This year, our team focused on working with the plant pathogen Agrobacterium. Agrobacterium, an Alphaproteobacterium, is widely used for plant transformation due to its innate ability to insert a fragment known as transfer DNA (T-DNA) from its root-inducing plasmid (Ri-plasmid) into the plant host. The species Agrobacterium rhizogenes is renowned for causing "hairy root disease" in plants, orchestrated by the expression of virulence genes (vir genes) located on the root-inducing plasmid. As the name suggests, this distinctive phenotype manifests through the formation of hairy roots on the transformed plant. Before infection, this soil bacterium detects predominantly phenolic compounds produced by wounded plants. This sensing mechanism involves a two-component sensory system consisting of the protein kinase VirA, which activates VirG through phosphorylation (Christie, 2009). This activation subsequently triggers the expression of nearly all other vir genes. To establish physical contact between the pathogen and the plant and to enable the transfer of T-DNA, Agrobacterium assembles a type IV secretion system, primarily composed of VirB proteins. Several other Vir proteins are responsible for processing the T-DNA , as well as translocating and integrating it into the plant's genome in the nucleus. In the context of achieving our engineering goals, biosafety is a fundamental and unwavering principle that underscores the core of our research endeavors. It is a responsibility that goes beyond our laboratory work; it is our commitment to safeguarding not only our team but also our environment and the communities we are a part of. Biosafety is crucial in preventing unintended releases, protecting ecosystems, and mitigating potential risks associated with genetically modified organisms. By adhering to strict safety guidelines, utilizing personal protective equipment, and implementing robust containment measures, we aim to ensure that our research remains responsible, ethical, and safe. Our dedication to biosafety is a reflection of our unwavering commitment to science, innovation, and the well-being of the world around us.
All our experiments were conducted in German Biosafety Level 1 labs. We greatly benefited from the expertise of our PIs, Lars Voll and Anke Becker. The Voll lab has been deeply involved in researching plant-pathogen interactions since 2006 (Münch et al., 2011), while the Becker lab (Döhlemann et al., 2017; Williams et al., 2021) has numerous projects utilizing Agrobacterium for plant transformation. Our team received comprehensive safety training from PIs and experienced PhD students who have extensive experience working with genetically modified plants and Agrobacterium. Furthermore, safety officers provided guidance at the inception of our project and continued to advise us throughout our daily activities throughout the year. Regular exchanges with our PIs were instrumental in ensuring safety while working in the laboratory.
Given that our project aims to enhance Agrobacterium-mediated plant transformation and broaden the range of potential host plants, biosafety plays an indispensable role in our daily laboratory routines. It is crucial to recognize that an improved transformation efficiency can lead to heightened virulence, consequently increasing potential risks. Because of this we took all necessary general safety and biocontainment precautions. In addition to following established safety protocols, we maintain meticulous documentation of our work. This includes comprehensive records of experimental procedures, material handling, and waste disposal. This approach allows for thorough traceability and easy identification of any potential issues, ensuring that our commitment to safety is not only proactive but also transparent. Throughout the entire project timeline, we diligently adhered to the use of personal protective equipment and consistently employed autoclaving procedures to sterilize all items that had contact with Agrobacterium before their disposal.
To enhance Agrobacterium-mediated plant transformation, our approach involved assembling a plasmid containing various composite parts and transforming it into the Agrobacterium rhizogenes strains ARqua1 and K599. These composite parts included different versions of the transcriptional activator VirG, controlled by various inducible promoters. Our goal was to upregulate general virulence within a controlled timeframe in controlled laboratory conditions. To assess transformation efficiency, both strains also carried the RUBY reporter construct, which contains the coding regions for the cytochrome P450 enzyme CYP76AD1, DOPA dioxygenase (DODA) and a glycosyltransferase, all separated self-cleaving 2A linker peptides. Successful integration of this construct into plant cells catalyzes the production of betalains, making them visible to the naked eye as red colouration (He et al., 2020).
Although our workhorse for primary testing of the composite parts was the model plant Arabidopsis thaliana, we also examined various non-model plants to assess the host range of Agrobacterium. All plant cultures were carefully maintained within growth chambers situated in S1 areas or in S1 greenhouses, managed collaboratively by the university and the Max-Planck-Institute for Terrestrial Microbiology. These greenhouses are routinely used for microbiological work, specifically work with Agrobacterium, and thus adhere to stringent safety protocols to ensure that plant materials remain isolated from the external environment, thereby guaranteeing biocontainment.
Work on Arabidopsis and Setaria was conducted under strictly sterile conditions, utilizing biosafety cabinets. We meticulously cared for personal protection of team members. Safety lab coats play a crucial role in ensuring the safety of individuals working in laboratory settings. We strictly prevent any contaminations with plant material or Agrobacterium. All materials, whether contaminated with Agrobacterium or not, were carefully stored in S1 areas and subsequently autoclaved before disposal. This practice extended to soil, growth medium, and pots.
Engineered Agrobacterium strains have the potential to colonize and infect non-target plants, thereby disrupting natural ecosystems and potentially leading to unintended ecological consequences. Furthermore, the modified strains could potentially outcompete native strains of Agrobacterium rhizogenes, thereby impacting natural genetic diversity and ecological interactions within soil microbial communities. The heightened pathogenicity of these modified strains could also pose risks to crops or non-target plants, potentially resulting in economic losses or affecting the balance of local plant populations. Given our objective to enhance the virulence of the plant pathogen Agrobacterium rhizogenes, we placed paramount importance on implementing proper safety measures. Recognizing the potential environmental hazards posed by modified, more virulent Agrobacterium strains escaping from the laboratory or greenhouse, we took rigorous precautions as mentioned above.
In our unwavering commitment to safety and risk mitigation on our journey with Agrobacterium-mediated plant transformation, we looked into possibilities exploring the coupling of multiple auxotrophies into our research. This proactive approach, supported by established literature findings, showcases the potential of introducing several auxotrophic markers to significantly reduce escape frequencies, often to undetectable levels (below the limit of 3 × 10-11) (Lopez & Anderson, 2015). By strategically combining and optimizing multiple auxotrophies, we could proactively fortify our experiments, making them more resilient to any unforeseen circumstances, and ultimately fostering a safer laboratory environment. Possible targets for such an auxotrophy safety system are leucine, pantothenate, and arginine as these have been demonstrated before as an ideal target (Vilchèze et al., 2018).
Another possible strategy involves the adoption of phosphite synthetic auxotrophy. As we advance our Agrobacterium-mediated plant transformation techniques, the incorporation of phosphite as an essential nutrient offers a promising alternative. Phosphite auxotrophy emerges as a robust safeguard, significantly reducing the risk of unintended organism release, given that phosphite is naturally scarce in most environments (Asin-Garcia et al., 2022). Through the implementation of phosphite synthetic auxotrophy by incorporating the gene for a phosphite dehydrogenase together with a specific phosphite transporter into the genome of Agrobacterium and additionally deleting all genes which are necessary for the natural ability to transport phosphate, we would not only reinforce containment but also expand the potential applications of our research within controlled settings, further bolstering our biosafety protocols.
We have not only sought to enhance transformation efficiency but have done so while maintaining a steadfast commitment to biosafety. As we explore the potential of our modified strains, we remain aware of the environmental considerations and ecological impact that our work may entail. The responsibility of ensuring the safe and responsible advancement of plant transformation technologies remains at the forefront of our efforts. By adhering to strict safety guidelines, collaborating with experts, and exercising caution at every stage of our project, we aim to contribute positively to the field while safeguarding our laboratory, the well-being of our team and the broader environment.