We went through 4 rounds of the DBTL cycle for our "Best composite part”. This part encodes the master regulator for the plant transformation machinery of Agrobacterium. For the final composite part we used a characterized inducible promoter system to fine the expression of the master regulator in order to allow fine tuning for the individual plant species of choice.
Besides, we have worked on the implementations of the revolutionary cut-dip-budding method to make it low-cost and easy-to-implement for the Agrobacterium mediated transformation of non-model organisms.
In the field of plant biotechnology, several transformation methods have been developed that have the potential to introduce desired traits into plants. However, these methods present significant challenges. Three main problems are prominent in plant transformation: (1) they are time-consuming and require long cultivation times before results are available, (2) they are customized for model organisms and are not transferable to non-model species, and (3) they often involve complex and costly experimental setups. Nevertheless, the most promising avenue for introducing foreign DNA into a plant's genome is mediated by Agrobacterium. This plant pathogen naturally carries a virulence plasmid containing T-DNA (transfer-DNA) regions that are later transferred into plant cells upon infection.
In order to solve the above mention problems, our primary objective is to enhance Agrobacterium-mediated transformation and expand the spectrum of transformable plants. We are directing our focus towards Agrobacterium rhizogenes, a species renowned for inducing the formation of 'hairy roots' upon successful infection and plant tissue transformation. This infection mechanism relies on the sensory kinase VirA, which detects various plant molecules and subsequently activates the expression of virulence genes through the transcription factor VirG.
To accomplish this daunting task, we relied on several rounds of engineering, both to improve our Master Switch construct and to optimize existing plant transformation protocols. Ultimately, our efforts bore fruits and culminated with the first transformation of a bambara groundnut plant, opening up new avenues for crop engineering.
Controlling the expression of the master regulator VirG through its overexpression or the inclusion of extra copies from more potent strains has been shown to increase transformation efficiency and host range (Anand et al., 2019).
We propose the use of a helper plasmid that decouples the expression of VirG from the VirA two component system, skipping the need for including phenolic compounds that emulate the plant response in the growth medium. By fine-tuning the expression of this “Master-Switch” of Agrobacterium virulence, we aim to take control of the transformation machinery and tailor it to individual plant species. Here, we present the result of our engineering efforts and the multiple iterations of the design-build-test-learn cycle.
The initial design for our construct relied on maximizing the expression of a second copy of the endogenous VirG from A. rhizogenes ARqua1. This was based on the assumption that a maximum induction of the virulence genes would lead to the best transformation results. This approach was also used to improve transformation efficiency in celery and rice (Liu et al., 1992). As a backbone, we chose the pSRK L1 entry vector, which was provided by the lab of our PI Anke Becker, has a pBBR1 broad host range ori. Based on the data gathered from our Anderson Promoter characterization, we identified J23102 as the strongest constitutive promoter, and designed a construct that used this promoter to drive the expression of the endogenous VirG CDS basic part we amplified from A. rhizogenes ARqua1.
The endogenous VirG CDS was PCR amplified from gDNA extracted from A. rhizogenes ARqua1 and cloned in the level 0 entry vector from the Marburg Collection. The primers used were designed to also remove internal BsmBI cutting sites.
However, after more thorough research, and - most importantly - after consulting with Sebastian Cocioba, we found that strong virulence induction might be an extremely high metabolic burden for the cell, leading to slower growth and possibly even an overall decrease in transformation efficiency. This prompted us to return to the drawing board and rethink how our composite part could work.
Next, we decided to change the design by using an inducible promoter system. By doing that, not only could we delay the virulence response until it was actually needed, but also open up the potential for fine-tuning the virulence response for each plant species of interest. Here we faced another challenge, the lack of basic parts that are well characterized in Agrobacterium. While some efforts have been made in shedding light on the function of inducible systems in this organism, its volume still pales in comparison to other model organisms.
We selected 9 promoters from the “Marionette Collection”, which contains a number of inducible systems highly optimized (in E. coli) for high dynamic range and low leakiness (Meyer et al., 2019). Additionally, Ptrc and Ptau were also included (Mostafavi et al., 2014; Stukenberg et al., 2021). Another consideration made when selecting the promoter systems to characterize was to include ones that use non-phenolic compounds as inducers (Ptau, IPTG, Pbetl, and Pbad), in the hope of minimizing cross talk with the native VirA/VirG two component system.
The results in Figure 3 show relative luminescence (RLU) output from H2O mock induction and maximum induction. This experiment demonstrated that most promoters did not respond significantly to induction in A. rhizogenes ARqua1, notable exceptions were Ptac, Pvan, PnahR and Ptau. With first two showing the highest overall induction strength and Ptau the widest dynamic range, in fact, the baseline expression of Ptau was as low as the dummy promoter, both at the threshold of detection for the plate reader used in the experiment, this demonstrates that the expression of Ptau is tightly regulated and has virtually zero leakiness. Overall, PnahR appeared to have a good middle ground between expression strength and orthogonality, and was selected for driving the expression of VirG in our Master-Switch construct.
Unfortunately, we noticed in the previous experiment that despite the high luminescence output, cultures grown in 100 µM of sodium salicylate showed significantly slower growth rates (Figure 4A). This prompted us to investigate the issue further and record the growth curve of A. rhizogenes carrying the PnahR characterization plasmid used in the previous experiment in a medium containing a serial dilution of sodium salicylate (Figure 4B).
As shown in Figure 4, the maximum induction concentration of 100 µM and the first 1:10 dilution resulted in severe growth inhibition.
Agrobacterium are usually are equipped to tolerate such plant defense compounds as sodium salicylate and vanillin, and no toxicity was reported on previous characterizations using Agrobacterium tumefaciens C58, pointing at a possible strain specific behavior in A. rhizogenes ARqua1 (Colognori et al., 2023; Gelvin, 2018; Schuster & Reisch, 2021). Based on this data, we chose to streamline our VirG expression candidates to Ptac and Ptau, combining high expression potential with low leakiness.
In addition to the promoters, we also looked into literature for different variants of the VirG transcription factor, and built a combinatorial library of constructs. There is a multitude of Agrobacterium strains with differing characteristics and virulence strengths. The strain A281 in particular, is able to transform a broader range of plant species and has higher efficiency due to its pTiBo542 Ti plasmid. Introducing copies of its virG and virB operons in regular strains has been shown to recreate the improved efficiency. This heightened activity is primarily attributed to the existence of V7I and I106T mutations in the coding sequence of the variant. (Chen et al., 1991).
While the virulence of Agrobacterium usually depends on external signals for its activation through the VirA/VirG two component system, certain mutations in VirG may result in a “constitutive” phenotype, where VirG binds to vir gene promoters and triggers virulence independent of being activated by VirA. One of these mutations, the change of one amino acid at position 54 from an asparagine (N) to aspartate (D) has been shown to cause in enhanced transformation efficiency in many plants (Chen et al., 1991; De Saeger et al., 2021). However, no “constitutitve” variety of VirG(pTiBo542) has been produced so far. So, we used bioinformatic tools to identify the aminoacid in the longer VirG(pTiBo542) that is equivalent to the position 54 in VirG(N54D), and reproduced the mutation. Based on sequence alignments, we identified this site at position 80 of VirG(pTiBo542)
Constructs containing combinations of the endogenous A. rhizogenes ARqua1 VirG, VirG(pTiBo542), VirG(pTiBo542 N80D), and the promoters Ptau and Ptac. This combinatory library was then transformed in A. rhizogenes ARqua1 for determining if an increase of plant transformation efficiency could be detected.
After the initial observation of results three days post-transformation with Ptau_super80_pSRK (Figure 6A),Ptac_TiBo542_pSRK (Figure 6B) and Ptac_super80_pSRK (Figure 6C), there was a noticeable decline in transformation efficiency which went from previous 46% to a range between 29% to 37 (Figure 6A-C). To address this issue promptly and avoid any unnecessary delays, our team initiated troubleshooting procedures. This involved conducting stability assay tests to gain a deeper understanding of the factors contributing to the reduction in transformation efficiency.
The pSRK entry vector carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with E. coli (Antoine & Locht, 1992; Blázquez et al., 2023). However, after observing that strains carrying both 35S:RUBY:KanR and Master-Switch plasmids displayed lower transformation efficiency when compared to strains solely carrying the 35S:RUBY plasmid, we decided to investigate further. This led to the suspicion that the two plasmids might be unstable when co-existing in Agrobacterium, negatively affecting cell health and thus decreasing overall transformation efficiency. In order to verify this hypothesis, we conducted a stability assay in A. rhizogenes ARqua1 carrying 35S:RUBY:KanR and Ptau_super80_pSRK. Cultures were grown overnight and used to inoculate a new liquid culture, until 5 overnight cultures were obtained. Samples from all days were verified via colony PCR.
By the 4th overnight culture, the cell density in all cultures of Agrobacterium carrying the pSRK constructs was already visible low, meanwhile, Agrobacterium carrying pABCa constructs grew normally. The colony PCR revealed that after 4 days, both plasmids were lost in plain LB and LB (gen+strep) cultures. In LB (gen+spec), the 35S:RUBY:KanR plasmid was detected in the 4th day and lost in the 5th. Based on these results, we opted to use the pABCa backbone as part of our next DBTL cycle for our VirG expression constructs, despite its lower copy number when compared to pSRK (Antoine & Locht, 1992; Döhlemann et al., 2017).
Three days after transforming Arabidopsis with the two pABCa constructs,Ptac_super80_pABCa (Figure 8A) and Ptac_TiBo542_pABCa (Figure 8B), we observed transformation rates that were still notably low when compared to our baseline experiments. However, this outcome indicated a positive aspect of our work – our strains seemed not to lose our construct plasmid. This result aligned with our cultivation practices, as we only cultured Agrobacterium for 1-2 days, and plasmid loss seems to occur after 4 days. Moreover, the pABCa plasmid exhibited stability and demonstrated comparable transformation efficiency after 3 days when compared to constructs with the pSRK backbone.
Upon evaluating our most recent results 10 days post-transformation with constructs containing the pSRK backbone, we were surprised to witness an unexpected increase in the number of RUBY-positive plants compared to the 3-day post-transformation results. We were especially surprised by the Ptac_TiBo542_pSRK transformation results which showed a transformation efficiency gain of 9% in comparison to the 35S:RUBY:KanR baseline. The current efficiency levels now seem to be on par with the outcomes from the baseline experiments. With the assumption that pABCa behaves similarly, we anticipate obtaining comparable results ten days after transformation, and we anticipate these results to be available within the next week.
In our future endeavors, we plan to seek out a multi-copy ori that combines the advantageous traits of both the pSRK and pABCa backbones. This will ensure stability while allowing for high copy numbers in our plasmids. We are dedicated to testing all of our constructs, with particular emphasis on the most promising ones, in a variety of non-model plants such as the bambara groundnut. Our goal is to establish if we can achieve results comparable to, or even better than, those obtained in our baseline experiments.
Furthermore, our upcoming research will focus on refining the optimal concentration of taurine for Ptau. This fine-tuning process will help us create ideal virulence activity conditions for our transformations. With the current results at our disposal, along with forthcoming data and possible new experiments, we aim to develop a standardized toolkit. This toolkit will provide the iGEM community with the means to efficiently transform non-model plant species, enabling them to tackle local challenges using their native plant varieties.
Our initial approach involved exploring diverse T-DNA delivery methods and customizing them to suit our project's specific requirements. As we assessed the host range across various plant species, we sought a universally applicable plant transformation protocol. Early in our laboratory endeavors, we stumbled upon a recently published paper describing the seemingly simple yet remarkably effective cut-dip-budding delivery system (Cao et al., 2023). This method appeared ideal for evaluating our selection of plants due to its simple implementation and minimal equipment requirements. However, we soon realized the necessity of adapting the protocol to our unique needs as an iGEM team. We particularly focused on low-cost and easy-to-implement adaptations to facilitate access to synthetic plant biology for upcoming iGEM teams.
We examined the published cut dip budding protocol for factors that might reduce transformation efficiency and looked for creative, low-cost solutions that are particularly useful for iGEM teams. We examined each of these adaptations in a round of experiments: We transitioned to a greenhouse facility with controlled temperatures to ensure uniform germination conditions for our plants. Additionally, we optimized the bacterial culture by centrifugation it to achieve higher concentrations in the log phase. We extended the incubation period for our plant cultures and compared different strains to achieve optimal transformation efficiency. Moreover, we introduced vanillin as a cost-efficient inducer of virulence genes into the process and constructed affordable temporary greenhouses to maintain humidity during the initial stages, minimizing transpirational stress. Each round of improvement was rigorously tested on at least twelve plants per species.
Through several optimization cycles, we were able to create transgenic bambara groundnut plants. Our hard work bore
fruit when we observed a RUBY-positive root in Vigna subterranea, the bambara groundnut, 8 weeks after our final cut-dip
budding method refinement. With each progressive improvement, we transitioned from a lack of root induction to
discovering hairy roots and, ultimately, a RUBY-positive root. We are so proud to announce that we were able to
establish the first working method to transform the non model organism bambara groundnut via Agrobacterium rhizogenes,
marking a big step in non model crop transformation. If you want to read this protocol with all adaptations, see the
experiments page.
The currently established method involve removal of freshly rooted plants from vermiculite multiple times (Cao et al.,
2023). As this causes significant stress on the plants, we set out to improve the method with the aim of reducing stress
on the plant by using non-invasive technique. After conducting extensive research, we found a novel method shared by
plant biotechnology researcher Sebastian Cocioba via social media. This innovative approach involves using rockwool
saturated with bacterial culture. We adapted this process into our protocols, enabling us to directly observe root
growth through a transparent tube, streamlining the process and significantly improving survival rates. This protocol is
particularly designed to transform experimental plants in a non-sterile environment using simple, inexpensive means.
This will allow upcoming iGEM teams to cost-effectively transform local plants without access to sterile workstations.
You can read the whole protocol on our experiments page (link zur exp page).