Project
Human Practices
Dry Lab
Wet Lab
Jamboree
Faced with the untreatable fungal disease Verticillium Wilt, we began a journey driven by curiosity and a desire to find a solution. Recognizing the potential of bacterial-mediated RNA interference (bmRNAi), we utilized this technology to combat V. dahliae. Our efforts resulted in THAELIA, a modular and adaptable bacterial system that effectively fights the fungus using RNAi technology. As mentioned in the Project Description, THAELIA is a bacterial system that produces double-stranded RNA (dsRNA) molecules that specifically target essential genes in V. dahliae. The aim is to silence these genes and disrupt Verticillium’s growth and virulence by delivering dsRNA molecules directly to it. To ensure efficient delivery, the dsRNA molecules are encapsulated within Outer Membrane Vesicles (OMVs), which serve as carriers able to transport the dsRNA from the bacteria to the fungus. Once received by the fungus, the dsRNA triggers the fungus’s endogenous RNAi mechanism, leading to gene silencing. Thus, THAELIA provides a precise and sustainable defense against V. dahliae, offering a promising strategy for combating the devastating effects of Verticillium Wilt on the vulnerable olive groves.
Now two questions arise; Why RNAi? and Why bacterial-mediated RNAi?
In our efforts to address Verticillium wilt, we have explored various approaches to combat V. dahliae while preserving the environment. Dr Tsikou Daniela, Assistant Professor of Plant Molecular and Developmental Biotechnology, introduced us to the RNA interference (RNAi) strategy. She explained that dsRNAs could be produced and used to activate an existing mechanism within the fungus [1]. The RNAi pathway is a natural process within V. dahliae that we aim to harness in order to silence fungal genes vital for survival. This environmentally-friendly approach perfectly aligns with our goal to protect olive trees while minimizing ecological impact. Additionally, RNAi promises species-specific precision in defeating the deadly fungus, guiding the way towards a sustainable solution to this urgent challenge. This approach ensures that we can target species-specific loci, or species-specific genes that only exist in Verticillium. This is important because it ensures the preservation of biodiversity in our land, especially in our olive groves [2]. By utilizing various bioinformatics tools, we identified gene regions with high homology exclusive to Verticillium species. This precision ensures that our RNAi strategy is both safe and effective. The dsRNA molecules designed to trigger the RNAi mechanism will not affect other microorganisms, even if they are endocytosed by them. Moreover, dsRNA functions as a non-toxic pesticide, minimizing any negative environmental impact [3]. RNAi also enables us to target important genes involved in the fungus' growth during a vulnerable stage of its life cycle (hyphal development phase). By silencing these genes, we aim to halt the growth of Verticillium, offering a potent method for combating this devastating disease.
1.Processing of
dsRNA: The dsRNA that
enters the fungus is cleaved by the enzyme DICER, producing small interfering RNAs (siRNAs).
2.Methylation of siRNA: The
resulting
siRNAs are then methylated to enhance their stability.
3.Formation of RISC: The methylated
siRNAs are loaded into the Argonaute (AGO) protein, leading to the assembly of the
RNA-induced silencing complex (RISC) with the recruitment of additional proteins.
4.Activation and Targeting: Under
specific thermodynamic conditions, the dsRNA separates into single-stranded RNA. This
single-stranded RNA, together with the RISC complex, binds to the target mRNA, resulting in
one of two possible outcomes:
a) The mRNA is cleaved and degraded, leading to silencing.
b) The translation of the mRNA is blocked, preventing protein synthesis
Figure 2: RNAi mechanism. In the picture, the DICER protein (depicted in blue) is necessary to process the dsRNA. The AGO proteins (depicted in green and yellow) form the RISC complex with the siRNAs and target the fungus mRNA for silencing either with mRNA cleavage or translation blockage [4].
The dsRNA molecules are effective, but they degrade rapidly in the soil. BmRNAi involves using bacteria to produce and deliver the dsRNA, which shields it from environmental degradation and ensures that it can more effectively reach the target organism. The introduction of bacteria into the soil has the potential to establish direct communication with the fungus, enabling highly efficient delivery of dsRNA while protecting it from degradation. Moreover, employing bacteria presents a cost-effective solution, with significantly shorter development and testing timelines compared to the production of genetically modified Verticillium-resistant plants [5]. This method also leads to the production of substantial dsRNA quantities without necessitating constant re-application, thus serving as a preemptive measure and protection for the olive tree's defense against possible infection. This strategy functions as an advantage over the fungus, as it enables the dsRNA's delivery without additional external interference [6]. Additionally, bmRNAi is a sustainable alternative to chemical antifungal pesticides, as it is environmentally friendly and does not produce ecotoxic effects. Unlike chemical pesticides, which can harm the environment, degrade soil quality, and negatively impact plant and animal health, bmRNAi targets specific genes without contributing to antifungal resistance. Antifungal resistance is a heritable fungal trait which develops through natural selection when fungi are exposed to large quantities of antifungal chemicals. The pressure exerted on fungi by exposure to a fungicide selects resistant strains. Moreover, bmRNAi enables the creation of an adaptive bacterial system that can be easily modified to target different genes if resistance develops, offering a modular and customizable solution for managing various plant pathogens [7].
Before landing on bacterial-mediated RNAi, we carefully considered all the proposed available techniques for the implementation of RNAi. These included Spray-Induced Gene Silencing (SIGS), Host-Induced Gene Silencing (HIGS), and Virus-Induced Gene Silencing (VIGS). Each of them proved to be less than an ideal candidate for our approach. Firstly, SIGS, which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, would not be able to perform against our soil borne fungus, as it is not able to penetrate the soil in an adequate capacity. This limitation arises from the high degradation of dsRNA in the soil, reducing its efficacy [8]. Furthermore, implementing HIGS would necessitate the alteration of the olive plant itself, a process that is both time-consuming and labor-intensive, leading to a lack of cost-effectiveness. This approach would also require the introduction of a new genetically modified cultivar. This solution is not feasible for the current issue, as the existing cultivars in Greek olive groves would remain vulnerable to the harmful fungus. Moreover, the requisite timeframe for producing such modified cultivars provides sufficient opportunity for the fungus to develop resistance to the engineered modifications [9]. Finally, VIGS is a method that uses the plant RNAi-mediated antiviral defense mechanism. When plants are infected with unmodified viruses, this mechanism targets the viral genome. However, when virus vectors carry sequences from host genes, the process can also target the corresponding host mRNAs [10]. VIGS is not suitable for treating diseases that affect dead tissues, such as the affected xylem of the olive tree. In contrast, bacterial-mediated RNAi juxtaposes an advantage for each of the aforementioned disadvantages and more. Dr. Dalakouras Athanasios helped us understand that bmRNAi is the most suitable way to implement RNAi technology for our project, seamlessly connecting production, protection, and delivery of dsRNA to combat V. dahliae.
To effectively combat the fungus, we focused on identifying essential genes critical to its virulence and survival. Additionally, we aimed to pinpoint regions within these genes that lack homology with genes from other microorganisms, plants, or animals. This approach is imperative in preserving biodiversity, ensuring food source security, and preserving human health. After conducting a thorough literature review and consultation with specialists, we successfully narrowed our targets down to three important genes: VdRGS1 (Regulator of G protein Signaling) , VdAAC (ADP, ATP Carrier), and VdTHI20 (Phosphomethylpyrimidine kinase). We chose multiple genes to enhance the strategy against the fungus and minimize the likelihood of it developing resistance to RNAi. Our deliberate selection of three genes aimed at minimizing the potential for simultaneous resistance development to all three genes through natural selection [11].
You can learn more about our gene targets by clicking the follow buttons:
G protein signaling is one of the most important signaling mechanisms in fungi. G Protein Coupled Receptors (GPCRs) detect extracellular signals and relay them to heterotrimeric G proteins (Gα, Gβ/Gγ subunits), which then integrate them into intrinsic signal transduction pathways. In the inactive state, the Gα subunit binds to the Gβ/Gγ subunits and GDP. Upon activation by a signal, GPCR exchanges GDP with GTP, resulting in the detachment of the Gβ/Gγ complex from the Gα subunit. Both Gα and Gβ/Gγ subunits are now capable of activating downstream signaling pathways. The signal transduction is terminated when the Gα subunit hydrolyzes GTP back to GDP, returning the system to its inactive state [12]. Regulators of G Protein Signaling (RGS) proteins serve as negative regulators of signaling pathways by deactivating the Gα subunit. They accelerate GTP hydrolysis, thereby rapidly switching off G protein-coupled signaling pathways and blocking downstream signals, as shown in Figure 3 [13].
Figure 3: RGS1 in action: Signaling through G protein coupled receptors. RGS1 proteins accelerate the GTPase activity of Gα subunits, promoting the rapid conversion of GTP to GDP. This inactivation of Gα subunits effectively turns off G-protein signaling [13, 18]
RGS1 appears to play a significant role in the virulence of V. dahliae, as it shows increased expression in the roots of affected plants. It is crucial during various developmental stages of the fungus, including spore germination, hyphal development, spore production, and microsclerotia formation. VIGS in this gene has been shown to increase plant resistance to Verticillium wilt [13]. Sarmiento-Villamil et al. proposed a model in which RGS1 is crucial for the fungus's transition from the biotrophic phase, where it infects living plants, to the saprophytic phase, where it survives in the soil by feeding on dead plant material. According to their model, RGS1 is inactive during the biotrophic phase, where G protein signaling is necessary for interaction with the plant host, and becomes active later during infection, when the fungus shifts to the saprophytic stage, focusing on vascular colonization rather than proliferation [14].
The protein AAC (ADP, ATP carrier) is highly conserved and abundant, playing a major role in the energy metabolism of the fungus. It is essential for maintaining the ADP/ATP balance by mediating the exchange of ADP and ATP between the mitochondria and the cytoplasm [15].
Figure 4: Function of ADP/ ATP carrier. The ADP/ATP carrier is a protein that facilitates the exchange of ATP and ADP across the mitochondrial membrane. It transports ATP out of the mitochondria into the cytoplasm and brings ADP back in, maintaining the cell's energy balance. [16,19]
VdAAC contributes to fungal germination, development and sporulation. Silencing of this gene resulted in smaller colony diameter and fewer conidia. [16].
The kinase VdTHI20 is part of the thiamine biosynthesis pathway. Thiamine consists of two aromatic components, a pyrimidine (2-methyl-4-amino-5-hydroxymethylpyrimidine, HMP) and a thiazole (4-methyl-5-β-hydroxyethyl thiazole, HET), which are synthesised in independent branches of the thiamine synthesis pathway. VdTHI20 plays a role in the pyrimidine synthesis branch by phosphorylating HMP to HMP-P and HMP-P to HMP-PP [17].
VdTHI20 plays a role in colonisation, conidia germination and production, and hyphae morphology. Host-induced gene silencing (HIGS) of THI20 resulted in weak disease symptoms and impaired fungal growth [17].
The ultimate goal of our bacterial system is to produce dsRNAs targeting V. dahliae genes and deliver it to V. dahliae via Outer Membrane Vesicles (OMVs). The OMVs will reach out to the fungus, and the dsRNA will be released into the fungus cytoplasm to activate the RNAi mechanism. To achieve this, our system consists of three distinct pillars:
1.Production of dsRNA: The dsRNA molecules are a significant component of the system, as they are responsible for inducing gene silencing in V. dahliae.
2.Encapsulation of dsRNA: To secure the adequate incorporation of dsRNA molecules into the OMVs, a proactive approach was designed to guide and encapsulate the dsRNA molecules within the vesicles. This strategy increases the efficacy of dsRNA encapsulation, by making it less dependent on chance.
3.Production of OMVs: In order to make easier the effective delivery of dsRNA molecules to the fungus, our system is engineered to generate a substantial quantity of OMVs, increasing the probability of successful delivery .
In synthetic biology, the chassis is the cellular host that acts as a dynamic canvas for engineered biological systems. By combining biology with engineering, the chassis allows an organism to be used in any desired application [18]. Our project aims to use a bacterial system to protect olive trees from Verticillium dahliae, by colonizing both inside and around the roots. By utilizing engineered Pseudomonas putida for bacterial-mediated RNA interference (bmRNAi), we strive to combat this soil-borne pathogen before it infiltrates the olive tree. Chosen for its role as a Plant Growth-Promoting Rhizobacterium (PGPR), P. putida offers several advantages. First of all, this bacterium exhibits the colonization capabilities of olive tree roots and olive tree root hairs [19]. Moreover, through the utilization of a Ribonuclease III (RNAse III) deficient strain, it has the capacity to produce dsRNA. RNase III is an endoribonuclease enzyme that catalyzes the degradation of double-stranded RNA (dsRNA) molecules [20] . Additionally, it has the ability to generate OMVs that effectively make possible the transfer of dsRNA [21]. This innovative approach represents a significant advancement in our mission to protect olive tree health.
Initially, we explored the idea of using a fungal chassis for our project, drawn to fungi's ability to deliver small RNAs. Trichoderma seemed promising due to its capacity to colonize olive roots and act as a Biocontrol Agent (BCA) against Verticillium dahliae. However, consultations with Dr. Pateraki Chrysanthi, a professor at our university, revealed that our department’s limited resources and inexperience made fungal modification highly challenging. Furthermore, Dr Aliki Tzima raised concerns about the RNAi mechanism in the fungal chassis because if RNAi is active within this chassis, it could alter or degrade the small RNAs before they have a chance to specifically target Verticillium. This interference might reduce the efficacy of the RNAi-based strategy by preventing the precise and effective silencing of the fungal genes responsible for the disease. As a result, we redirected our focus to bacterial BCAs. Upon conducting a thorough literature review and discussion with Dr. Dalakouras Athanasios, we have chosen to direct our focus towards Plant Growth-Promoting Rhizobacteria (PGPRs), specifically Bacillus and Pseudomonas spp. The indispensable role of Bacillus' RNAse III in its survival mechanisms, suggests a tough challenge to obtain a Bacillus RNAse III deficient strain that results in a substantial production of double-stranded RNA [22]. After careful consideration, we have determined that Pseudomonas spp. presents as the optimal selection for our intended purposes. Pseudomonas spp. are non-pathogenic, gram-negative bacteria that thrive in soil and are easily manipulated in the lab [23]. Our next step was to select a specific strain. We required a strain that was RNAse III-deficient, capable of producing Outer Membrane Vesicles (OMVs), had endophytic properties in olive roots [24]. After consulting with Dr. Jesus Mercado Blanco, who highlighted the endophytic abilities of Pseudomonas putida and its potential as a biological control agent, we identified P. putida as the most suitable chassis for our needs.
1.Production of dsRNA
When considering the induction of gene silencing in the fungus, we knew that we needed to produce the dsRNA molecules efficiently within a bacterial system using
an orthogonal approach. This approach had to ensure that the dsRNA structure would be suitable for delivery via
OMVs. The effectiveness of our system is influenced by the production
yield, stability, and successful
encapsulation of these RNA molecules [25].
To address those factors we firstly selected the bacteriophage T7 RNA
polymerase to produce the dsRNA molecules, aiming for higher
yield and greater orthogonality. Secondly, because both dsRNA
and hairpin RNA (hpRNA)
can be effectively processed by the RNA interference (RNAi) pathway, the
choice between dsRNA and hpRNA ultimately depends on which molecule can be more efficiently produced and delivered by our
bacterial host. After an extensive literature review and consultation with researchers, we concluded
that dsRNA is the optimal choice [26].
The bacteriophage T7 RNA polymerase is a widely used tool for expressing
foreign genes in
E. coli. This enzyme is extremely efficient, resulting in much
higher transcription rates than those achieved by the cell's own RNA polymerases. It also
has a low transcription termination frequency and is highly specific to its T7RNAP promoters
[27]. This specificity helps maintain the system's
orthogonality, as the cell relies solely
on T7 polymerase to transcribe the introduced gene.
Recent work by Beentjes et al. has optimized the system for
Pseudomonas putida by replacing the T7 bacteriophage TΦ
terminator with a more effective terminator downstream of the gene
of interest. This enhancement improves transcriptional termination, ensuring that the gene
of interest is expressed efficiently and that transcription does not continue into adjacent
sequences. The use of a stronger terminator reduces the likelihood of read-through
transcription and stabilizes the expression of the target gene. Additionally, Beentjes et al. implemented a weaker ribosome binding site (RBS)
upstream of the T7 RNA polymerase gene. This adjustment prevents the overproduction of T7
RNA polymerase, avoiding unnecessary accumulation within the cell and reducing potential
metabolic burden. These modifications collectively enhance the efficiency and precision of
gene expression in the system [28]. Inspired by their work, we
decided to test the T7 system
ourselves. We decided to use the synthetic terminator T7 terminator
hyb6 identified by Calvopina-Chavez et al., which
have shown high termination efficiency [29].
After discussing our approach with Dr. Jeffrey E. Barrick, we decided to integrate the T7 RNA polymerase gene into the chromosome because with many copies
of the T7 polymerase the system would be unstable. By doing that we also establish a modular
system where the T7 polymerase and the dsRNA sequence are maintained in distinct genetic
locations.
In bacteria, hpRNA is cleaved into smaller dsRNA fragments, even in RNase III-deficient E. coli [30]. This poses a challenge for our system, as we aim to use the fungal RNAi pathway to produce siRNAs of specific lengths [31]. Unintended cleavage of hpRNA into shorter dsRNAs could interfere with this process. Furthermore, after discussing with Dr. Rennos Fragkoudis, we concluded that producing hpRNA the same length as a dsRNA molecule would consume more cellular resources and take up more space inside OMVs. This would increase cellular stress and make encapsulation more difficult, ultimately lowering efficiency. Lastly, the production of dsRNA using T7 polymerase and two oppositely oriented T7 promoters is a common method used both in vivo and in vitro [32].
2.Encapsulation of the
dsRNA
Encapsulating
dsRNA within OMVs presented our team with a fascinating challenge:
how to ensure that the dsRNA is effectively and efficiently delivered inside these
vesicles? Our breakthrough came through exploring the innovative Targeted and Modular
Extracellular Vesicles Loading TAMEL approach,
pioneered by Professor Joshua N. Leonard and his team. Traditionally, RNA was loaded into
Extracellular Vesicles (EVs) passively, a method that often lacked precision and control. TAMEL
revolutionizes this by actively directing RNA into EVs using a sophisticated fusion protein with two
crucial domains: one that embeds into the EV membrane and another that specifically binds the RNA
[33].
Inspired by TAMEL, we designed a chimeric protein with two key components. The first is a dsRNA binding domain (dsRBD) designed to tightly bind to dsRNA, ensuring that the RNA is securely attached. The second domain is able to integrate with the OMV membrane, positioning the RNA-binding domain in close proximity to the vesicle interior. This dual-domain approach not only enhances the precision of RNA loading but also maximizes the efficiency of dsRNA delivery, paving the way for more effective therapeutic applications.
Selecting a dsRNA binding domain for encapsulation holds a revolutionary advantage due to its universal and reliable binding properties. Unlike RNA motifs, which are often destined for specific sequences and can be unpredictable, a dsRNA binding domain interacts with dsRNA with precision, no matter the sequence. This approach aligns with synthetic biology's principles: modularity and standardization. By employing dsRNA binding domains, we eliminate the uncertainty associated with sequence-specific motifs, ensuring that dsRNA is encapsulated within OMVs with consistent efficiency. This approach simplifies the design of RNA delivery systems and reflects the core of synthetic biology, enabling more reliable and scalable RNA delivery solutions [34]. So, dsRNA binding domains (dsRBDs) can be used to bind double-stranded RNA (dsRNA) due to their ability to recognize RNA based on its structure. Their binding is shape-dependent rather than sequence-specific, allowing them to interact with a broad range of dsRNA molecules [35]. Following Dr. Rodolfo Rasia's recommendation, we chose to use the RNase III dsRB, a protein that naturally binds and processes double-stranded RNA in bacteria [36]. To find the best candidate, we tested several options using a tool developed by our dry lab.
Incorporating a linker is essential to connect the dsRNA binding domain and the domain responsible for transporting dsRNA into OMVs. The linker ensures proper spacing and alignment, allowing both domains to function efficiently without interfering with each other. This improves the overall stability and ensures that the dsRNA is effectively bound and delivered into OMVs. After consulting with Dr. Rodolfo Rasia, it was recommended that the linker should be at least 40-50 bp long to allow the domains to adopt their proper conformations. In cases where the linker is too short, it can be repeated, but care must be taken not to use the exact same sequence to avoid the risk of it forming a self-loop. It's also important to avoid serines and lysines, as they tend to perform poorly in this context. An already existing linker within the cell could offer an effective solution [37]. The RNase III protein naturally contains a linker that separates its catalytic domain from its dsRNA binding domain, allowing the protein to maintain flexibility and function properly. This RNase III linker, given its native role, could be an effective choice for our design, providing the right spacing and flexibility between the two domains to optimize performance [38].
To select an effective domain for targeting dsRNA into outer membrane vesicles (OMVs), it is needed to focus on outer membrane proteins (OMPs) and lipoproteins. [39] Research shows that proteins not directly connected to the peptidoglycan (PG) layer, such as lipoproteins, are more likely to be incorporated into OMVs. OMPs, which are beta-barrel proteins that extend across the outer membrane, are also commonly found in OMVs and are strong candidates for our purpose. However, one challenge with OMPs is that they can migrate within the membrane, potentially limiting their inclusion in OMVs [40]. Our goal is to find a protein that can efficiently dock dsRNA to the outer membrane and localize well within OMVs. Dr. Meta Kuehn advised us to consider porins, which are transmembrane proteins that are abundant in cells and have the right structural properties for this role. Among the available options, OMPs such as OprC, OprD, OprE, OprF, OprH, and OprG from P. putida KT2440 are potential candidates. Additionally, for our design, we need a protein small enough to fit into OMVs and be reliably localized in P. putida vesicles. Based on these criteria, OprD and OprF stand out as the best candidates. These proteins are well-suited for targeting dsRNA into OMVs and offer the necessary characteristics for successful integration into our system [41].
3.Production of OMVs
To enhance the production of outer membrane vesicles (OMVs) in our bacteria, we target the
protein TolB for degradation. We modify TolB by adding TEV protease
cleavage sites, allowing it to be cut by the TEV protease. By using a TolB
mutant and introducing a new TolB variant with TEV sites, we
can effectively regulate the Tol B’s degradation. We chose to work with TolB because it plays a
crucial role in OMV formation, and its targeted degradation can significantly increase OMV
production.
TolB plays a key role in maintaining the connection between the
bacterium's outer membrane and its cell wall. TolB protein is found in the periplasm, the space
between the outer and the inner membrane [42].
When TolB levels are reduced, the outer membrane becomes less stable, which leads to the formation of more OMVs. To achieve this reduction, we will introduce TEV protease recognition sites into the TolB protein. These sites allow TEV protease to specifically target and break down TolB, reducing its levels in the cell [43]. By doing so, we can trigger increased OMV production in a controlled manner. This approach is inspired by the controlled hypervesiculation methods used in the iGEM UZurich 2021 project.
These OMVs will transport dsRNA to fungi, providing protection from degradation and facilitating its uptake. Once inside the fungal cells, the OMVs will trigger an RNAi mechanism to silence fungal genes and combat the disease [44].
As suggested by Dr Andrea Masotti, there are two possible mechanisms for the release of dsRNA into Verticillium using OMVs. The first mechanism is that OMVs behave like exosomes, where the vesicle membrane fuses with the fungal cell membrane and releases the dsRNA directly into the cytoplasm [45]. This process is complex, as it involves interactions between vesicle surface proteins and receptors on the fungal cell, initiating a signaling cascade. The second mechanism involves OMVs entering the fungal cell through endocytosis. Once inside the cell, the vesicle could potentially burst due to the acidic environment, releasing its dsRNA contents into the cytoplasm [46].
OMVs are small spherical structures produced by Gram-negative bacteria that form from the outer membrane and contain materials from inside the cell. These vesicles help bacteria interact with their environment, support their survival under stress, and aid the communication within bacterial communities [47]. OMVs have the ability to transport a variety of biomolecules, including nucleic acids, proteins, and lipids. These tiny vesicles can carry their cargo over significant distances [48]. Additionally, OMVs show promise for delivering biological molecules, including RNA-based therapeutics. Their ability to encapsulate and protect dsRNA molecules, combined with the potential for receptor-mediated targeting, makes them ideal candidates for specific delivery to pathogens [49]
TEV protease is a good choice for cleaving TolB because of its high sequence specificity, recognizing the ENLYFQS sequence and cutting precisely between Q and S. This reduces off-target effects compared to proteases like factor Xa or thrombin [50]. Its catalytic mechanism involves a cysteine, making it resistant to many common protease inhibitors. This precision and reliability make it suitable for reducing TolB levels and increasing outer membrane vesicle production, while maintaining the orthogonality of the system [51].
Our bacterium has been engineered to perform multiple tasks, including the production of dsRNA, the formation of Outer Membrane Vesicles (OMVs), and the encapsulation of dsRNA within OMVs. To optimize efficiency and minimize cellular stress, we have strategically distributed the regulation of these procedures across different phases of the bacterial life cycle. Using specialized auto-inducible promoters, we regulate gene expression in two phases: during the exponential phase, T7 polymerase and dsRNA are expressed, while the stationary phase triggers the production of TEV protease, promoting OMV formation, as it is shown in Figure 16. This division distributes the workload, ensuring optimal performance at each stage. Additionally, this modular system can be easily adapted to target various fungal pathogens by altering the dsRNA sequence, making it highly versatile for combating multiple fungal diseases.
Exponential phase
The BG37 promoter was chosen for producing T7 polymerase in our system due
to its unique ability to auto induced gene expression during the exponential
phase of the bacterial life cycle. This engineered promoter had been previously tested on
P. putida, making it a promising candidate for our needs. Since the
exponential phase is when bacterial cells are most active and capable of high metabolic output, we
needed a promoter that could take advantage of this window for efficient production of T7 polymerase
[52]. To ensure BG37 was the right fit for our system, we conducted
tests to evaluate both its
strength and the precise phase of activation. These tests confirmed that BG37 effectively drives
expression during the exponential phase, offering strong and reliable gene activation without
requiring external inducers. This characteristic made it ideal for our project, as it allowed for a
natural, self-regulated expression system perfectly timed with the bacterial growth cycle,
optimizing the production of T7 polymerase while minimizing additional stress on the cells [53].
The T7 polymerase production results in the simultaneous production of dsRNA (Figure 18) and the chimeric protein (Figure 19), as both are under the control of the T7 promoter. Placing both the dsRNA and the chimeric protein under the control of the same T7 promoter ensures synchronized expression, allowing both components to be produced simultaneously and in proportionate amounts. This coordination is crucial for efficient packaging of the dsRNA into outer membrane vesicles (OMVs) by the chimeric protein. As both elements are required for successful delivery, their co-regulation simplifies the system's design and ensures that sufficient quantities of each are available at the same time, optimizing vesicle formation and dsRNA delivery.
Stationary phase
The use of the P3.1 promoter, which is auto-inducible during the stationary phase, offers a
strategic advantage for outer membrane vesicle (OMV) production by reducing cellular stress and
optimizing dsRNA encapsulation [54]. By inducing OMV formation in the
stationary phase, we allow the
cell to prioritize the earlier production of dsRNA and the chimeric protein, both of which are
driven by the T7 promoter during the exponential phase. This ensures that when OMV production is
activated, the vesicles will efficiently encapsulate the already synthesized dsRNA. Furthermore, by
limiting OMV production to the stationary phase, we mitigate the stress associated with continuous
vesicle formation, preserving cell viability and maximizing OMV yield for effective delivery of
dsRNA to the target organism.
The whole system
Our system is designed to efficiently produce dsRNA and form Outer Membrane Vesicles (OMVs) in P. putida through a regulation strategy, based on auto inducible
promoters, that ensures each function occurs at the optimal time, minimizing stress on the bacterial
cells and enhancing overall performance. To further enhance stability and consistency, and following
a suggestion from Jeffrey E. Barrick, we integrated the T7 polymerase and eTEV protease
transcription units directly into the chromosome. This ensures stable and
consistent expression of these critical components without the need for external plasmids, reducing the risk of plasmid loss. Furthermore, this approach enhances the
system’s reliability by reducing cell-to-cell variability, leading to
more uniform expression levels across the bacterial population [55]. In
contrast, the dsRNA and
chimeric protein production units are maintained on plasmid vectors, giving the system modularity
and flexibility. By using plasmids for these elements, we can easily swap or modify the dsRNA targets or chimeric proteins depending on the fungal
disease we aim to combat. This modular design allows for quick adaptation
of the system to different targets without needing to alter the core components which are integrated
into the chromosome. Overall, this dual approach, combining chromosomal integration for core
functions with plasmid-based modularity for targeting, creates a highly flexible
and adaptable bacterial system.
Searching for an inducer
To ensure our system survives long enough in the plant root to effectively combat V. dahliae, we searched for an inducer, a fungal signal, that would be able to activate our system. This ensures that
the bacteria stay dormant when the pathogen is absent, conserving energy and extending their lifespan. Additionally, this pathogen-specific regulation
enhances biosecurity, as the bacteria only reproduce in the presence of
Verticillium, reducing the risk of unintended environmental impact.
For this purpose, we designed an innovative approach: a gate that controls
bacterial replication, ensuring that our bacteria remain in a state of minimal metabolic activity,
unless activated by the presence of the pathogen. Once the bacterial formula has been implemented
in the plant roots, the bacteria stop reproducing and remain in a lag phase, with their replication
triggered only when they detect the Ave1 protein, an effector secreted by
V. dahliae [56]. This creates a tightly
regulated system where the
bacteria conserve energy and remain inactive until they encounter the pathogen, extending their
lifespan within the rhizosphere.
In plants, Ave1 binds to the Ve1 receptor, triggering an immune response that leads to resistance to Verticillium species [57]. So, we decided to design a chimeric receptor that combines the Ve1 extracellular domain with the intracellular signaling domain of EnvZ, a histidine kinase. The chimeric Ve1-EnvZ receptor is expressed in P. putida constitutively under the J23119 promoter, ensuring its continuous presence on the bacterial membrane.
When Ave1 binds to the Ve1 receptor expressed in the engineered bacteria, it triggers a signaling cascade, as it is shown in Figure 23. The binding activates the EnvZ kinase, which transfers a phosphate group to the response regulator OmpR. By expressing E. coli’ s EnvZ-OmpR two-component system, that is not naturally present in P. putida, we ensure the preservation of orthogonality in our system, as it avoids interference with any native signal transduction pathways in the bacterium [58]. Phosphorylated OmpR binds to the ompC promoter, which we engineered to control the production of DnaA. By placing the ompC promoter upstream of the endogenous DnaA gene, we ensure that DnaA, a key regulator for initiating the bacterial cell cycle which is needed to start DNA replication, is only produced when Ave1 is detected [59]. This precise system allows P. putida to transition from lag phase to exponential phase in response to the presence of V. dahliae. So, without the presence of the fungus, the bacterial population wouldn’t be able to grow and spread conserving resources and ensuring they don’t activate prematurely. This allows the bacteria to enter the exponential phase and activate further gene expression only when needed, ensuring both efficiency and safety in the application of the engineering P. putida.
Biocontainment
To ensure the safety of our design, we incorporated another biocontainment approach alongside the
gate: double auxotrophy. This requires the bacteria to rely on two
external nutrients for survival, preventing uncontrolled growth in the absence of those essential
compounds in the environment. For more information, you can visit our Safety page.
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