A. tumefaciens is a Gram-negative, rod shape (0.6–1.0 × 1.5–3.0 μm), aerobic bacterium with 1-4 peritrichous flagella, which belongs to Alphaproteobacteria (John M. Young, 2015). Type strain C58 contains one circular chromosome, one linear
Servent et al., 1993; Goodner et al., 2001; Wood et al., 2001).
A. tumefaciens was first identified as a causal agent of plant crown gall in 1907
(Smith and Townsend, 1907), and named as Bacterium tumefaciens at that time. It was surprising that once the pathogen infects a plant, agrobacteria are no longer required for tumor generation (White and Braun, 1941). Therefore, a term “tumor inducing principle (TIP)” was created to indicate that the tumor is caused by something produced by the bacteria but not the bacteria itself. The tumor cells isolated from “secondary tumor”, tumor tissue with absence of agrobacteria, were further found to be able to grow on a medium without plant hormones, auxin and cytokinin, which are necessary for normal plant cells to grow on a medium (Braun, 1958). In 1960s, the virulence factor of A.
tumefaciens was found to be transferred between virulent strains and avirulent strains
(Kerr, 1969). In 1970s, a megaplasmid found to be exclusively present in virulent A.
tumefaciens strain is isolated (Zaenen et al., 1974; Watson et al., 1975). This plasmid
was further named as tumor inducing, or Ti plasmid. A small portion of the plasmid was found to be present in the tumor tissue, suggesting that TIP is indeed this fragment of DNA (Chilton et al., 1977). This DNA fragment was named as T-DNA for transferred DNA. Soon after the identification of T-DNA, it is further shown that T-DNA is
transcribed in tumor (Drummond et al., 1977).
In addition, several types of opine were found to be produced in tumor, and the type of opine is consistent with the A. tumefaciens strain capable of acquiring this opine (Lippincott et al., 1972). Later, a genome-wide Tn5 transposon mutagenesis analysis in A. tumefaciens strain Ach5 was conducted (Garfinkel and Nester, 1980). It was found
that insertion in the T-DNA region may result in defect of opine production or altered tumorigenesis but did not completely lost virulence. Insertion mutants with affected virulence were mapped to three major regions, two on Ti plasmid (T-DNA region and vir genes), one on circular chromosome, named as chromosomal virulence (chv) genes.
DNA region responsible for triggering auxin or cytokinin synthesis in plant was mapped onto T-DNA by mutagenesis (Garfinkel et al., 1981; Ooms et al., 1981). At the same period of time, T-DNA was shown be inserted into the plant genome but not maintained as a plasmid form in tumor cells (Thomashow et al., 1980). The fact that vir gene expression is only observed in planta suggests that plant may play a role to induce vir gene transcription; and plant exudates, including phenolic compounds acetosyringone (AS), were found to induce vir genes expression (Stachel et al., 1985; Stachel et al., 1986a). VirA/VirG two-component is responsible for sensing AS (Lee et al., 1995).
Other than phenolic compounds, several monosaccharides were also found to enhance vir gene expression via sugar binding protein ChvE. ChvE-monosaccharide complex
interacts with VirA sensor kinase and results in activation of the transcription of vir genes (Ankenbauer and Nester, 1990; Cangelosi et al., 1990; Shimoda et al., 1990).
Another environment signal associated with vir gene expression is pH. virG expression is also induced by acidic signal regulated by ChvG/ChvI two-component system (Charles and Nester, 1993; Mantis and Winans, 1993). Interestingly, ChvG/ChvI is not only used to induce vir gene expression but also several acid-inducible genes including T6SS genes (Wu et al., 2012).
The active research from the late 20th to 21st century has greatly advanced our understandings of the molecular mechanism underlying Agrobacterium-mediated transformation. This process can be divided into five major steps as 1) attachment: A.
tumefaciens cells attach to the plant cell; 2) signal sensing and gene expression: plant
signals are sensed by A. tumefaciens followed by activation of vir gene transcription and generating Vir proteins; 3) DNA processing and transport: DNA is processed and T-DNA and effector proteins are transported via VirD4/VirB T4SS into plant cells; 4) cytoplasmic trafficking and nuclear import: T-DNA and several effector proteins traffic
in cytoplasm and enter the nucleus of plant cell; 5) T-DNA integration: T-DNA is integrated into plant genome for tumorigenesis and opine production (Hwang et al., 2017). The key factors involved in each step are briefly described as follows.
Production of cyclic ß-1,2-glucan (Thomashow et al., 1987; Zorreguieta et al., 1988; Cangelosi et al., 1989; O'Connell and Handelsman, 1989), cellulose (Matthysse et al., 1981; Matthysse, 1983) and unipolar polysaccharide (UPP) produced at the pole of an A. tumefaciens cell (Tomlinson and Fuqua, 2009) are involved in the attachment of A. tumefaciens to the plant cell. Plant or environment signals able to induce the
expression of virulence associated genes include phenolic compounds, monosaccharide and acidic pH (Nester, 2014). After expression of virulence-associated genes, T-DNA was cleaved and nicked by endonuclease VirD2 with assistance of VirD1, and the 5’ end of the single-stranded T-DNA is bound with VirD2 to form relaxosome (Stachel et al., 1986b; Albright et al., 1987; Jayaswal et al., 1987; Wang et al., 1987; Filichkin and Gelvin, 1993). T-DNA and its associated VirD2 protein and several effectors (VirE2, VirE3, VirF, ORF5) are then recognized by VirD4, a T4SS coupling protein, and transferred independently through T4SS into plant cell (Christie et al., 2014). During T-DNA translocation, T-T-DNA sequentially interact with T4SS proteins as the order of
VirD4-VirB11-VirB6/VirB8-VirB2/VirB9 (Cascales and Christie, 2004). It is generally believed that VirE2, single-stranded DNA binding protein may bind to ssT-DNA-VirD2 complex inside plant cytoplasm to form a mature T-complex, which then enters into the nucleus via nuclear pore (Herrera-Estrella et al., 1990; Howard et al., 1992). The mechanism of T-DNA insertion is controversial (Hwang et al., 2017). Whether non-homologous end joining machinery is required for T-DNA differs from different reports.
Recently, it is shown that polymerase-theta-mediated DNA repair may be the key player responsible for T-DNA insertion into plant genome (van Kregten et al., 2016).