirer, N., and Fuqua, C. ( 2014). Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium. Front. Plant Sci. 5, 176. Google Scholar
  131. Hellens, R., Mullineaux, P., and Klee, H. ( 2000). Technical focus: Aguide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451. Google Scholar
  132. Hepburn, A.G., White, J., Pearson, L., Maunders, M.J., Clarke, L.E., Prescott, A.G., and Blundy, K.S. ( 1985). The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Tiplasmids. J. Gen. Microbiol. 131, 2961–2969. Google Scholar
  133. Herrera-Estrella, A., Chen, Z.M., Van Montagu, M., and Wang, K. ( 1988). VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA-protein complex at the 5′ terminus of T-strand molecules. EMBO J. 7, 4055–4062. Google Scholar
  134. Herrera-Estrella, L., Simpson, J., and Martinez-Trujillo, M. ( 2005). Transgenic plants: an historical perspective. Methods Mol. Biol. 286: 3–32. Google Scholar
  135. Hilson, P., Allemeersch, J., Altmann, T., Aubourg, S., Avon, A., Beynon, J., Bhalerao, R.P., Bitton, F., Caboche, M., Cannoot, B., Chardakov, V., Cognet-Holliger, C., Colot, V., Crowe, M., Darimont, C., Durinck, S., Eickhoff, H., de Longevialle, A.F., Farmer, E.E., Grant, M., Kuiper, M.T., Lehrach, H., Leon, C., Leyva, A., Lundeberg, J., Lurin, C., Moreau, Y., Nietfeld, W., Paz-Ares, J., Reymond, P., Rouze, P., Sandberg, G., Segura, M.D., Serizet, C., Tabrett, A., Taconnat, L., Thareau, V., Van Hummelen, P., Vercruysse, S., Vuylsteke, M., Weingartner, M., Weisbeek, P.J., Wirta, V., Wittink, F.R., Zabeau, M., and Small, I. ( 2004). Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genome Res 14, 2176–2189. Google Scholar
  136. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., and Schilperoort, R.A. ( 1983). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303, 179–180. Google Scholar
  137. Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M., Dhaese, P., Depicker, A., Inzé, D., Engler, G., Villarroel, R., Van Montagu, M., and Schell, J. ( 1980). The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3, 212–230. Google Scholar
  138. Hood, E.E., Gelvin, S.B., Melchers, L.S., and Hoekema, A. ( 1993). New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218. Google Scholar
  139. Hood, E.E., Helmer, G.L., Fraley, R.T., and Chilton, M.D. ( 1986). The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–1301. Google Scholar
  140. Hooykaas, P.J.J. ( 2004). Transformation mediated by Agrobacterium tumefaciens. In Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, J.S. Tkacz and L. Lange , eds, (Boston, MA: Springer US), pp. 41–65. Google Scholar
  141. Howard, E.A., Winsor, B.A., De Vos, G., and Zambryski, P. ( 1989). Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5′ ends of T-strands. Proc. Natl. Acad. Sci. USA 86, 4017–4021. Google Scholar
  142. Hu, X., Zhao, J., DeGrado, W.F., and Binns, A.N. ( 2012). Agrobacterium tumefaciens recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals. Proc. Natl. Acad. Sci. USA 110, 678–683. Google Scholar
  143. Huala, E., Dickerman, A.W., Garcia-Hernandez, M., Weems, D., Reiser, L., LaFond, F., Hanley, D., Kiphart, D., Zhuang, M., Huang, W., Mueller, L.A., Bhattacharyya, D., Bhaya, D., Sobral, B.W., Beavis, W., Meinke, D.W., Town, C.D., Somerville, C., and Rhee, S.Y. ( 2001). The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant. Nucleic Acids Res. 29, 102–105. Google Scholar
  144. Huang, H., and Mā, H. ( 1992). An improved procedure for transforming Arabidopsis thaliana (Landsbergerecta) root explant. Plant Mol. Biol. Reporter 10, 372–383. Google Scholar
  145. Hwang, H.H., and Gelvin, S.B. ( 2004). Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell 16, 3148–3167. Google Scholar
  146. Hwang, H.H., Liu, Y.T., Huang, S.C., Tung, C.Y., Huang, F.C., Tsai, Y.L., Cheng, T.F., and Lai, E.M. ( 2015). Overexpression of the HspL promotes Agrobacterium tumefaciens virulence in Arabidopsis under heat shock conditions. Phytopathology 105, 160–168. Google Scholar
  147. Hwang, H.H., Wang, M.H., Lee, Y.L., Tsai, Y.L., Li., Y.H., Yang, F.J., Liao, Y.C., Lin, S.K., and Lai, E.M. ( 2010). Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation. Mol. Plant Pathol. 11, 677–690. Google Scholar
  148. Hwang, H.H., Wu, E.T., Liu, S.Y., Chang, S.C., Tzeng, K.C., and Kado, C.l. ( 2013a). Characterization and host range of five tumorigenic Agrobacterium tumefaciens strains and possible application in plant transient transformation assays. Plant Pathol. 62, 1384–1397. Google Scholar
  149. Hwang, H.H., Yang, F.J., Cheng, T.F., Chen, Y.C., Lee, Y.L., Tsai, Y.L., and Lai, E.M. ( 2013b). The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens. Phytopathology 103, 888–899. Google Scholar
  150. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R.J., and Joung, J.K. ( 2013c). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotech. 31, 227–229. Google Scholar
  151. Inan, G. Zhang, Q., Li, P., Wang, Z., Cao, Z., Zhang, H., Zhang, C., Quist, T.M., Goodwin, S.M., Zhu, J., Shi, H., Damsz, B., Charbaji, T., Gong, Q., Ma, S., Fredricksen, M., Galbraith, D.W., Jenks, M.A., Rhodes, D., Hasegawa, P.M., Bohnert, H.J., Joly, R.J., Bressan, R.A., and Zhu, J.K. ( 2004). Salt Cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol. 135, 1718–1737. Google Scholar
  152. Jakubowski, S.J., Cascales, E., Krishnamoorthy, V., and Christie, P.J. ( 2005). Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-Pilus biogenesis. J. Bacteriol. 187, 3486–3495. Google Scholar
  153. Jakubowski, S.J., Kerr, J.E., Garza, I., Krishnamoorthy, V., Bayliss, R., Waksman, G., and Christie, P.J. ( 2009). Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis. Mol. Microbiol. 71, 779–794. Google Scholar
  154. Jakubowski, S.J., Krishnamoorthy, V., and Christie, P.J. ( 2003). Agrobacterium tumefaciens VirB6 protein articipates in formation of VirB7 and VirB9 complexes required for type IV secretion. J. Bacteriol. 185, 2867–2878. Google Scholar
  155. Jakubowski, S.J., Krishnamoorthy, V., Cascales, E., and Christie, P.J. ( 2004). Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system. J. Mol. Biol. 341, 961–977. Google Scholar
  156. Jayaswal, R.K., Veluthambi, K., Gelvin, S.B., and Slightom, J.L. ( 1987). Double-stranded cleavage of T-DNA and generation of singlestranded T-DNA molecules in Escherichia coli by a virD-encoded border-specific endonuclease from Agrobacterium tumefaciens. J. Bacterio I. 169, 5035–5045. Google Scholar
  157. Jia, Q., Bundock, P., Hooykaas, P.J.J., and de Pater, S. ( 2012). Agrobacterium tumefaciens T-DNA integration and gene targeting in Arabidopsis thaliana non-homologous end-joining mutants. J. Bot. 2012, 1–13. Google Scholar
  158. Jin, S., Roitsch, T., Ankenbauer, R.G., Gordon, M.P., and Nester, E.W. ( 1990a). The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation. J. Bacteriol. 172, 525–530. Google Scholar
  159. Jin, S.G., Prusti, R.K., Roitsch, T., Ankenbauer, R.G., and Nester, E.W. ( 1990b). Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J. Bacteriol. 172, 4945–4950. Google Scholar
  160. Jin, S.G., Roitsch, T., Christie, P.J., and Nester, E.W. ( 1990c). The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes. J. Bacteriol. 172, 531–537. Google Scholar
  161. Jones, H.D., Doherty, A., and Sparks, C.A. ( 2009). Transient transformation of plants. Methods Mol. Biol. 513, 131–152. Google Scholar
  162. Judd, P.K., Kumar, R.B., and Das, A. ( 2004). The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole. Mol. Microbiol. 55, 115–124. Google Scholar
  163. Judd, P.K., Kumar, R.B., and Das, A. ( 2005). Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus. Proc. Natl. Acad. Sci. USA 102, 11498–11503. Google Scholar
  164. Kalogeraki, V.S., Zhu, J., Eberhard, A., Madsen, E.L., and Winans, S.C. ( 1999). The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid. Mol. Microbiol. 34, 512–522. Google Scholar
  165. Kanemoto, R.H., Powell, A.T., Akiyoshi, D.E., Regier, D.A., Kerstetter, R.A., Nester, E.W., Hawes, M.C., and Gordon, M.P. ( 1989). Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens. J. Bacteriol. 171, 2506–2512. Google Scholar
  166. Katavic, V., Haughn, G.W., Reed, D., Martin, M., and Kunst, L. ( 1994). In planta transformation of Arabidopsis thaliana. Mol. Gen. Genet. 245, 363–370. Google Scholar
  167. Kelly, B.A., and Kado, C.l. ( 2002). Agrobacterium-mediated T-DNA transfer and integration into the chromosome of Streptomyces lividans. Mol. Plant Pathol. 3, 125–134. Google Scholar
  168. Kemper, E., Grevelding, C., Schell, J., and Masterson, R. ( 1992). Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance. Plant Cell Rep. 11, 118–121. Google Scholar
  169. Kerr, A. ( 1971). Acquisition of virulence by non-pathogenic isolates of Agrobacterium radiobacter. Physiol. Plant Pathol. 1, 241–246. Google Scholar
  170. Kerr, J.E., and Christie, P.J. ( 2010). Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 Pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system. J. Bacteriol. 192, 4923–4934. Google Scholar
  171. Kikkert, J.R., Vidal, J.R., and Reisch, B.l. ( 2004). Stable transformation of plant cells by particle bombardment/biolistics. Methods Mol. Biol. 286: 61–78. Google Scholar
  172. Kim, M.J., Baek, K., and Park, C.M. ( 2009). Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Rep. 28, 1159–1167. Google Scholar
  173. Kleinboelting, N., Huep, G., Kloetgen, A., Viehoever, P., and Weisshaar, B. ( 2012). GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database. Nucleic Acids Res. 40, D1211–D1215. Google Scholar
  174. Komari, T., Takakura, Y., Ueki, J., Kato, N., Ishida, Y., and Hiei, Y. ( 2006). Binary vectors and super-binary vectors. Methods Mol. Biol. 343: 15–41. Google Scholar
  175. Koncz, C., and Schell, J. ( 1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396. Google Scholar
  176. Koncz, C., Németh, K., Rédei, G.P., and Schell, J. ( 1994). Homology recognition during T-DNA integration into the plant genome. In Homologous Recombination and Gene Silencing in Plants (Springer Nature), pp. 167–189. Google Scholar
  177. Kovacs, L.s.G., and Pueppke, S.G. ( 1994). Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain. Mol. Gen. Genet. 242, 327–336. Google Scholar
  178. Krall, L., Wiedemann, U., Unsin, G., Weiss, S., Domke, N., and Baron, C. ( 2002). Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 99, 11405–11410. Google Scholar
  179. Kumar, R.B., and Das, A. ( 2001). Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8. J. Bacteriol. 183, 3636–3641. Google Scholar
  180. Kumar, R.B., Xie, Y.-H., and Das, A. ( 2002). Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore. Mol. Microbiol. 36, 608–617. Google Scholar
  181. Kumar, S.V., Misquitta, R.W., Reddy, V.S., Rao, B.J., and Rajam, M.V. ( 2004). Genetic transformation of the green alga—Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 166, 731–738. Google Scholar
  182. Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., and Citovsky, V. ( 2001). Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. USA 98, 1871–1876. Google Scholar
  183. Kwok, T., Zabler, D., Urman, S., Rohde, M., Hartig, R., Wessler, S., Misselwitz, R., Berger, J., Sewald, N., König, W., and Backert, S. ( 2007). Helicobacter exploits integrin for type IV secretion and kinase activation. Nature 449, 862–866. Google Scholar
  184. Lacroix, B., and Citovsky, V. ( 2011). Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant. PLoS ONE 6, e25578. Google Scholar
  185. Lacroix, B., and Citovsky, V. ( 2013). The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation. Int. J. Dev. Biol. 57, 467–481. Google Scholar
  186. Lacroix, B., and Citovsky, V. ( 2016). Afunctional bacterium-to-plant DNA transfer machinery of Rhizobium etli. PLoS Pathog. 12, e1005502. Google Scholar
  187. Lacroix, B., Loyter, A., and Citovsky, V. ( 2008). Association of the Agrobacterium T-DNA-protein complex with plant nucleosomes. Proc. Natl. Acad. Sci. USA 105, 15429–15434. Google Scholar
  188. Lacroix, B., Tzfira, T., Vainstein, A., and Citovsky, V. ( 2006). A case of promiscuity: Agrobacterium's endless hunt for new partners. Trends Genet. 22, 29–37. Google Scholar
  189. Lacroix, B.t., Vaidya, M., Tzfira, T., and Citovsky, V. ( 2005). The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. EMBO J. 24, 428–437. Google Scholar
  190. Lai, E.M., and Kado, C.l. ( 1998) Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. J. Bacteriol. 180, 2711–2717. Google Scholar
  191. Lai, E.M., and Kado, C.l. ( 2000). The T-pilus of Agrobacterium tumefaciens. Trends Microbiol. 8, 361–369. Google Scholar
  192. Lai, E.M., Chesnokova, O., Banta, L.M., and Kado, C.l. ( 2000). Genetic and environmental factors affecting T-Pilin export and T-Pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens. J. Bacteriol. 182, 3705–3716. Google Scholar
  193. Lai, E.M., Eisenbrandt, R., Kalkum, M., Lanka, E., and Kado, C.l. ( 2002). Biogenesis of T pili in Agrobacterium tumefaciens requires precise VirB2 propilin cleavage and cyclization. J. Bacteriol. 184, 327–330. Google Scholar
  194. Lai, E.M., Shih, H.-W., Wen, S.R., Cheng, M.W., Hwang, H.H., and Chiu, S.H. ( 2006). Proteomic analysis of Agrobacterium tumefaciens response to the vir gene inducer acetosyringone. Proteomics 6, 4130–4136. Google Scholar
  195. Lamesch, P., Berardini, T.Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., Muller, R., Dreher, K., Alexander, D.L., Garcia-Hernandez, M., Karthikeyan, A.S., Lee, C.H., Nelson, W.D., Ploetz, L., Singh, S., Wensel, A., and Huala, E. ( 2012). The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40, D1202–D1210. Google Scholar
  196. Lazo, G.R., Stein, P.A., and Ludwig, R.A. ( 1991). A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat. Biotechnol. 9, 963–967. Google Scholar
  197. Lee, C.W., Efetova, M., Engelmann, J.C., Krameil, R., Wasternack, C., Ludwig-Muller, J., Hedrich, R., and Deeken, R. ( 2009). Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana. Plant Cell 21, 2948–2962. Google Scholar
  198. Lee, K., Dudley, M.W., Hess, K.M., Lynn, D.G., Joerger, R.D., and Binns, A.N. ( 1992). Mechanism of activation of Agrobacterium virulence genes: identification of phenol-binding proteins. Proc. Natl. Acad. Sci. USA 89, 8666–8670. Google Scholar
  199. Lee, L.Y., and Gelvin, S.B. ( 2008). T-DNA binary vectors and systems. Plant Physiol. 146, 325–332. Google Scholar
  200. Lee, L.Y., Fang, M.J., Kuang, L.Y., and Gelvin, S.B. ( 2008). Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant Methods 4, 24. Google Scholar
  201. Lee, Y.W., Jin, S., Sim, W.S., and Nester, E.W. ( 1995). Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 92, 12245– 12249. Google Scholar
  202. Lee, Y.W., Jin, S., Sim, W.S., and Nester, E.W. ( 1996). The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic Inducers by the VirA protein. Gene 179, 83–88. Google Scholar
  203. Lenser, T., and Theißen, G. ( 2014). Floral dip transformation in Lepidium campestre. Bio-protocol 4: e1201. Google Scholar
  204. Li, D., Dreher, K., Knee, E., Brkljacic, J., Grotewold, E., Berardini, T.Z., Lamesch, P., Garcia-Hernandez, M., Reiser, L., and Huala, E. ( 2014a). Arabidopsis database and stock resources. Methods Mol. Biol. 1062, 65–96. Google Scholar
  205. Li, F., Alvarez-Martinez, C., Chen, Y., Choi, K.J., Yeo, H.J., and Christie, P.J. ( 2012). Enterococcus faecalis PrgJ, a VirB4-like ATPase, mediates pCF10 conjugative transfer through substrate binding. J. Bacteriol. 194, 4041–4051. Google Scholar
  206. Li, G., Brown, P.J.B., Tang, J.X., Xu, J., Quardokus, E.M., Fuqua, C., and Brun, Y.V. ( 2011). Surface contact stimulates the just-in-time deployment of bacterial adhesins. Mol. Microbiol. 83, 41–51. Google Scholar
  207. Li, J., Krichevsky, A., Vaidya, M., Tzfira, T., and Citovsky, V. ( 2005a). Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc. Natl. Acad. Sci. USA 102, 5733–5738. Google Scholar
  208. Li, J., Vaidya, M., White, C., Vainstein, A., Citovsky, V., and Tzfira, T. ( 2005b). Involvement of KU80 in T-DNA integration in plant cells. Proc. Natl. Acad. Sci. USA 102, 19231–19236. Google Scholar
  209. Li, J.F., Park, E., von Arnim, A.G., and Nebenfuhr, A. ( 2009). The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5, 6. Google Scholar
  210. Li, L., Jia, Y., Hou, Q., Charles, T.C., Nester, E.W., and Pan, S.Q. ( 2002). A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and Ti plasmid. Proc. Natl. Acad. Sci. USA 99, 12369–12374. Google Scholar
  211. Li, X., Yang, Q., Tu, H., Lim, Z., and Pan, S.Q. ( 2014b). Direct visualization of Agrobacterium -delivered VirE2 in recipient cells. Plant J. 77, 487–495. Google Scholar
  212. Li, X., and Pan, S.Q. ( 2017). Agrobacterium delivers VirE2 protein into host cells via clathrin-medlated endocytosis. Sci. Adv. 3, e1601528. Google Scholar
  213. Li, Y., Rosso, M.G., Viehoever, P., and Weisshaar, B. ( 2007). GABI-Kat Simple Search: an Arabidopsis thaliana T-DNA mutant database with detailed information for confirmed insertions. Nucleic Acids Res. 35, D874–D878. Google Scholar
  214. Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., and Geurts, R. ( 2004). RNA Interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J. Exp. Bot. 55, 983–992. Google Scholar
  215. Lin, B.C., and Kado, C.l. ( 1977). Studies on Agrobacterium tumefaciens. VII. a virulence Induced by temperature and ethidium bromide. Can. J. Microbiol. 23, 1554–1561. Google Scholar
  216. Lin, Y.-H., Gao, R., Binns, A.N., and Lynn, D.G. ( 2008). Capturing the VirA/VirG TCS of Agrobacterium tumefaciens. Adv. Exp. Med. Biol. 631, 161–177. Google Scholar
  217. Liu, F., Cao, M.Q., Yao, L., Li, Y., Robaglia, C., and Tourneur, C. ( 1998). In planta transformation of pakchoi (Brassica campestris L. ssp. Chinensis) by Infiltration of adult plants with Agrobacterium. Acta Hort. 467, 187–192. Google Scholar
  218. Liu, P., and Nester, E.W. ( 2006). Indoleacetic acid, a product of transferred DNA, inhibits vir gene expression and growth of Agrobacterium tumefaciens C58. Proc. Natl. Acad. Sci. U.S.A. 103, 4658–4662. Google Scholar
  219. Liu, P., Wood, D., and Nester, E.W. ( 2005). Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens. J. Bacteriol. 187, 6039–6045. Google Scholar
  220. Llosa, M., Zupan, J., Baron, C., and Zambryski, P. ( 2000). The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis. J. Bacteriol. 182, 3437–3445. Google Scholar
  221. Lloyd, A.M., Barnason, A.R., Rogers, S.G., Byrne, M.C., Fraley, R.T., and Horsch, R.B. ( 1986). Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science 234, 464–466. Google Scholar
  222. Lorence, A., and Verpoorte, R. ( 2004). Gene transfer and expression in plants. Methods Mol. Biol. 267, 329–350. Google Scholar
  223. Low, H.H., Gubellini, F., Rivera-Calzada, A., Braun, N., Connery, S., Dujeancourt, A., Lu, F., Redzej, A., Fronzes, R., Orlova, E.V., and Waksman, G. ( 2014). Structure of a type IV secretion system. Nature 508, 550–553. Google Scholar
  224. Loyter, A., Rosenbluh, J., Zakai, N., Li, J., Kozlovsky, S.V., Tzfira, T., and Citovsky, V. ( 2005). The plant VirE2 Interacting Protein 1. A molecular link between the Agrobacterium T-Complex and the host cell chromatin? Plant Physiol. 138, 1318–1321. Google Scholar
  225. Lu, B., Wang, J., Zhang, Y., Wang, H.C., Liang, J.S., and Zhang, J.H. ( 2012). AT14A mediates the cell wall-plasma membrane-cytoskeleton continuum in Arabidopsis thaliana cells. J. Exp. Bot. 63, 4061–4069. Google Scholar
  226. Ma, L.S., Hachani, A., Lin, J.S., Filloux, A., and Lai, E.M. ( 2014). Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16, 94–104. Google Scholar
  227. Magori, S., and Citovsky, V. ( 2011). Agrobacterium counteracts hostinduced degradation of Its effector F-Box protein. Sci. Signal. 4, ra69. Google Scholar
  228. Magori, S., and Citovsky, V. ( 2012). The role of the ubiquitin-proteasome system in Agrobacterium tumefaciens-mediated genetic transformation of plants. Plant Physiol. 160, 65–71. Google Scholar
  229. Mangano, S., Gonzalez, C.D., and Petruccelli, S. ( 2014). Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves. Methods Mol. Biol. 1062, 165–173. Google Scholar
  230. Mantis, N.J., and Winans, S.C. ( 1992). The Agrobacterium tumefaciens vir gene transcriptional activator virG is transcriptionally induced by acid pH and other stress stimuli. J. Bacteriol. 174, 1189–1196. Google Scholar
  231. Maresh, J., Zhang, J., and Lynn, D.G. ( 2006). The innate immunity of maize and the dynamic chemical strategies regulating two-component signal transduction in Agrobacterium tumefaciens. ACS Chem. Biol. 1, 165–175. Google Scholar
  232. Marion, J., Bach, L., Bellec, Y., Meyer, C., Gissot, L., and Faure, J.D. ( 2008). Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J. 56, 169–179. Google Scholar
  233. Martinez-Trujillo, M., Limones-Briones, V., Cabrera-Ponce, J.L., and Herrera-Estrella, L. ( 2004). Improving transformation efficiency of Arabidopsis thaliana by modifying the floral dip method. Plant Mol. Biol. Rep. 22, 63–70. Google Scholar
  234. Mateos-Gomez, P.A., Gong, F., Nair, N., Miller, K.M., Lazzerini-Denchi, E., and Sfeir, A. ( 2015). Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257. Google Scholar
  235. Matthysse, A.G. ( 1983). Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection. J. Bacteriol. 154, 906–915. Google Scholar
  236. Matthysse, A.G. ( 1987). Characterization of nonattaching mutants of Agrobacterium tumefaciens. J.Bacteriol. 169, 313–323. Google Scholar
  237. Matthysse, A.G., Holmes, K.V., and Gurlitz, R.H. ( 1981). Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells. J. Bacteriol. 145, 583–595. Google Scholar
  238. Matthysse, A.G., Marry, M., Krall, L., Kaye, M., Ramey, B.E., Fuqua, C., and White, A.R. ( 2005). The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens. Mol. Plant Microbe. Interact. 18, 1002–1010. Google Scholar
  239. McBride, K.E., and Summerfelt, K.R. ( 1990). Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 14, 269–276. Google Scholar
  240. McCullen, C.A., and Binns, A.N. ( 2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macro molecular transfer. Annu. Rev. Cell Dev. Biol. 22, 101–127. Google Scholar
  241. McHale, M., Eamens, A.L., Finnegan, E.J., and Waterhouse, P.M. ( 2013). A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis. Plant J. 76, 519–529. Google Scholar
  242. Mehrotra, S., and Goyal, V. ( 2012). Agrobacterium-mediated gene transfer in plants and biosafety considerations. Appi. Biochem. Biotechnol. 168, 1953–1975. Google Scholar
  243. Melchers, L.S., Maroney, M.J., den Dulk-Ras, A., Thompson, D.V., van Vuuren, H.A.J., Schilperoort, R.A., and Hooykaas, P.J.J. ( 1990). Octopine and nopaline strains of Agrobacterium tumefaciens differ in virulence; molecular characterization of the virF locus. Plant Mol Biol 14, 249–259. Google Scholar
  244. Mestiri, I., Norre, F., Gallego, M.E., and White, C.l. ( 2014). Multiple hostcell recombination pathways act in Agrobacterium-mediated transformation of plant cells. Plant J. 77, 511–520. Google Scholar
  245. Michielse, C.B., Hooykaas, P.J.J., van den Hondel, C.A., and Ram, A.F. ( 2005). Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr. Genet. 48, 1–17. Google Scholar
  246. Michielse, C.B., Ram, A.F., Hooykaas, P.J.J., and van den Hondel, C.A. ( 2004). Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures. J. Bacteriol. 186, 2038–2045. Google Scholar
  247. Mushegian, A.R., Fullner, K.J., Koonin, E.V., and Nester, E.W. ( 1996). A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals. Proc. Natl. Acad. Sci. USA. 93, 7321–7326. Google Scholar
  248. Mysore, K.S., Bassuner, B., Deng, X.B., Darbinian, N.S., Motchoulski, A., Ream, W., and Gelvin, S.B. ( 1998). Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration. Mol. Plant Microbe Interact. 11, 668–683. Google Scholar
  249. Mysore, K.S., Kumar, C.T., and Gelvin, S.B. ( 2000a). Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation. Plant J. 21, 9–16. Google Scholar
  250. Mysore, K.S., Nam, J., and Gelvin, S.B. ( 2000b). An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration. Proc. Natl. Acad. Sci. USA 97, 948–953. Google Scholar
  251. Nam, J., Matthysse, A.G., and Gelvin, S.B. ( 1997). Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration. Plant Cell 9, 317–333. Google Scholar
  252. Nam, J., Mysore, K.S., and Gelvin, S.B. ( 1998). Agrobacterium tumefaciens transformation of the radiation hypersensitive Arabidopsis thaliana mutants uvh1 and rad5. Mol. Plant Microbe Interact. 11, 1136–1141. Google Scholar
  253. Nam, J., Mysore, K.S., Zheng, C., Knue, M.K., Matthysse, A.G., and Gelvin, S.B. ( 1999). Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium. Mol. Gen. Genet. 261, 429–438. Google Scholar
  254. Newell, C.A. ( 2000). Plant transformation technology: Developments and applications. Mol. Biotechnol. 16, 53–66. Google Scholar
  255. Nishizawa-Yokoi, A., Nonaka, S., Saika, H., Kwon, Y.-l., Osakabe, K., and Toki, S. ( 2012). Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice. New Phytol. 196, 1048–1059. Google Scholar
  256. Niu, X., Zhou, M., Henkel, C.V., van Heusden, G.P., and Hooykaas, P.J.J. ( 2015). The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF. Plant J. 84, 914– 924. Google Scholar
  257. Nonaka, S., Yuhashi, K.-l., Takada, K., Sugaware, M., Minamisawa, K., and Ezura, H. ( 2008). Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens. New Phytol. 178, 647–656. Google Scholar
  258. O'Connell, K.P., and Handelsman, J. ( 1989). chvA locus may be involved in export of neutral cyclic β-1,2-linked D-Glucan from Agrobacterium tumefaciens. Mol. Plant Microbe Interact. 2, 11–16. Google Scholar
  259. Ooms, G., Hooykaas, P.J.J., Van Veen, R.J.M., Van Beelen, P., Regensburg-Tuïnk, T.J.G., and Schilperoort, R.A. ( 1982). Octopine Tiplasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7, 15–29. Google Scholar
  260. Padavannil, A., Jobichen, C., Qinghua, Y., Seetharaman, J., VelazquezCampoy, A., Yang, L., Pan, S.Q., and Sivaraman, J. (2014). Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants. PLoS Pathog. 10: e1003948. Google Scholar
  261. Palanichelvam, K., Oger, P., Clough, S.J., Cha, C., Bent, A.F., and Farrand, S.K. ( 2000). A second T-Region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid. Mol. Plant Microbe Interact. 13, 1081–1091. Google Scholar
  262. Pan, X., Liu, H., Clarke, J., Jones, J., Bevan, M., and Stein, L. ( 2003). ATIDB: Arabidopsis thaliana insertion database. Nucleic Acids Res. 31, 1245–1251. Google Scholar
  263. Park, S.Y., Vaghchhipawala, Z., Vasudevan, B., Lee, L.-Y., Shen, Y., Singer, K., Waterworth, W.M., Zhang, Z.J., West, C.E., Mysore, K.S., and Gelvin, S.B. ( 2015). Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins. Plant J. 81, 934–946. Google Scholar
  264. Patil, B.L., and Fauquet, C.M. ( 2015). Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies. Mol. Plant Pathol. 16, 484–494. Google Scholar
  265. Paulsson, M., and Wadstrom, T. ( 1990). Vitronectin and type-l collagen binding by Staphylococcus aureus and coagulase-negative staphylococci. FEMS Microbiol. Immunol. 2, 55–62. Google Scholar
  266. Pawlowski, W.P., and Somers, D.A. ( 1996). Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotechnol. 6, 17–30. Google Scholar
  267. Pelczar, P., Kalck, V., Gomez, D., and Hohn, B. ( 2004). Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells. EMBO Rep. 5, 632–637. Google Scholar
  268. Pena, A., Matilla, I., Martin-Benito, J., Valpuesta, J.M., Carrascosa, J.L., de la Cruz, F., Cabezon, E., and Arechaga, I. ( 2012). The hexameric structure of a conjugative VirB4 protein ATPase provides new insights for a functional and phylogenetic relationship with DNA translocases. J. Biol. Chem. 287, 39925–39932. Google Scholar
  269. Piers, K.L., Heath, J.D., Liang, X., Stephens, K.M., and Nester, E.W. ( 1996). Agrobacterium tumefaciens-mediated transformation of yeast. Proc. Natl. Acad. Sci. USA 93, 1613–1618. Google Scholar
  270. Pitzschke, A. ( 2013). Agrobacterium infection and plant defense-transformation success hangs by a thread. Front. Plant Sci. 4, 519. Google Scholar
  271. Powell, G.K. Hommes, N.G., Kuo, J., Castle, L.A., and Morris, R.O. ( 1988). Inducible expression of cytokinin biosynthesis in Agrobacterium tumefaciens by plant phenolics. Mol. Plant Microbe Interact. 1, 235– 242. Google Scholar
  272. Regensburg-Tuïnk, A.J.G., and Hooykaas, P.J.J. ( 1993). Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens. Nature 363, 69–71. Google Scholar
  273. Rhee, S.Y., Beavis, W., Berardini, T.Z., Chen, G., Dixon, D., Doyle, A., Garcia-Hernandez, M., Huala, E., Lander, G., Montoya, M., Miller, N., Mueller, L.A., Mundodi, S., Reiser, L., Tacklind, J., Weems, D.C., Wu, Y., Xu, I., Yoo, D., Yoon, J., and Zhang, P. ( 2003). The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 31, 224–228. Google Scholar
  274. Richardson, K., Fowler, S., Pullen, C., Skelton, C., Morris, B., and Putterill, J. ( 1998). T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis. Aust. J. Plant Physiol. 25, 125–130. Google Scholar
  275. Rivero, L., Scholl, R., Holomuzki, N., Crist, D., Grotewold, E., and Brkljacic, J. ( 2014). Handling Arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses. Methods Mol. Biol. 1062, 3–25. Google Scholar
  276. Rosas-Díaz, T., Cana-Quijada, P., Amorim-Silva, V., Botella, M.A., Lozano-Durán, R., Bejarano, E.R. ( 2017). Arabidopsis NahG plants as a suitable and efficient system for transient expression during Agrobacterium tumefaciens. Mol. Plant 10: 353–356. Google Scholar
  277. Rossi, L., Hohn, B., and Tinland, B. ( 1996). Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. U.S.A. 93, 126–130. Google Scholar
  278. Sagulenko, E., Sagulenko, V., Chen, J., and Christie, P.J. ( 2001). Role of Agrobacterium VirB11 ATPase in T-Pilus assembly and substrate selection. J. Bacteriol. 183, 5813–5825. Google Scholar
  279. Sakalis, P.A., van Heusden, G.P.H., and Hooykaas, P.J.J. ( 2014). Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells. Microbiology 3, 104–117. Google Scholar
  280. Salman, H., Abu-Arish, A., Oliel, S., Loyter, A., Klafter, J., Granek, R., and Elbaum, M. ( 2005). Nuclear localization signal peptides induce molecular delivery along microtubules. Biophys. J. 89, 2134–2145. Google Scholar
  281. Sanford, J.C. ( 2000). The development of the biolistic process. In Vitro Cell. Dev. Biol.-Plant 36, 303–308. Google Scholar
  282. Sangwan, R., Bourgeois, Y., Brown, S., Vasseur, G.r., and Sangwan-Norreel, B. ( 1992). Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 188, 439–456. Google Scholar
  283. 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. Google Scholar
  284. Sardesai, N., Lee, L.Y., Chen, H., Yi, H., Olbricht, G.R., Stirnberg, A., Jeffries, J., Xiong, K., Doerge, R.W., and Gelvin, S.B. (2013). Cytokinins secreted by Agrobacterium promote transformation by repressing a plant Myb transcription factor. Sci. Signal. 6, ra100. Google Scholar
  285. Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P.C., and Baron, C. ( 1999). Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens. J. Bacteriol. 181, 7485–7492. Google Scholar
  286. Scholl, R.L., May, S.T., and Ware, D.H. ( 2000). Seed and molecular resources for Arabidopsis. Plant Physiol. 124, 1477–1480. Google Scholar
  287. Schrammeijer, B., den Dulk-Ras, A., Vergunst, A.C., Jurado Jácome, E., and Hooykaas, P.J.J. ( 2003). Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3. Nucleic Acids Res. 31, 860–868. Google Scholar
  288. Schrammeijer, B., Risseeuw, E., Pansegrau, W., Regensburg-Tuïnk, T.J.G., Crosby, W.L., and Hooykaas, P.J.J. ( 2001). Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein. Curr. Biol. 11, 258–262. Google Scholar
  289. Sheikholeslam, S.N., and Weeks, D.P. ( 1987). Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol. Biol. 8, 291–298. Google Scholar
  290. Shi, Y., Lee, L.Y., and Gelvin, S.B. ( 2014). Is VIP1 important for Agrobacterium -mediated transformation? Plant J. 79, 848–860. Google Scholar
  291. Shimoda, N., Toyoda-Yamamoto, A., Aoki, S., and Machida, Y. ( 1993). Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. J. Biol. Chem. 268, 26552–26558. Google Scholar
  292. Shou, H., Frame, B.R., Whitham, S.A., and Wang, K. ( 2004). Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol. Breed. 13, 201–208. Google Scholar
  293. Shurvinton, C.E., Hodges, L., and Ream, W. ( 1992). A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. Proc. Natl. Acad. Sci. USA 89, 11837–11841. Google Scholar
  294. Stachel, S.E., Messens, E., Van Montagu, 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. Google Scholar
  295. Stachel, S.E., Timmerman, B., and Zambryski, P. ( 1986). Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322, 706–712. Google Scholar
  296. Stachel, S.E., Timmerman, B., and Zambryski, P. ( 1987). Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5′ virD gene products. EMBO J. 6, 857–863. Google Scholar
  297. Subramoni, S., Nathoo, N., Klimov, E., and Yuan, Z.-C. ( 2014). Agrobacterium tumefaciens responses to plant-derived signaling molecules. Front. Plant Sci. 5, 322. Google Scholar
  298. Swarbreck, D., Wilks, C., Lamesch, P., Berardini, T.Z., Garcia-Hernandez, M., Foerster, H., Li, D., Meyer, T., Muller, R., Ploetz, L., Radenbaugh, A., Singh, S., Swing, V., Tissier, C., Zhang, P., and Huala, E. ( 2008). The Arabidopsis Information Resource (TAIR): gene structure and function annotation. Nucleic Acids Res. 36, D1009–D1014. Google Scholar
  299. Tague, B.W. ( 2001). Germ-line transformation of Arabidopsis lasiocarpa. Transgenic Res. 10, 259–267. Google Scholar
  300. Tague, B.W., and Mantis, J. ( 2006). In planta Agrobacterium mediated transformation by vacuum infiltration. Methods Mol. Biol. 323, 215–223. Google Scholar
  301. Tao, Y., Rao, P.K., Bhattacharjee, S., and Gelvin, S.B. ( 2004). Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2. Proc. Natl. Acad. Sci. USA 101, 5164–5169. Google Scholar
  302. Tenea, G.N., Spantzel, J., Lee, L.Y., Zhu, Y., Lin, K., Johnson, S.J., and Gelvin, S.B. ( 2009). Overexpression of several Arabidopsis histone genes increases Agrobacterium-mediated transformation and transgene expression in plants. Plant Cell 21, 3350–3367. Google Scholar
  303. Thomashow, M.F., Karlinsey, J.E., Marks, J.R., and Hurlbert, R.E. ( 1987). Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment. J. Bacteriol. 169, 3209–3216. Google Scholar
  304. Tinland, B., Schoumacher, F., Gloeckler, V., Bravoangel, A.M., and Hohn, B. ( 1995). The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J. 14, 3585–3595. Google Scholar
  305. Tomlinson, A.D., and Fuqua, C. ( 2009). Mechanisms and regulation of polar surface attachment in Agrobacterium tumefaciens. Curr. Opin. Microbiol. 12, 708–714. Google Scholar
  306. Travella, S., Ross, S.M., Harden, J., Everett, C., Snape, J.W., and Harwood, W.A. ( 2005). A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep. 23, 780–789. Google Scholar
  307. Trieu, A.T., Burleigh, S.H., Kardailsky, I.V., Maldonado-Mendoza, I.E., Versaw, W.K., Blaylock, L.A., Shin, H., Chiou, T.-J., Katagi, H., Dewbre, G.R., Weigel, D., and Harrison, M.J. ( 2000). Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 22, 531–541. Google Scholar
  308. Tsai, Y.L., Chiang, Y.R., Narberhaus, F., Baron, C., and Lai, E.M. ( 2010). The small heat-shock protein HspL is a VirB8 chaperone promoting type IV secretion-mediated DNA transfer. J. Biol. Chem. 285, 19757–19766. Google Scholar
  309. Tsai, Y.L., Wang, M.H., Gao, C., Klusener, S., Baron, C., Narberhaus, F., and Lai, E.M. ( 2009). Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens. Microbiology 155, 3270–3280. Google Scholar
  310. Tsuda, K., Qi, Y., Nguyen le, V., Bethke, G., Tsuda, Y., Glazebrook, J., and Katagiri, F. ( 2012). An efficient Agrobacterium-mediated transient transformation of Arabidopsis. Plant J 69, 713–719. Google Scholar
  311. Tzfira, T. ( 2006). On tracks and locomotives: the long route of DNA to the nucleus. Trends Microbiol. 14, 61–63. Google Scholar
  312. Tzfira, T., Li, J., Lacroix, B., and Citovsky, V. ( 2004a). Agrobacterium T-DNA integration: molecules and models. Trends Genet. 20, 375–383. Google Scholar
  313. Tzfira, T., Vaidya, M., and Citovsky, V. ( 2001). VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J. 20, 3596–3607. Google Scholar
  314. Tzfira, T., Vaidya, M., and Citovsky, V. ( 2002). Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1. Proc. Natl. Acad. Sci. USA 99, 10435-10440. Google Scholar
  315. Tzfira, T., Vaidya, M., and Citovsky, V. ( 2004b). Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431, 87–92. Google Scholar
  316. Uchimiya, H., Fushimi, T., Hashimoto, H., Harada, H., Syono, K., and Sugawara, Y. ( 1986). Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.). Mol. Gen. Genet. 204, 204–207. Google Scholar
  317. Vaghchhipawala, Z.E., Vasudevan, B., Lee, S., Morsy, M.R., and Mysore, K.S. ( 2012). Agrobacterium may delay plant nonhomologous end-joining DNA repair via XRCC4 to favor T-DNA integration. Plant Cell 24, 4110–4123. Google Scholar
  318. Valvekens, D., Montagu, M.V., and Van Lijsebettens, M. ( 1988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536–5540. Google Scholar
  319. van Attikum, H., Bundock, P., and Hooykaas, P.J.J. ( 2001). Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J. 20, 6550–6558. Google Scholar
  320. van Attikum, H., Bundock, P., Overmeer, R.M., Lee, L.Y., Gelvin, S.B., and Hooykaas, P.J.J. ( 2003). The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA. Nucleic Acids Res. 31, 4247–4255. Google Scholar
  321. van den Eede, G., Aarts, H.J., Buhk, H.J., Corthier, G., Flint, H.J., Hammes, W., Jacobsen, B., Midtvedt, T., van der Vossen, J., von Wright, A., Wackernagel, W., and Wilcks, A. ( 2004). The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants. Food Chem. Toxicol. 42, 1127–1156. Google Scholar
  322. van den Elzen, P.J.M., Townsend, J., Lee, K.Y., and Bedbrook, J.R. ( 1985). A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 5, 299–302. Google Scholar
  323. van Kregten, M., de Pater, S., Romeijn, R., van Schendel, R., Hooykaas, P.J.J., and Tijsterman, M. ( 2016). T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nat. Plants 2, 16164. Google Scholar
  324. Vergunst, A.C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C.M., Regensburg-Tuink, T.J., and Hooykaas, P.J.J. ( 2000). VirB/D4-Dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982. Google Scholar
  325. Vergunst, A.C., van Lier, C.M., den Dulk Ras, A., and Hooykaas, P.J.J. ( 2003). Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1. Plant Physiol. 133, 978–988. Google Scholar
  326. Vergunst, A.C., van Lier, M.C., den Dulk-Ras, A., Grosse Stuve, T.A., Ouwehand, A., and Hooykaas, P.J.J. ( 2005). Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc. Natl. Acad. Sci. USA 102, 832–837. Google Scholar
  327. Vergunst, A.C., Waal, E.C., and Hooykaas, P.J.J. (1998). Root transformation by Agrobacterium tumefaciens. Methods Mol. Biol., 227–244. Google Scholar
  328. Wagner, V.T., and Matthysse, A.G. ( 1992). Involvement of a vitronectinlike protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells. J. Bacteriol. 174, 5999–6003. Google Scholar
  329. Waksman, G., and Fronzes, R. ( 2010). Molecular architecture of bacterial type IV secretion systems. Trends Biochem. Sci. 35, 691–698. Google Scholar
  330. Wallden, K., Rivera-Calzada, A., and Waksman, G. ( 2010). Microreview: Type IV secretion systems: versatility and diversity in function. Cell. Microbiol. 12, 1203–1212. Google Scholar
  331. Wallden, K., Williams, R., Yan, J., Lian, P.W., Wang, L., Thalassinos, K., Orlova, E.V., and Waksman, G. ( 2012). Structure of the VirB4 ATPase, alone and bound to the core complex of a type IV secretion system. Proc. Natl. Acad. Sci. U.S.A. 109, 11348–11353. Google Scholar
  332. Wang, H., Yang, H., Shivalila, Chikdu S., Dawlaty, Meelad M., Cheng, Albert W., Zhang, F., and Jaenisch, R. ( 2013). One-Step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918. Google Scholar
  333. Wang, K., Stachel, S.E., Timmerman, B., Van Montagu, M., and Zambryski, P.C. ( 1987). Site-specific nick in the T-DNA border sequence as a result of Agrobacterium vir gene expression. Science 235, 587–591. Google Scholar
  334. Wang, W.C., Menon, G., and Hansen, G. ( 2003). Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants. Plant Cell Rep. 22, 274–281. Google Scholar
  335. Wang, Y., Peng, W., Zhou, X., Huang, F., Shao, L., and Luo, M. ( 2014). The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus. New Phytol. 203, 1266–1281. Google Scholar
  336. Ward, E.R., and Barnes, W.M. ( 1988). VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5′ end of T-strand DNA. Science 242, 927–930. Google Scholar
  337. Watson, B., Currier, T.C., Gordon, M.P., Chilton, M.D., and Nester, E.W. ( 1975). Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacteriol. 123, 255–264. Google Scholar
  338. Weaver, J., Goklany, S., Rizvi, N., Cram, E.J., and Lee-Parsons, C.W. ( 2014). Optimizing the transient fast Agro-mediated seedling transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep. 33, 89–97. Google Scholar
  339. Wendt, T., Doohan, F., and Mullins, E. ( 2012). Production of Phytophthora infestans-resistant potato (Solanum tuberosum) utilising Ensifer adhaerens OV14. Transgenic Res. 21, 567–578. Google Scholar
  340. Wroblewski, T., Tomczak, A., and Michelmore, R. ( 2005). Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259–273. Google Scholar
  341. Wu, C.F., Lin, J.S., Shaw, G.C., and Lai, E.M. (2012). Acid-induced type VI secretion system is regulated by ExoR-ChvG/Chvl signaling cascade in Agrobacterium tumefaciens. PLoS Pathog. 8: e1002938. Google Scholar
  342. Wu, H.Y., Chen, C.Y., and Lai, E.M. ( 2014a). Expression and functional characterization of the Agrobacterium VirB2 amino Acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency. PLoS ONE 9, e101142. Google Scholar
  343. Wu, H.Y., Chung, P.C., Shih, H.W., Wen, S.R., and Lai, E.M. ( 2008). Secretome analysis uncovers an Hcp-family protein secreted via a yype VI secretion system in Agrobacterium tumefaciens. J. Bacteriol. 190, 2841–2850. Google Scholar
  344. Wu, H.Y., Liu, K.H., Wang, Y.C., Wu, J.F., Chiu, W.L., Chen, C.Y., Wu, S.H., Sheen, J., and Lai, E.M. ( 2014b). AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10, 19. Google Scholar
  345. Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., Li, Y., Tang, X., Zhu, L., Chai, J., and Zhou, J.M. ( 2008). Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr. Biol. 18, 74–80. Google Scholar
  346. Xu, J., Kim, J., Danhorn, T., Merritt, P.M., and Fuqua, C. ( 2012). Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis. Res. Microbiol. 163, 674–684. Google Scholar
  347. Xu, J., Kim, J., Koestler, B.J., Choi, J.-H., Waters, C.M., and Fuqua, C. ( 2013). Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motileto-sessile switch. Mol. Microbiol. 89, 929–948. Google Scholar
  348. Yang, Q.H., Li, X.Y., Tu, H., and Pan, S.Q. ( 2017). Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K-powered ER/actin network. Proc. Natl. Acad. Sci. USA 114, 2982– 2987. Google Scholar
  349. Yanofsky, M.F., and Nester, E.W. ( 1986). Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens. J. Bacteriol. 168, 244–250. Google Scholar
  350. Ye, G.N., Stone, D., Pang, S.Z., Creely, W., Gonzalez, K., and Hinchee, M. ( 1999). Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J. 19, 249–257. Google Scholar
  351. Yi, H., Mysore, K.S., and Gelvin, S.B. ( 2002). Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. Plant J. 32, 285–298. Google Scholar
  352. Yi, H., Sardesai, N., Fujinuma, T., Chan, C.W., Veena, and Gelvin, S.B. ( 2006). Constitutive expression exposes functional redundancy between the Arabidopsis histone H2A gene HTA1 and other H2A gene family members. Plant Cell 18, 1575–1589. Google Scholar
  353. Young, C., and Nester, E.W. ( 1988). Association of the virD2 protein with the 5′ end of T strands in Agrobacterium tumefaciens. J. Bacteriol. 170, 3367–3374. Google Scholar
  354. Yuan, Z.C., Edlind, M.P., Liu, P., Saenkham, P., Banta, L.M., Wise, A.A., Ronzone, E., Binns, A.N., Kerr, K., and Nester, E.W. ( 2007b). The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium. Proc. Natl. Acad. Sci. U.S.A. 104, 11790–11795. Google Scholar
  355. Yuan, Z.C., Liu, P., Saenkham, P., Kerr, K., and Nester, E.W. ( 2007a). Transcriptome profiling and functional analysis of Agrobacterium tumefaciens reveals a general conserved response to acidic conditions (pH 5.5) and a complex acid-mediated signaling involved in Agrobacterium-Plant interactions. J. Bacteriol. 190, 494–507. Google Scholar
  356. Zale, J.M., Agarwal, S., Loar, S., and Steber, C.M. ( 2009). Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens. Plant Cell Rep. 28, 903–913. Google Scholar
  357. Zaltsman, A., Krichevsky, A., Kozlovsky, S.V., Yasmin, F., and Citovsky, V. ( 2010a). Plant defense pathways subverted by Agrobacterium for genetic transformation. Plant Signal Behav. 5, 1245–1248. Google Scholar
  358. Zaltsman, A., Krichevsky, A., Loyter, A., and Citovsky, V. ( 2010b). Agrobacterium induces expression of a host F-Box protein required for tumorigenicity. Cell Host Microbe 7, 197–209. Google Scholar
  359. Zaltsman, A., Lacroix, B., Gafni, Y., and Citovsky, V. ( 2013). Disassembly of synthetic Agrobacterium T-DNA-protein complexes via the host SCFVBF ubiquitin-ligase complex pathway. Proc. Natl. Acad. Sci. U.S.A. 110, 169–174. Google Scholar
  360. Zambre, M., Terryn, N., De Clercq, J., De Buck, S., Dillen, W., Van Montagu, M., Van Der Straeten, D., and Angenon, G. ( 2003). Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells. Planta 216, 580–586. Google Scholar
  361. Zambryski, P., Joos, H., Genetello, C., Leemans, J., Vanmontagu, M., and Schell, J. ( 1983). Ti-plasmid vector for the Introduction of DNA into plant-cells without alteration of their normal regeneration capacity. EMBO J. 2, 2143–2150. Google Scholar
  362. Zechner, E.L., Lang, S., and Schildbach, J.F. ( 2012). Assembly and mechanisms of bacterial type IV secretion machines. Philos. Trans. R Soc. Lond. B: Biol. Sci. 367, 1073–1087. Google Scholar
  363. Zhang, J., Boone, L., Kocz, R., Zhang, C., Binns, A.N., and Lynn, D.G. ( 2000). At the maize/Agrobacterium interface: natural factors limiting host transformation. Chem. Biol. 7, 611–621. Google Scholar
  364. Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W., and Chua, N.H. ( 2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646. Google Scholar
  365. Zhang, Y., Yin, X., Yang, A., Li, G., and Zhang, J. ( 2005). Stability of inheritance of transgenes in maize (Zea mays L.) lines produced using different transformation methods. Euphytica 144, 11–22. Google Scholar
  366. Zhang, Z., Mao, Y., Ha, S., Liu, W., Botella, J.R., and Zhu, J.K. ( 2015). A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep. 35, 1519–1533. Google Scholar
  367. Zhou, X.R., and Christie, P.J. ( 1997). Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins. J. Bacteriol. 179, 5835–5842. Google Scholar
  368. Zhu, J.K., Damsz, B., Kononowicz, A.K., Bressan, R.A., and Hasegawa, P.M. ( 1994). A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-1 alpha. Plant Cell 6, 393–404. Google Scholar
  369. Zhu, J.K., Shi, J., Singh, U., Wyatt, S.E., Bressan, R.A., Hasegawa, P.M., and Carpita, N.C. ( 1993). Enrichment of vitronectin- and fibronectin-like proteins in NaCI-adapted plant cells and evidence for their involvement in plasma membrane-cell wall adhesion. Plant J. 3, 637–646. Google Scholar
  370. Zhu, Y., Nam, J., Carpita, N.C., Matthysse, A.G., and Gelvin, S.B. ( 2003a). Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene. Plant Physiol. 133, 1000–1010. Google Scholar
  371. Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.Y., Cao, H., Valentine, L., Li, J., Kaiser, A.D., Kopecky, A.L., Hwang, H.H., Bhattacharjee, S., Rao, P.K., Tzfira, T., Rajagopal, J., Yi, H., Veena, Yadav, B.S., Crane, Y.M., Lin, K., Larcher, Y., Gelvin, M.J., Knue, M., Ramos, C., Zhao, X., Davis, S.J., Kim, S.I., Ranjith-Kumar, C.T., Choi, Y.J., Hallan, V.K., Chattopadhyay, S., Sui, X., Ziemienowicz, A., Matthysse, A.G., Citovsky, V., Hohn, B., and Gelvin, S.B. ( 2003b). Identification of Arabidopsis rat Mutants. Plant Physiol. 132, 494–505. Google Scholar
  372. Ziemienowicz, A., Tinland, B., Bryant, J., Gloeckler, V., and Hohn, B. ( 2000). Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol. Cell Biol. 20, 6317–6322. Google Scholar
  373. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T., and Felix, G. ( 2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749–760. Google Scholar
  374. Zorreguieta, A., Geremia, R.A., Cavaignac, S., Cangelosi, G.A., Nester, E.W., and Ugalde, R.A. ( 1988). Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene. Mol. Plant Microbe Interact. 1, 121–127. Google Scholar
  375. Zupan, J., Hackworth, C.A., Aguilar, J., Ward, D., and Zambryski, P. ( 2007). VirB1* promotes T-pilus formation in the vir-Type IV secretion system of Agrobacterium tumefaciens. J. Bacteriol. 189, 6551–6563. Google Scholar
© 2017 American Society of Plant Biologists
Hau-Hsuan Hwang, Manda Yu and Erh-Min Lai "Agrobacterium-Mediated Plant Transformation: Biology and Applications," The Arabidopsis Book 2017(15), (20 October 2017).

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