Transient transformation of Tobacco

Agrobacterium infiltration
PPCB lab course presentation

Adithya Acharya | 28.10.2022

Source- Zheng, L et al;2021 1

 Why is N benthamnia (tobacco) used as a model plant ?

• Tobacco is a model plant for Agrobacterium-

mediated genetic transformation.

• Tobacco plants can be easily regenerated

from tobacco leaf pieces through
organogenesis.

• Short acclimatization time and a high

transpotting survival rate.

Source - Kavas, M.et al; 2016 2

up to 100% of the in vitro- raised plants transferred from lab to greenhouse condition were successfully established ex vitro.

 What is transient transformation and why do we use it ?

• Temporary expression of genes most frequently plasmid DNA encoding an expression

cassette.

• Usually the rst choice for investigating the gene function.

• Possible to do in a few days or weeks.
• Gene function studied by transiently silencing and/or overexpressing a gene of interest
in the plant.

• Most popular way for transient transformation in plants is Agrobacterium in ltration.

Di erence between transient and transgene expression - explain

ff
fi
 Agrobacterium tumefaciens mediated infection
T-DNA transfer and Ti plasmid

Source - Plant cell expression systems script page 4-5 4

Agrobacterium tumefaciens is a soil plant pathogenic bacterium which naturally infects the wound sites in dicotyledonous plants causing the formation of the crown gall tumors.

A. tumefaciens has the ability to transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is then stably integrated into the host genome and transcribed

The transferred DNA (T-DNA) is part of the Ti-plasmid (Figure 2) and contains two types of genes: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins and the genes encoding
for enzymes involved in the synthesis of novel plant metabolites (opine).The expression of the virulence genes (vir-genes) and therefore the process of T-DNA transfer is stimulated by phenolic compounds (like
acetosyringone) exuded by wounded plants.

Apart from the T-DNA border sequences, most of the genes of the T-DNA can be replaced by genes of interest to be transferred into the plant. A wide spectrum of plants is susceptible to transformation by this bacterium

 Agrobacterium transformation and infiltration

Basic overview

Agrobacterium transformation with

Transformed Agrobacterium in ltration in tobacco

gene of interest and cultivation and further plant cultivation

5
Source - Spiegel, H.et al; 2022

Preparation of Electrocompetent A. tumefaciens Cells

Electroporation of A. tumefaciens

Cultivation of Recombinant A. tumefaciens Cells

Preparation of the In ltration Solution

Cultivation of N. benthamiana Plants

fi
fi
 Agrobacterium transformation
Things to consider before designing the experiment

• Cloning strategies to be used.

• Choice of the Agrobacterium strain.

• Reporter genes to be used.

• Levels of protein expression.

• Inclusion of antibiotic resistance gene in the vector

cassette

6
Source - Sparkes, I. A.et al,2022

Promoter choice and the number of T-DNA inserts present in the cell are directly correlated with the level of expression.

Binary vectors are notoriously di cult to manipulate owing to their large size (∼10–14 kbp).

Adding an antibiotic resistance gene to the plasmid solves both problems at once – it allows a scientist to easily detect plasmid-containing bacteria when the cells are
grown on selective media, and provides those bacteria with a pressure to keep your plasmid.

There are various Agrobacterium strains available, some more virulent than others , P19 protein including helps The p19 protein of tomato bushy stunt virus (TBSV),
prevents the onset of post-transcriptional gene silencing (PTGS) in the in ltrated tissues and allows high level of transient expression

ffi
fi
 Agrobacterium transformation
Expression cassette- Binary vectors

• Engineered strains of A. tumefaciens are used to deliver

genetic material to plants.

• Utilizes the natural mechanism of T-DNA transfer.

• Binary vectors are genetic constructs with a disarmed Ti

plasmid lacking a functional T-DNA but instead contains a
second plasmid.

• Transformation of the second plasmid with A. tumefaciens

carrying the T-DNA left and right border sequences anking
a selectable marker.

Source - Plant cell expression systems script page 21 7

The expression cassette between the T-DNA borders can be tailored to control expression levels and subcellular targeting in plant cells.

The Ti plasmid , which in its natural form is very large (up to 235 kb) and di cult to manipulate in the laboratory, therefore provides the T-DNA transfer functions in trans
to the binary vector , which is small and suitable for cloning.

Multiple tandem expression cassettes here are several available strategies including the placement of multiple tandem expression cassettes within the same T-DNA and
the preparation of separate binary vectors (and separate A. tumefaciens strains) for each subunit delivered by co-in ltration [15, 16]. The tandem expression cassette
strategy ensures that all transgenes co-integrate at the same locus and is therefore preferred for stable transformation, because it avoids the risk of meiotic segregation.
ffi
fi
fl
 Agrobacterium transformation
Reporter gene

• Reporter genes allow selection and visual

detection of transient gene expression.

• Proven easy and convenient to use in

transient expression and stable transformation
of plant cells.

• GFP (Green uorescent protein) is the most

common reporter gene used in Agrobacterium
mediated transformation in tobacco.

Source - Qi, J.et al,2014 8

Wild type tobacco uorescent detection - none GFP detection - image B

successful transformation - GFP detection in transient tobacco leaf cells - Image D

fl
fl
 Agrobacterium infiltration
Basic overview

Source - Smith R. H. 2012 9

Two di erent in ltration and incubation procedures can be applied according to the number of expression constructs to be tested and the scale: (A) syringe-based
in ltration of single or multiple leaves on intact plants, and (B) vacuum-based in ltration of entire intact plants.
fi
ff
fi
fi
 Agrobacterium infiltration
Two methods

Syringe-Based In ltration Vaccum -Based In ltration

10
Source - Spiegel, H.et al; 2022

For injection, this solution is then placed in a syringe (without a needle) The tip of the syringe is pressed against the underside of a leaf while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium suspension is then injected
into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf.

Vaccum based -

Vacuum in ltration is another way to introduce Agrobacterium deep into plant tissue. In this procedure, leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied,
forcing air out of the intercellular spaces within the leaves via the stomata. When the vacuum is released, the pressure di erence forces the "Agrobacterium" suspension into the leaves through the stomata into the mesophyll tissue. This can result in nearly all of the
cells in any given leaf being in contact with the bacteria.
fi
fi
fi
ff
Summary
• Tobacco is the preferred model plant for Agrobacterium in ltration studies because of its
easy establishment from in-vitro to ex-vitro.

• Transient gene expression in plants could be best expressed by Agrobacterium in ltration

method.

• The whole process of Transformation in tobacco via Agrobacterium contains two steps
Agrobacterium transformation and Agrobacterium in ltration in the desired plant.

• Choice of Agrobacterium strain is very important as they should be able to protect

themselves from the PTGS (Post transcriptional gene silencing) mechanism of plants.

• Syringe based in ltration is more preferred in cases where in ltration is to be done on

multiple leaves of a plant rather than several intact plants.

11
fi
fi
References
Sparkes, I. A., Runions, J., Kearns, A., & Hawes, C. (2006). Rapid, transient expression of uorescent fusion
proteins in tobacco plants and generation of stably transformed plants. Nature protocols, 1(4), 2019-2022

Smith R. H. 2012. Plant tissue culture Techniques and experiments. Third edition. Harcourt B. J., Publishers.
Academic press, inc, San Diego.

Kavas, M., BALOĞLU, M. C., Yücel, A. M., & Öktem, H. A. (2016). Enhanced salt tolerance of transgenic tobacco
expressing a wheat salt tolerance gene. Turkish Journal of Biology, 40(4), 727-735

Spiegel, H., Schillberg, S., & Nölke, G. (2022). Production of recombinant proteins by Agrobacterium-mediated
transient expression. In Recombinant Proteins in Plants (pp. 89-102). Humana, New York, NY.

Qi, J., Li, G., Dong, Z., & Zhou, W. (2014). Transformation of tobacco plants by Yali PPO-GFP fusion gene and
observation of subcellular localization. Guangxi Zhiwu/Guihaia, 34(3), 369-374.

Zheng, L., Yang, J., Chen, Y., Ding, L., Wei, J., & Wang, H. (2021). An improved and efficient method of
Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function
in poplar. BMC plant biology, 21(1), 1-19.

12

13