Project Inspiration

This year's project idea came through after a great amount of consideration and the rejection of many preliminary ideas. It was a priority for our team to decide on a project that would have a considerable impact on our community and worldwide, but also ensure the preservation of the biodiversity of species and biological communities.

We were particularly concerned about the unequal distribution of products in developing countries, especially African countries which are entitled to 11,9% of the tomato product share, and the loss of crops due to pests. Furthermore, we considered that unrestricted plant exportation worldwide increases the risk of pathogen transmission and causes microbiome disruption, which is responsible for the loss of native species and thus biodiversity. Taking into account the recent increase in tospoviruses spread in Greece and especially in Crete, we decided the ideal project for this year’s competition is to create a diagnostic tool that detects the plant pathogen TSWV and other tospoviruses to prevent viruses transmission and destruction of inland tomato produce.


Profile of the virus and other tospoviruses

Tomato spotted wilt virus (TSWV) is a pathogen with one of the broadest known host ranges among RNA viruses. It belongs to the species Tospovirus, the genus Orthotospovirus , the family Tospovirdae, and the order Bunyaviridae . TSWV is a negative-stranded RNA virus in the and is a major constraint on the production worldwide of tomatoes and other solanaceous crops. Of note, TSWV has a described host range of over 1000 plant species distributed across more than 90 families of angiosperms. Its genome is composed of three viral genomic RNAs (large, medium, and small) encoding five proteins, three conserved among all bunyaviruses and two that likely reflect specific adaptations to plants and insects.

The genus Tospovirus is unique within the other four genera (Orthobunyavirus, Hantavirus, Nairovirus, and Phlebovirus) of the family Bunyaviridae because it consists of viruses that infect plants. In general, tospoviruses are recognized globally as an emerging agricultural threat posing a grave concern for global food security. Mature virions range in size from 80 to 100 nm and they show considerable variation between and within species in terms of symptoms caused, virulence, and ability to overcome host resistance.

TSWV is predominantly transmitted by western flower thrips (F. occidentalis) which is also the primary driver of the worldwide emergence of tospoviruses. Last but not least, western flower thrips is one of the most important vectors due to its wide plant host range and expansive distribution across North and South America, Australia, Europe, and the Middle East.

Impact on tomato cultivation

Solanum Lycopersicum (tomato) is one of the most important economic vegetable crops. However, TSWV is one of the most destructive diseases affecting tomato cultivation and production worldwide which leads to huge economic losses. Infected tomato plants are usually dwarfed and have necrotic streaks and dark-brown flecks on their leaves, stems, and fruits. The first symptoms in tomato seedlings are inhibited growth points and copper-colored rolls of young leaves. Subsequently, many small dark-brown flecks form, and leaf veins become purple (Shiming et al., 2021).

Concept of LRR diagnostic tools

LRRs and plant immunity

Leucine-rich repeats, also known as LRRs, are protein domains evolutionarily conserved among plants, vertebrates, and invertebrates. Abundant with leucine residues, they form a pattern that ranges from 20 to 30 amino acids and fold into a horseshoe shape. Their main purpose is to provide a framework for protein-protein interactions. Involved with the innate immune system, LRR-containing proteins are responsible for the recognition of numerous bacterial, fungal and viral effectors as well as molecules indicating damaged-self.

During the infection, plant pathogens inject virulence-related proteins, the effectors, that interfere with the host’s physiology and immune responce. Fortunately, plants have developed receptors to detect these virulent molecules. Intracellular NLR receptors (nucleotide-binding leucine-rich repeat receptors), are employed to sense effector presence or activity and trigger a robust immune response. This response usually leads to the death of the compromised cell which halts the colonization of the pathogen resulting in disease resistance.

Our novel diagnostic tool

The innovation that differentiates our diagnostic test from classic antibody tests is that we pursue a whole-cell diagnostic approach. E.coli cells display the NB-LRR domains and bind the movement protein of several viruses of the genus tospovirus that is recognized by the Sw-5b resistance gene of Solanum peruvianum . The movement protein contributes to the pathogenicity of the viruses that serves as an effector protein. For our project we introduce Agrobacterium tumefaciens cells, transformed in order to express the effector domain that is fused with fluorescent YFP, into Nicotiana sylvestris. Our E.coli cells that are surface-displaying the novel chimeric protein are tested for their binding efficacy. In the case that the NB-LRR and effector domains bind, fluorescence is observed after several rounds of cell washes and we have a positive result.

Advantages over Antibody diagnostic tests

A whole-cell LRR-based diagnostic tool is a novel idea with numerous advantages over traditional Antibody diagnostic tests. For instance, the NLRs of a plant are binding proteins that can be successfully expressed in prokaryotic cells and be functional, as they are single peptides. Conventional antibodies that are used for diagnosis are typically expressed in eukaryotic cell systems to ensure post translational modifications and correct folding of the proteins. Thus, expression of the proteins of interest in bacterial expression systems exhibits significant advantages.

The economical advantage of our initiative should also be considered. Protein purification is not required due to the fact that whole cells are used to express the binder. Thus the cost of production is minimal. The use of whole cells is also advantageous because it eliminates the need for the purified proteins to be be subdued to gold-conjugation for pathogen detection. For the visualization of the result we propose a chromoprotein expressed in the bacterial cells. Last but not least, since immunization and experimentation on lab animals are not required, this method of developing detection tools is eliminating any ethical concerns coupled with the conventional antibody-based diagnostic tool.


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