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.
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.
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).
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.
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.
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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