Project
Human Practices
Dry Lab
Wet Lab
Jamboree
Olive trees, celebrated by poets, writers, and artists, since ancient times, hold a significant role in Greek mythology like no other tree. They are a major crop and a cornerstone of the agricultural economy, reflecting their enduring importance and cultural heritage. In Greece, the olive is a pillar of our diet and our economy with olive groves comprising approximately 79% of the total land dedicated to tree crops. In Thessaly alone, 5.8% of the region is occupied by olive trees, highlighting the olive industry's impact on a local level [1]. Despite their extensive cultivation, olive trees are often infected by several microorganisms, which affect their productivity and longevity. Such a microorganism is the fungus Verticillium dahliae, that has become notorious as the bane of Mediterranean farmers [2]. The disease is currently the most serious threat to olive cultivation, globally, with profound consequences for the growers [3].
With a deep-rooted connection to rural life, our team understands the profound significance of the olive tree and the severe impact of Verticillium dahliae . We have witnessed firsthand the extensive damage this pathogen inflicts on the livelihoods of team members who are dependent on olive tree cultivation and their surrounding communities. Through conversations with specialists and farmers, we have gained valuable insights into how Verticillium dahliae targets more than 300 different crops such as tomatoes, cotton, and eggplants, with its most significant impact felt in olive cultivation. This fungal disease poses a significant threat not only locally but also globally, deeply concerning growers,nursery companies, and the olive oil industry worldwide. Verticillium wilt affects olive groves in Asia, North and South America, Oceania, and South Africa [4]. For the Mediterranean basin especially, this threat is imminent, as the olive species under cultivation are highly susceptible to the fungus resulting in yield losses and tree mortality [5].
Local olive growers and professors have confirmed the significance of this issue, noting the disease's persistent presence in orchards, which drastically reduces tree productivity and jeopardizes their survival. Moreover, according to the growers, there is no recovery for infected trees, resulting in significant economic losses. Despite their efforts, there is currently no permanently effective treatment for the disease. Given that most cultivated olive trees lack resistance to this pathogen and with climate change exacerbating conditions, Verticillium wilt continues to pose a growing challenge in our communities and beyond.
The fungus survives as structures named microsclerotia in dead plant tissues in the soil. Microsclerotia germinate, producing hyphae, which penetrate and colonize the roots. Colonization of the xylem follows, as well as the production of conidia, which causes xylem vessel occlusion. As a result, the presence of conidia blocks the transport of water and nutrients, and symptoms start to develop. Lastly, microsclerotia are formed in dying tissues and are released into the soil via the incorporation of the infected tissues [6]. Microsclerotia can remain dormant in the soil for up to 20 years, patiently waiting for the opportunity to infect the next tree.
Regarding the symptoms, leaf discoloration, drying, leaf drop, and some individual branches of the tree dry up, and sometimes half of its sides, are characteristics of the disease [6].
The only measures available are chemical treatment, planting healthy seedlings, avoiding planting and intercropping in spots with previously infected trees, soil solarization, pruning symptomatic branches and burning them immediately, and finally disinfecting the tools used [6]. Our team learned through communication with farmers, producers, and professors that the current measures are ineffective and expensive for the management of this dangerous plant pathogen, which continues to trouble the olive oil community [8]. Recognizing the limitations of current disease management methods, we believe it is imperative to explore alternative approaches. Creating a biological system, using synthetic biology principles, represents a promising solution to address this seemingly untreatable problem.
Our project aims to develop a bacterial formulation to protect olive trees from Verticillium dahliae . The treatment involves using engineered P. putida to facilitate bacterial-mediated RNA interference (bmRNAi). In particular, we will deploy a bacterial system designed to produce double - stranded RNA (dsRNA) and deliver it to Verticillium, resulting in specific gene silencing [9]. To ensure the dsRNA reaches Verticillium effectively and withstands the harsh conditions of the soil, we are utilizing Outer Membrane Vesicles (OMVs) as the transport mechanism. These OMVs will protect the dsRNA during transit, ensuring it remains intact and functional upon reaching its target [10]. P. putida strain we plan to use functions as a Plant Growth Promoting Rhizobacterium (PGPR) [11] and will colonize the roots of olive trees. This bacterial colonization will enable the bacteria to protect the trees both around and within the root system. Should Verticillium appear, the P. putida will act to prevent the infection, safeguarding the olive trees from this pathogen.
Our engineered bacteria will have three distinct goals:
1. The dsRNA production
2. The encapsulation of the dsRNA into the OMVs
3. The production of the OMVs. To promote controlled hypervesiculation in P.
putida,
we have taken inspiration from iGEM
UZurich's 2021 project.
Firstly, dsRNA production relies on an expression system based on T7 polymerase, derived from the T7 phage, which has proven to be highly effective [12]. Additionally, by incorporating the T7 polymerase, we ensure that our system remains orthogonal, avoiding any unwanted effects on other cellular processes [13], [14]. Secondly, the dsRNA will be delivered near the bacterial membrane using a chimeric protein that combines an RNA Binding Domain (RBD) with an Outer Membrane-associated protein. Thus, the produced dsRNA will bind to the protein and be transferred near the membrane, ensuring its encapsulation into the OMVs.
Finally, to produce OMVs, we aim to selectively disrupt the bacterial membrane. An effective approach involves reducing the levels of the periplasmic protein TolB, which plays a crucial role in maintaining membrane integrity [15]. To achieve this, we will employ a mutant TolB protein with TEV cleavage sites and express TEV protease during the stationary phase of bacterial growth. So, during the stationary phase, the TEV protease cleaves the TolB protein, triggering the release of a substantial quantity of OMVs.
1.TEV Protease Production
2.TEV Protease Cleavage of TolB
3.Disruption of Bacterial Membrane
Stability:
The cleavage of TolB leads to destabilization of the bacterial membrane.
4.Overproduction of Outer Membrane Vesicles (OMVs)
These OMVs will be the mode of transport of the dsRNA in order to reach and be taken up by the fungus [16], whilst also acting as a form of protection from degradation [17].
To identify our gene targets, we conducted an extensive literature review and consulted with several field experts. Our goal was to pinpoint essential genes in Verticillium dahliae to silence, focusing on those crucial for the fungus's growth and virulence while ensuring the targets are species- specific to avoid unintended effects on other organisms. Through our research, we identified three genes critical for the early stages of fungal growth:
By silencing these essential genes, critical for Verticillium's survival, we effectively ensure that the pathogen cannot persist.
The engineered P. putida strain will be applied into the soil as part of our solution, to colonize the root of olive trees and establish its endosymbiotic capability before infection from Verticillium dahliae occurs, effectively preventing it. We envision our project as a strategic intervention during crucial periods when olive trees are most vulnerable. To ensure the successful implementation of our project, we actively engaged with local olive producers, whose lives and livelihoods are deeply affected by Verticillium wilt. Based on their valuable feedback, we identified the optimal times to apply our irrigation formula:
By aligning our strategy with these critical times, we aim to provide robust protection for olive trees, ensuring their health and longevity against the threat of Verticillium wilt.
Our innovative system presents an effective solution for combating an otherwise untreatable fungus. Utilizing non-toxic dsRNA molecules that target specific sequences, it ensures biodiversity protection without harming plants, human health, or the environment [21]. Modular and adaptable, this system can easily be tailored to treat various pathogens through RNAi and OMV technology. As an autonomous biological system, it independently produces both dsRNA and OMVs. This approach is cost-effective, by avoiding the complexities and costs associated with industrial production of dsRNA and OMVs, allowing bacteria to grow and function autonomously [22],[23].
Studying microbe-plant interactions has provided researchers with invaluable information on the inner workings of the plant holobiont [24]. However, the complexity of plant microbiomes has made it challenging for researchers to fully understand their dynamics and functions. In a similar manner, genetically modified organisms need to be studied extensively before being tested and utilized in real settings, due to their highly complicated nature.
We faced similar concerns when designing our approach for the biocontainment of our engineered P. putida. With the goal to reinforce the biosecurity of our project we studied already existing methods for testing Bacteria-Plant interactions inside the lab. We discovered platforms that can simulate field conditions [25]. These devices enable biotechnologists to experiment on plants that have been grown alongside both mutualistic and antagonistic microbes, as well as engineered bacteria
While studying these systems we noticed a pattern: they are usually highly abstract in the sense that they do not fully replicate the complexity and variability of real-world environments. At the same time, as our team comprises undergraduate and over graduate students we quickly became daunted with the complexity of some of these platforms - conducting even the simplest experiments required significant expertise and effort. There is an apparent need for a platform that will succeed in a “global meta-omic approaches for quantifying all possible changes” (Ke et al., 2016) [25] while also maintaining an accessibility angle.
Smart Bacteria - Plant Monitoring (SB-PM) is a greenhouse type device that sets specific growth conditions, such as temperature and lighting, and automatically collects data on the plant and microbes - with an emphasis on bacteria. This device can provide scientists with the much-needed data for plant-microbe interactions under various conditions, while also enabling manual sampling for standard experiments. The SB-PM is separated in two distinct parts: the sensors and the controllers. Both the measured and controlled parameters were decided after consulting with professors specialized in plant biotechnology. The sensors log crucial parameters related to plant-bacteria interactions to a database designed for open, public access. These measurements include temperature, moisture, CO2 / O2 concentration, and fluorescence, among others. The controllers set the conditions in which the plants and bacteria will grow. Inside this smart greenhouse there will be variable temperature, lighting, humidity (via a watering system), nutrient supply and more.
In conclusion, our team embarked on the Thaelia project, driven by the need to provide a solution to the widespread problem of Verticillium wilt, faced by many olive growers who suffer from the continuous loss of their crops. Through our innovative biological solution, we aim to not only protect olive crops but also contribute to a more sustainable approach to agriculture and biodiversity conservation. Our engagement with affected farmers has reinforced the demand for such a solution, with many expressing a willingness to invest in our formulation. Currently, there is no effective chemical treatment available for this disease. Extensive communication with farmers, producers, and educators revealed a significant gap in effective solutions for Verticillium dahliae , a persistent and devastating plant pathogen impacting the olive oil community. This emphasized the urgent need for innovative approaches to combat this disease.
To raise awareness, we developed an educational outreach initiative highlighting the severity of Verticillium wilt and showcasing the potential of RNAi technology as a novel biocontrol strategy. Recognizing the importance of inclusivity, we tailored our educational materials and activities to diverse audiences, ensuring that scientific knowledge was accessible to individuals of a wide spectrum of ages, backgrounds, and social groups. For instance, this included using 3D models and live cultures of microorganisms, when engaging with students, fostering a deeper understanding of Verticillium dahliae and the importance of our project. Therefore, we prioritized interactive and relatable learning experiences, capturing the interest of our audience, recognizing the value of active participation and collaboration in the learning journey. These events demonstrate the potential of science communication to inspire future generations of researchers and innovators and help us learn, connect, and work toward a better future.
[1] ELSTAT, 2022.
www.statistics.gr (accessed June 2024).
[2] Godena, S., Ivić, D., Ban, D., & Goreta Ban, S. (2022). Characterization of
Verticillium dahliae isolates from olive and susceptibility of local olive
cultivars to Verticillium wilt in Istria, Croatia. In Scientia Horticulturae (Vol. 292, p. 110630). Elsevier
BV.
https://doi.org/10.1016/j.scienta.2021.110630
[3] Panagopoulos Ch. G. (2007). Diseases of fruit-bearing trees and vines.
Publications Ath. Stamoulis, p.: 206-217.
[4] Acharya, B., Ingram, T. W., Oh, Y., Adhikari, T. B., Dean, R. A., & Louws,
F.
J.
(2020). Opportunities and Challenges in Studies of Host-Pathogen Interactions and Management of
Verticillium dahliae in Tomatoes. In Plants (Vol. 9, Issue 11, p. 1622). MDPI
AG.
[5] Requena-Mullor, J. M., García-Garrido, J. M., García, P. A., & Rodríguez, E.
(2020). Climatic drivers of
Verticillium dahliae occurrence in Mediterranean olive-growing areas of
southern
Spain. PloS one, 15(12), e0232648. https://doi.org/10.1371/journal.pone.0232648
[6] Jamos E. K., 2017. Phytopathology, 2nd edition. UNIBOOKS IKE, Athens.
[7]
https://www.canolacouncil.org/canola-encyclopedia/diseases/verticillium-stripe/
[8] Kowalska, B. (2021). Management of the soil-borne fungal pathogen – Verticillium dahliae Kleb. causing vascular wilt diseases. In Journal of Plant
Pathology (Vol. 103, Issue 4, pp. 1185–1194). Springer Science and Business Media LLC. https://doi.org/10.1007/s42161-021-00937-8
[9] Mann, C. W. G., Sawyer, A., Gardiner, D. M., Mitter, N., Carroll, B. J., &
Eamens, A. L. (2023). RNA-Based Control of Fungal Pathogens in Plants. International journal of molecular
sciences, 24(15), 12391. https://doi.org/10.3390/ijms241512391
[10] Li, R., & Liu, Q. (2020). Engineered Bacterial Outer Membrane Vesicles as
Multifunctional Delivery Platforms. In Frontiers in Materials (Vol. 7). Frontiers Media SA. https://doi.org/10.3389/fmats.2020.00202
[11] Prieto, P., Schilirò, E., Maldonado-González, M. M., Valderrama, R.,
Barroso-Albarracín, J. B., & Mercado-Blanco, J. (2011). Root hairs play a key role in the endophytic
colonization of olive roots by Pseudomonas spp. with biocontrol activity. Microbial ecology, 62(2), 435–445.
https://doi.org/10.1007/s00248-011-9827-6
[12] Hashiro, S., Chikami, Y., Kawaguchi, H., Krylov, A. A., Niimi, T., &
Yasueda,
H. (2021). Efficient production of long double-stranded RNAs applicable to agricultural pest control by
Corynebacterium glutamicum equipped with coliphage T7-expression system. Applied microbiology and
biotechnology,
105(12), 4987–5000. https://doi.org/10.1007/s00253-021-11324-9
[13] Liang, T., Sun, J., Ju, S., Su, S., Yang, L., & Wu, J. (2021). Construction
of
T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level.
Frontiers
in chemistry, 9, 664967. https://doi.org/10.3389/fchem.2021.664967
[14] Sutaria, D. S., Badawi, M., Phelps, M. A., & Schmittgen, T. D. (2017).
Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading.
Pharmaceutical research, 34(5), 1053–1066. https://doi.org/10.1007/s11095-017-2123-5
[15] Li, Q., Li, Z., Fei, X., Tian, Y., Zhou, G., Hu, Y., Wang, S., & Shi, H.
(2022). The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella
Choleraesuis. In AMB Express (Vol. 12, Issue 1). Springer Science and Business Media LLC.
[16] Wu, M., Li, Q., Xia, G., Zhang, Y., & Wang, F. (2022). New insights into
defense responses against
Verticillium dahliae infection revealed by a quantitative proteomic analysis
in
Arabidopsis thalianaIn C. Wilson (Ed.), Functional Plant Biology (Vol. 49, Issue 11, pp.980–994). CSIRO.
[17] Yoon, J.-S., Kim, K., & Palli, S. R. (2020). Double-stranded RNA in
exosomes:
Potential systemic RNA interference pathway in the Colorado potato beetle, Leptinotarsa decemlineata. In
Journal
of Asia-Pacific Entomology (Vol. 23, Issue 4, pp. 1160–1164). Elsevier BV.
[18] Su, X., Rehman, L., Guo, H., Li, X., Zhang, R., & Cheng, H. (2017). AAC as
a
Potential Target Gene to Control
Verticillium dahliae . In Genes (Vol. 8, Issue 1, p. 25). MDPI AG. https://doi.org/10.3390/genes8010025
[19] Xu, J. Wang, et al (2018). Host‐induced gene silencing of a regulator of G
protein signalling gene (VdRGS1) confers resistance to Verticillium wilt in cotton. Plant Biotechnology
Journal,
16(9), 1629–1643.
[20] in, T., et al (2020).
Verticillium dahliae VdTHI 20, Involved in Pyrimidine Biosynthesis, Is
Required
for DNA Repair
[21] Koeppe, S., Kawchuk, L., & Kalischuk, M. (2023). RNA Interference Past and
Future Applications in Plants. International journal of molecular sciences, 24(11), 9755. https://doi.org/10.3390/ijms24119755
[22] Goodfellow, S., Zhang, D., Wang, M. B., & Zhang, R. (2019).
Bacterium-Mediated
RNA Interference: Potential Application in Plant Protection. Plants (Basel, Switzerland), 8(12), 572. https://doi.org/10.3390/plants8120572
[23] Verdonckt, T.-W., & Vanden Broeck, J. (2022). Methods for the
Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies. In
Frontiers
in Physiology (Vol. 13). Frontiers Media SA. https://doi.org/10.3389/fphys.2022.836106
[24] Mitter, B., Pfaffenbichler, N., & Sessitsch, A. (2016).
Plant-microbe partnerships in 2020. Microbial biotechnology, 9(5), 635–640. https://doi.org/10.1111/1751-7915.12382
[25] Ke, J., Wang, B., & Yoshikuni, Y. (2021). Microbiome
engineering:
synthetic biology of plant-associated microbiomes in sustainable agriculture. Trends in Biotechnology,
39(3),
244-261. https://doi.org/10.1016/j.tibtech.2020.07.008