Team:Bielefeld-CeBiTec/Tobacco

Abstract

In our project, we created a system for the detection of chemical degradation products. When our modified plant gets in contact with a precise chemical, its leaf color changes to red, signaling the presence of the chemical. Therefore, we used the model organism Nicotiana benthamiana since it is suitable for screening of synthetically built constructs by applying agroinfiltration. The detection system is based on a receptor to bind specifically a chemical, a signaling cascade for transmission of the binding and a reporter to display it. We used agroinfiltration to test the functionality of the plant signaling cascade and the reporter systems. We were able to successfully prove the transfection of the RUBY Reporter in Nicotiana benthamiana, its time stability and that its expression can be induced.

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Tobacco

Introduction

Stable transformation of plants is time-consuming due to long generation times. However, it is possible to transiently express genes of interest in Nicotiana benthamiana (tobacco) plants. Therefore, we decided to test the different elements of our detection system by transient expression in tobacco leaves for faster analysis of its functionalities.

Nicotiana benthamiana

N. benthamiana originates from Australia and belongs to the family of Solanaceae which also includes important agricultural plants like tomato, pepper, and potato. It rose in popularity as a model plant due to its capability to express foreign genes from plant virus vectors, and therefore is often used in the field of plant virology [1]. Due to its thin, yet relatively big leaves, it is an exceptionally suitable plant for Agrobacterium mediated transformation in the form of agroinfiltration.

Agrobacterium mediated transformation and agroinfiltration

The soil bacterium Agrobacterium tumefaciens (A. tumefaciens) has the natural ability to transform host plants by incorporating transfer DNA (T-DNA) into the host genome [2]. Therefore, it carries a tumor inducing (Ti) plasmid that contains the T-DNA and vir genes that are, besides others, required to integrate the T-DNA into the host plant genome. To transform plants, a binary vector system is used, where the functions of the Ti-plasmid are distributed onto two plasmids. In this system, the Ti-plasmid is modified to a plasmid which does not contain the T-DNA region anymore, but only the vir genes, making it a disarmed Ti-helper plasmid. The T-DNA region can be found on a second plasmid [3]. This plasmid contains the gene(s) of interest, flanked by T-DNA borders and thereby enabling incorporation into the plant genome. These T-DNA borders consist of 25 bp comprising repeats and are sufficient for the recognition of the genes of interest as T-DNA [4]. The plasmid also contains further features like selection markers and origins of replications [3] (Fig. 1).



Figure 1: binary vector system consisting of Ti-helper plasmid and plasmid
with gene of interest in between T-DNA borders.

The most common indirect method to transform plants or single plant cells is agrobacterium-mediated transformation [5]. For transient expression, agroinfiltration is our method of choice. Agrobacteria are injected into the underside of the leaf of 4–6-week-old tobacco plants with a needleless syringe. Two days after infiltration, the gene expression should be visible and experiments can be performed. Agroinfiltration is an easy method for screening constructs very quickly in comparison to stable transformation of plants [6]. Therefore, we use agroinfiltration with the Agrobacterium tumefaciens strain GV3101 to test our signaling cascade and the reporter systems that we want to use as an output signal.



Figure 2: principle of agroinfiltration in N. benthamiana. A needleless syringe is used to infiltrate agrobacteria
into the underside of the tobacco leaf. After two days, the RUBY reporter should be detectable.


Plasmid construction

We constructed the signaling cascade split on two pMAP plasmids The first comprising the receptor ssTNT.R3, the membrane protein AtFLS-Trg-PhoB, and the second plasmid, comprising the transcription factor PhoB-VP64 and the reporter RUBY. Our initial approach was to clone the signaling cascade with Gibson Assembly. However, after several rounds of cloning, we were not able to assemble the parts into the plasmid. Therefore, we tried a different cloning approach. In our second phototrophs event, Dr. Andreas Andreou presented Mobius Assembly [7], a versatile Golden Gate cloning system developed at the Institute of Molecular Plant Sciences at the University of Edinburgh. We reached out to him and decided to assemble our signaling cascade using Mobius Assembly instead of Gibson Assembly.



Figure 3: carrying parts of the signaling cascade

The first plasmid (Fig. 3A) contains a 35S promoter (BBa_K3900015), the TNT-receptor (BBa_K3900011), a NOS terminator (BBa_K788001) and the transmembrane protein AtFLS-Trg-PhoR (BBa_K3900008), while the second plasmid (Fig. 3B) consists of a FMV promoter (BBa_K3900012), the transcription factor PhoB-VP64 (BBa_K3900013), a NOS terminator (BBa_K788001), the reporter RUBY (BBa_K3900028) under control of the synthetic PlantPho Promoter (BBa_K3900014) and a G7 terminator (BBa_P10402).

N. benthamiana agroinfiltration

Expression of RUBY reporter in tobacco leaves

To test the functionality of the recently published RUBY Reporter [8] in N. benthamiana, we infiltrated tobacco plants with agrobacteria, carrying a pHDE plasmid with RUBY under control of a 35S promotor (BBa_K3900049). We were able to show that RUBY is expressed and is clearly visible two days after infiltration (Fig. 4A). Furthermore, we also showed that RUBY can be induced by using an estrogen-based induction system by using RUBY under the control of the estrogen inducible lexA promoter (BBa_K3900048) (Fig. 4B). A slight red coloration of the leaves was visible. Consequently, RUBY is suitable as an inducible reporter in plants.



Figure 4: A: Red coloration of the tobacco leaf two days after infiltration with 35S:RUBY.
B: Slightly visible red coloration of the lexA:RUBY infiltrated tobacco leaf two days after estrogen induction.

Co-transformation

The distribution of the signaling cascade on two plasmids required to transform two plasmids into N. benthamiana. Hence, one of our first steps was to test whether co-transformation can be applied with agroinfiltration. We transformed tobacco plants with the plasmid pK7FWG2 carrying an EGFP (BBa_K1875003) and the RUBY reporter plasmid. Thereby, we were able to prove that RUBY as well as EGFP were expressed in the infiltrated spots by confocal laser scanning microscope (CLSM) (Fig. 7). Thereby, a co-transformation in N. benthamiana was successfully proven.



Figure 5:: A: CLSM microscopy showed EGFP expression in infiltrated leaf,
B: negative control which shows that a non-infiltrated leaf did not show any EGFP-signal; C: visible RUBY signal in infiltrated spot.

Testing of the signaling cascade in N. benthamiana leaves

For testing of the signaling cascade, the corresponding ligand of the TNT receptor is TNT. For us, working with TNT was not possible due to safety requirements. However, we used benzenetricarboxylic acid (BTCA) as a receptor ligand due to its similar chemical structure to TNT. BTCA is less dangerous in handling and less toxic than TNT. Beforehand, we tested computationally if BTCA interacts with the TNT receptor. We could show that docking of BTCA to the TNT-receptor results in multiple hydrogen bonds (Fig. 6).


Figure 6:: Docking of BTCA to the TNT-receptor shows multiple hydrogen bonds.,
suggesting a possible receptor-ligand interaction.

Our hydroculture experiments showed that BTCA is taken up by tobacco plants and can be intracellularly detected using GC/MS. Altogether, BTCA seemed to be a good alternative to TNT for testing the functionality of the plant signaling cascade. We applied BTCA in 1 mM, 500 µM, 100 µM, 10 µM and 100 nM concentrations to our tobacco plants in four different approaches:

1. Hydroculture of tobacco plants containing BTCA starting one day before initiating the experiment.
2. Infiltration with agrobacteria in combination with BTCA and additional infiltration of BTCA in the following two days.
3. Infiltration with agrobacteria and infiltration with BTCA the following two days.
4. Infiltration with agrobacteria and incubation of leaves in BTCA solution one day after starting the experiment.



Figure 7: A: N. benthamiana Hydroculture with different concentrations of BTCA. B: Infiltration with BTCA. C: Incubation with BTCA.

Unfortunately, after several days of incubation, the leaves of our test plants did not turn red and we conclude that the signaling cascade and induction of RUBY expression was not activated (Fig. 7). This can be due to several reasons: the ligand-receptor interaction might not work well enough, there could be problems in the signal transduction or the reporter activation. Due to the lack of time, we were no longer able to verify these assumptions. To examine the affinity of the ligand, we would need to test TNT instead of BTCA, but as stated prior, this is not possible due to safety requirements. However, we proved that the computational engineered BTCA receptor was functional in bacteria and we could test it in plants as well. To exclude that the transformation was not successful, the next step would be the genotyping of our plants to see if the signaling cascade was integrated into the genome We could also perform a transcript analysis using qRT-PCR to confirm the transcription of the signal cascade genes.
Once the signaling cascade is working in plants, further receptors that were designed computationally could be tested on its functionality.

References

1. Goodin, M. M., Zaitlin, D., Naidu, R. A. & Lommel, S. A. Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact 21, 1015–1026 (2008).

2. Gelvin, S. B. Integration of Agrobacterium T-DNA into the Plant Genome. Annual Review of Genetics 51, 195–217 (2017).

3. Lee, L.-Y. & Gelvin, S. B. T-DNA Binary Vectors and Systems. Plant Physiology 146, 325–332 (2008).

4. Kempken, F. Gentechnik bei Pflanzen: Chancen und Risiken. (Springer Berlin Heidelberg, 2020). doi:10.1007/978-3-662-60744-2.

5. Hwang, H.-H., Yu, M. & Lai, E.-M. Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book 15, e0186 (2017).

6. Bally, J. et al. The Rise and Rise of Nicotiana benthamiana : A Plant for All Reasons. Annu. Rev. Phytopathol. 56, 405–426 (2018).

7. Andreou, A. I. & Nakayama, N. Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly. PLOS ONE 13, e0189892 (2018).

8. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res 7, 1–6 (2020).