~Inspiration~

‘’If you deconstruct Greece, you will in the end see an olive tree, a grapevine, and a boat remain” - Odysseus Elytis

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].

Map I: Distribution map of Verticillium dahliae [4].

~Problem~

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.

~Life cycle and symptoms~

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.

Fig 1. Life cycle of Verticillium dahliae [7].

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].

Fig 2. (a) Unilateral wilting (hemiplegia) of an olive tree, a symptom of Verticillium dahliae infection, is shown in this photograph taken in the village of Elia on May 18, 2024. The right side of the tree is affected, as evidenced by dried and yellowing leaves, (b) Healthy olive tree.

Treatment

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 aim~

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.

Fig 5: The encapsulation of the dsRNA 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.

Fig 6: Reducing the levels of Tol B with the usage of TEV proteas

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].

Fig 7: Verticillium's uptake of OMVs

Our target genes

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:

1. VdAAC (ADP/ATP Carrier): critical for energy production [18]
2. VdRGS1 (Regulator of G Protein Signaling): controlling spore germination, hyphal growth, and conidiospore production [19]
3. VdTHI20 (Thiamine Biosynthesis Pathway Protein): vital for thiamine biosynthesis, protecting the fungus from UV damage and supporting hyphal growth [20]

By silencing these essential genes, critical for Verticillium's survival, we effectively ensure that the pathogen cannot persist. Bringing Our Project to Life 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:

• For saplings intended for transplantation to an olive grove, our solution will be introduced in nurseries. This proactive approach aims to protect young saplings, as a potential Verticillium infection could cause immediate death.
• For established olive groves, we plan to deploy our system during the two most crucial times of the year: spring and autumn. These seasons were chosen based on insights from farmers, who informed us that Verticillium wilt is more prevalent during these periods due to the high temperatures and humidity that favor the pathogen's growth.

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.

Fig 8: Guardians of the Roots: Bacterial Colonization to Shield Olive Trees from Verticillium

bmRNAi: A novel strategy against Verticillium dahliae

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].

The Challenge (We faced) when Studying Microbe-Plant Interactions:

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 bacter

Fig 9: Platforms for Phytomicrobiome Applications [25]

Problem With Simulation Platforms

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.

Enter Smart Bacteria-Plant Monitoring

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.

~Stakeholders & EDU~

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.

~References~

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