One of the key factors influencing an Agrobacterium-mediated gene transformation is the vector selection. However, sometimes you feel like you’re swimming in an ocean of vector options without really knowing which vector to choose, how to choose a specific vector and which criteria apply in making the selection.

No worries! This article provides you with information about how to select a suitable vector for Agrobacterium-mediated plant transfer. You will find in this article the Agrobacterium vector features, criteria to help with selection, advantages, disadvantages, and ease in cloning vectors.


What is an Agrobacterium vector?

In cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and expressed.

A vector allows you to create transgenic organisms (not only plants) with improved traits (e.g., disease resistance) or to understand a gene or protein function.

More than thirty years ago, the concept of using Agrobacterium tumefaciens as a vector to create transgenic plants was viewed as a prospect and a "wish." However, today, many agricultural and horticultural important species are routinely transformed using this bacterium, and the list of Agrobacterium-mediated transformation susceptible species grows at an accelerated rate.

Researchers explored and understood the mechanism of how wild Agrobacterium strains transferred DNA into the plants. The Agrobacterium tumor-inducing plasmid or Ti-plasmid does the DNA transfer. A plasmid is a small extrachromosomal DNA molecule within the bacteria that is physically separated from the chromosomal DNA and can replicate independently.

Later, to use Agrobacterium as a "tool" for plant genetic engineering, scientists modified the transferred DNA or T-DNA region to avoid getting sick plants while introducing a gene of interest.

Illustration of the agrobacterium cell with its components - the chromosome and the Ti-plasmid which is crucial for agrobacterium mediated transformation

Figure 1. Components of the wild Agrobacterium.



Features of the different Agrobacterium vectors

Two key advances made Agrobacterium transformation the method of choice for plant transformation.

  • Removal of all the T-DNA genes does not impede the Agrobacterium ability to transfer DNA but does prevent the formation of tumors. This allowed scientists to produce "disarmed strains."
  • The two main components for an Agrobacterium plasmid, the T-DNA and the virulence (vir) region can reside on separate plasmids. These components form the basis of modern Ti plasmid vectors, termed binary Ti vectors.


The T-DNA is approximately 10 to 30 kbp in size (about 10% of the plasmid size). Some plasmids can contain more than one T-DNA region. Furthermore, the plasmid contains about 35 virulence genes responsible for processing the T-DNA and its subsequent export from the bacterium to the plant cell.

Most in vitro gene manipulation techniques use E. coli to increase the plasmid yield, as it is a fast-growing bacterium. Consequently, one advantage of working with binary Ti vectors is their capability to replicate in both E. coli and Agrobacterium.

Let's talk about the different existing Agrobacterium vectors used in plant transformation.


Binary vector

Although the term binary vector literally refers to the entire system that consists of two replicons (DNA molecules), one for the T-DNA and the other for the virulence genes, the plasmid that carries the T-DNA is frequently called a binary vector. For convenience, I follow the literal definition of two plasmids to give more details to the reader.

A binary vector consists of two plasmids: a disarmed Ti plasmid carrying the T-DNA region and a helper plasmid containing the virulence genes.


The disarmed Ti plasmid:

The disarmed Ti-plasmid consists of a T-DNA and the vector backbone (Figure 2A). Both components have different genes that together determine the DNA sequence to be transferred and help in the identification of the transformed tissue or plant. Below I described each of these two components present in the disarmed Ti-plasmid.

Table 1. Components of the disarmed Ti-plasmid.

T-DNA

Vector backbone

Element

Description

Element

Description

The border sequences

the right border (RB) and the left border (LB) can come from Octapine and Nopaline Ti plasmids.

Plasmid replication functions

It is an origin replication to be replicated in E. coli and A. tumefaciens. For A. tumefaciens we have IncP, IncW, pVS1 and pRi. For E. coli, we have pUC, ColE1, IncP, F factor, and Phage P1.

Multiple cloning sites:

pUC and pBluescript plasmids contain multiple cloning sites.


Bacterial selectable marker

It can be kanamycin, ampicillin, gentamicin, spectinomycin, chloramphenicol, and tetracycline.

Plant selectable marker

It can be kanamycin, hygromycin, phosphinothricin, glyphosate, phosphomannose isomerase

Plasmid mobilization functions

They support T-DNA mobilization functions such as OriT and Bom

Reporter gene

It can be a GUS, LUC, GFP fluorescent proteins



Promoter

Constitutive promoters usually drive the selectable marker genes. They include CamV 35S, T-DNA genes, ubiquitin, and actin genes.




The helper plasmid:

There are several virulence genes, named virA, virB, virC, etc., all producing different proteins that have different roles in the transfer of T-DNA from Agrobacterium to its host (Figure 2B). Below a chart describing the main functions performed for each gene.

Table 2. Description of the virulence genes functions in the Agrobacterium helper plasmid.

Vir gene

Function

Vir A, Vir G

Sense phenolic compounds and induces expression of virulence genes

Vir B, Vir D4

Important in the secretion system. Required for export of T-complex into the cell

Vir C

Promotes high-efficiency T-strand synthesis

Vir D1, Vir D2

Topoisomerase and endonuclease functions, respectively

Vir E1, Vir E2

Chaperone function. Protect T-strand from nuclease attack.

components of the binary vector for agrobacterium transformation consist of the disarmed Ti-plasmid and the helper plasmid

Figure 2. Components of the binary vector. A) Disarmed Ti-plasmid. B) Helper plasmid. B marker: bacterial selectable marker; LB: Left border; Mob: Mobilization function; MSC: Multiple cloning sites; Ori A: replication function for Agrobacterium; Ori E: replication function for E. coli; P marker: Plant selectable marker; Pro: Promoter; RB: Right border; Rep: Reporter gene.


Superbinary vector

The finding that some of the virulence genes exhibited gene dosage effects led to the development of a superbinary vector, which carried additional virulence genes. The superbinary vector system has the same backbone as the binary vector, but it also has an additional DNA segment that contains virulence genes like virB, virC, and virG from pTiBo542, and it is introduced into the small T-DNA-carrying plasmid. This region is also called "S vir". The additional vir genes have led to high efficiency in transforming various plants, especially recalcitrant plants, such as important cereals.

illustration of the superbinary vector for agrobacterium mediated transformation. Comes with the s. vir gene or virulence

Figure 3. Components of the superbinary vector. S vir (in red) is the additional segment carrying virulence genes and it this differentiates with the binary vector.



Ternary vector

The ternary vector is a new system proposed by Anand et al. (2018) using a third plasmid— termed the “accessory plasmid” or the “virulence helper plasmid” —to carry an additional virulence gene cluster. The ternary vector system is a three-component system with a disarmed Ti plasmid, a helper plasmid, and an accessory virulence plasmid.


  • The disarmed Ti plasmid:

The disarmed Ti plasmid is composed of the T-DNA region flanked by the RB and LB, an origin of replication (ori) for Agrobacterium (e.g., pVS1 or pRiA4) and another ori for E. coli (pUC) and a plant selectable marker (usually kanamycin).


  • The helper plasmid:

The helper plasmid has a vir region, a bacterial selectable marker (str, streptomycin), tra/trb regions that function in conjugational transfer of the Ti plasmids, and, if present, a repABC/repAʹBʹCʹ region with function in replication of the Ti plasmids.


  • The accessory virulence plasmid:

The accessory virulence plasmid consists of an ori for Agrobacterium (e.g., pVS1 or pRiA4) and an ori for E. coli (pUC ori) and a bacterial selectable marker (spe, spectinomycin), and a large virulence region.

The ternary vector system nearly doubles the transformation efficiency in recalcitrant maize inbred lines (Anand et al., 2018). The ternary system also facilitated efficient Agrobacterium transformation of sorghum (Sorghum bicolor) and was used to develop transformation protocols for popular but recalcitrant African varieties (Che et al., 2018).

Illustration of the Trenary Vector for agrobacterium-mediated gene transfer. This vector system includes 3 plasmids - a helper plasmid, a disarmed TI plasmid and an accessory plasmid

Figure 4. Components of the ternary vector. The accessory plasmid is the new component in the ternary vector and this differentiates with previous vectors. Tra/trb: transcriptional activators; rep ABC: ABC replication origin.


Table 3. Commonalities and differences between the Agrobacterium vectors.


Binary

Superbinary

Ternary

Commonalities

Modified Ti-plasmid

Modified Ti-plasmid

Modified Ti-plasmid

Differences

It has a helper plasmid containing virulence genes

It works well in dicots

It has additional virulence genes in the T-DNA-carrying plasmid

It works well in monocots and recalcitrant plants

It has an accessory helper plasmid containing a large virulence region

Causes an intense infection in some monocots and recalcitrant plants


How to choose an appropriate Agrobacterium vector for my protocol?

A wide range of Agrobacterium vectors are available now. Unfortunately, there is no vector that is good for all purposes, but many of the vectors currently available are quite versatile. No matter how difficult the choice is, you have to make a decision. Here I can provide you with some criteria to guide your vector selection.


Determine the genotype to work:

You should define if you are planning to work with dicots or monocots species. Within monocots, there are the cereals group. Within dicots, you have Arabidopsis and most of the fruit crops

Agrobacterium naturally infects dicots species. In this sense, specialized vectors (e.g., superbinary and ternary) were developed to cause intense infection in monocot plants, including rice, wheat, barley, and sorghum.


Determine the study goal:


If you plan to produce transgenic plants stably, a selectable marker must be present in the disarmed Ti-plasmid.
Suppose you want to understand a gene or protein's function, a common approach in functional studies. In that case, a selectable marker is not necessary as you only wish to see the gene expression. The transgenic tissues can be differentiated using a fluorescent protein such as GUS, GFP, or LUC.
Some other vectors were designed for specific purposes, e.g., vectors for suppressing plant genes by RNA interference technology may also be chosen.


Revise literature regarding your target plant:


It is possible that your target plant already has an Agrobacterium transformation protocol reported. You can use this previous report as a base to start optimizing all the key factors influencing the Agrobacterium-mediated gene transformation.
For instance, a good review for commonly used binary and superbinary vectors in plant transformation is available in Komari et al. 2006.



Determine the size of your gene of interest to introduce:


Key criteria for delivery of transgene fragments smaller than 15 kb are (1) compatibility of selectable markers with the experiments and (2) availability of convenient cloning sites.
For delivering DNA fragments larger than 15 kb, the top consideration is whether the large DNA fragments can be cloned efficiently to the vectors and maintained stably in E. coli and A. tumefaciens. Large DNA pieces in certain vectors, e.g., high-copy number vectors, may sometimes cause low efficiency of transformation of bacteria or rearrangement of the inserts. If the DNA fragments are larger than 15 kb, IncP, BIBAC, and TAC vectors are recommended.



Perform infection tests using different vectors:


If you have access to different strains harboring different Ti plasmids, you can perform an infection test and quantify the infection efficiency.
For instance, Bakhsh et al. 2014 working with a dicot species like tobacco, evaluated five different strains (GV2260, LBA4404, AGL1, EHA105, and C58C1) where LBA4404 was selected as the best strain. For a monocot plant like wheat, McCormac et al. 1998 evaluated LBA4404 and EHA101 with the latter as the best strain.

You can calculate the infection efficiency by using our calculator here.



How to choose the right agrobacterium vector for plant transformation


Advantages and disadvantages of the Agrobacterium vectors


Binary

Superbinary

Ternary





Advantages

Replication in E. coli and Agrobacterium

User-friendly system

A wide selection of cloning sites,

High copy numbers in E. coli

High cloning capacity,

Improved compatibility with strains of choice

A wide pool of selectable markers for plants,

Increased efficiency of plant transformation.

Replication in E. coli and Agrobacterium

User-friendly system

A wide selection of cloning sites,

High copy numbers in E. coli

High cloning capacity,

Improved compatibility with strains of choice

A wide pool of selectable markers for plants,

Increased efficiency of plant transformation.

Replication in E. coli and Agrobacterium

Double infection efficiency compared to the superbinary vector






Disadvantages

Works well only in dicots

Works well only in monocots

Works well only in monocots

Ease of cloning

High

High-Medium

Medium-Low

Cost considerations

Cheap

Cheap

Unknown

Former/latest technology

Old (90s)

Old (90s)

Recent (2018)


References

Anand, A., Bass, S. H., Wu, E., Wang, N., McBride, K. E., Annaluru, N., Miller, M., Hua, M., & Jones, T. J. (2018). An improved ternary vector system for Agrobacterium-mediated rapid maize transformation. Plant Mol Biol, 97(1-2), 187-200. https://doi.org/10.1007/s11103-018-0732-y

Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research, 12(22), 8711-8721.

Che, P., Anand, A., Wu, E., Sander, J. D., Simon, M. K., Zhu, W., Sigmund, A. L., Zastrow-Hayes, G., Miller, M., Liu, D., Lawit, S. J., Zhao, Z. Y., Albertsen, M. C., & Jones, T. J. (2018). Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application. Plant Biotechnol J, 16(7), 1388-1395. https://doi.org/10.1111/pbi.12879

Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev, 67(1), 16-37, table of contents. https://doi.org/10.1128/mmbr.67.1.16-37.2003

Hellens, R., Mullineaux, P., & Klee, H. (2000). A guide to Agrobacterium binary Ti vectors. Trends in plant science, 5(10), 446-451.

Klee, H., Yanofsky, M., & Nester, E. (1985). Vectors for transformation of higher plants. Biotechnology, 3, 637-642.

Komari, T., Takakura, Y., Ueki, J., Kato, N., Ishida, Y., & Hiei, Y. (2006). Binary Vectors and Super-binary Vectors. In K. Wang (Ed.), Methods in Molecular Biology (Vol. 1). Humana Press Inc.

Komori, T., Imayama, T., Kato, N., Ishida, Y., Ueki, J., & Komari, T. (2007). Current status of binary vectors and superbinary vectors. Plant Physiol, 145(4), 1155-1160. https://doi.org/10.1104/pp.107.105734

Lee, L. Y., & Gelvin, S. B. (2008). T-DNA binary vectors and systems. Plant Physiol, 146(2), 325-332. https://doi.org/10.1104/pp.107.113001

McBride, K. E., & Summerfelt, K. R. (1990). Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol, 14, 269-276.

Nonaka, S., Someya, T., Kadota, Y., Nakamura, K., & Ezura, H. (2019). Super-Agrobacterium ver. 4: Improving the Transformation Frequencies and Genetic Engineering Possibilities for Crop Plants. Front Plant Sci, 10, 1204. https://doi.org/10.3389/fpls.2019.01204

Pandya, I. Y. (2017). Agrobacterium Tumifaciens: Fundamental concepts, and Application in Plant biotechnology. Slideshare presentation. Available in https://www.slideshare.net/DrIshanYPandya/agrobactreium-natures-genetic-engineer. Downloaded 18 of March 2021.

Polóniová, Z., Libantová, J., Gálová, Z., & Matusikova, I. (2013). Plant transformation vectors and their stability in Agrobacterium tumefaciens. Journal of Microbiology, Biotecnology and Food Sciences(1), 1559-1568.

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Zhang, Y., Zhang, Q., & Chen, Q. J. (2020). Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems. Sci China Life Sci, 63(10), 1491-1498. https://doi.org/10.1007/s11427-020-1685-9.