Abstract

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
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.

Keywords agrobacterium tumefince, T-DNA

1 Introduction

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.
2 The Biology Of Agrobacterium-Plant Interactions And T-DNA
Transformation
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 The current knowledge and recent findings regarding the key events
of Agrobacterium-mediated plant transformation process are reviewed in the following sections

[Link] 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.

 Figure 1. Major steps of the Agrobacterium tumefaciens-mediated plant transformation
process.

2.1.1 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.

2.1.2 Sensing And Regulation Of Virulence Genes Of A. Tumefaciens In Response To

Plant Signals

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.
2.1.3 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).

2.1.4 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)

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)

2.1.5 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).
3 Applications Of Agrobacterium-Mediated Transformation
3.1 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.

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.

3.1.1 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 explants 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 eco-types,
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 explants 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 explant coincubation, different
antibiotics, such as carbenicillin, cefotaxime, vancomycin HCl, 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 β-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).
 3.1.2. 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 (Bl-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).

3.2 Transient transformation

In general, months are required to generate a transgenic line, and even longer if a
homozygous genotype is desirable. Agrobacterium-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 (i.e. promoter-of-interest fused with a GUS/luciferase/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.

3.2.1 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 virus-induced 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 (Burch-Smith 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 (Patil 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).
 3.2.2 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 de-fence 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-0 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).
4 Conclusions

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

Acknowledgements
References
Agarwal, S., Loar, S., Steber, C., and Zale, J. ( 2008). Floral transformation of wheat.
Methods Mol. Biol. 478, 105–113. Google Scholar

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

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

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

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

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

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

Alonso, J.M. and Stepanova, A.N. ( 2003). T-DNA mutagenesis in Arabidopsis. Methods

Mol. Biol. 236, 177–188. Google Scholar

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

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
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

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

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

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

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

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

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 Agrobacteriumtumefaciens. Plant Physiol. 146, 703–715. Google Scholar

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

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

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