Page 143 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

TYMV is a single-stranded RNA virus that mainly infects crops from the Brassicaceae family, and its
infection can be severe due to its viral protease (TYMV PRO). TYMV PRO is essential for virus replication
as it processes the viral polyprotein. Additionally, it acts as a deubiquitinase (DUB) that may interrupt the
antiviral mechanisms in plants that are mediated by ubiquitin. A promising strategy for combating TYMV is
to use a highly selective protein-based inhibitor called ubiquitin variant (UbV), which blocks the activity of
TYMV PRO. Using phage display, we identified UbVs that selectively bind to the DUB domain of TYMV
PRO. TYMV PRO and UbVs were expressed and purified to assess their binding abilities; UbV3 was the
tightest binding inhibitor against TYMV PRO, with an IC50 of 19±2.7nM and EC50 of 0.3nM. To ensure
the specificity, UbVs were tested against a panel of A. thaliana plant DUBs representing four prominent
families. None of the UbVs are significantly bound with the plant DUBs. To confirm the ability of UbV3 to
block the TYMV replication, transgenic A. thaliana expressing UbV3 was generated using the
Agrobacterium tumefaciens-based transformation. The PCR, Western blot, ELISA, and subcellular colour
localization analysis were assessed to determine the successful transformation and expression of UbV3
in A. thaliana. The seed germination percentage, plant height, the days at bolting and the dry weight of
both wild type A. thaliana Col-0 and transgenic A. thaliana were tested using the One-way ANOVA and
confirmed no significant change in plant growth and flowering. Based on the Pearson correlation
coefficient between the amount of UbV3 and TYMV detection after the infection trial, the transgenic plants
(r=-0.8) were identified to continue viral infection studies to determine if UbV3 expression protects the
plant against TYMV infection.
Keywords: TYMV, protein inhibitors, viral protease, Arabidopsis thaliana, infection studies

*[O84] DECIPHERING TETRANYCHUS URTICAE - ARABIDOPSIS THALIANA INTERACTIONS:
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UNVEILING DETOXIFICATION MECHANISMS AND PLANT RESISTANCE STRATEGIES. Michele
Antonacci, Jorden Maglov, Julia Pastor Fernandez, Chetan Sharma, Vladimir Zhurov, Brendan
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Abiskaroon, Maksymilian Chruszcz, and Vojislava Grbic. The University of Western Ontario,
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Department of Biology, 1151 Richmond Street, London, ON, Canada, N6A 5B7; and Michigan State
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University, Department of Biochemistry & Molecular Biology, 288 Farm Lane, East Lansing, MI, USA,
48824
Correspondence to: mantonac@uwo.ca

The co-evolutionary dynamics between herbivorous pests and their plant hosts represent a complex
interplay of biochemical and molecular mechanisms. This study elucidates the interaction between
Tetranychus urticae, a detrimental herbivore worldwide, and Arabidopsis thaliana, an extensively used
model plant. T. urticae, also known as the two-spotted spider mites (TSSM), is able to overcome a wide
array of plant defences and to rapidly adapt to new host plants. The key mechanism enabling TSSM’s
adaptation to new host environments is its ability to modify its enzymatic detoxification system and
develop metabolic resistance in just 25 generations after a host shift. Upon feeding on Arabidopsis
leaves, TSSM encounters the accumulation of complex bland of defensive compounds including indole
glucosinolates. Indole glucosinolates are secondary metabolites specific to Arabidopsis’ botanic family
(Brassicaceae) — which provide a chemical barrier against TSSM herbivory through their breakdown
products. In the comprehensive study of Arabidopsis and TSSM interaction, the primary objective is to
determine which indole glucosinolate derivatives (IGDs) in Arabidopsis provide resistance to TSSM
herbivory and to investigate which detoxification patterns are adopted in TSSM to ward off these plant
barriers. The analysis involves the use of Arabidopsis mutants with altered endogenous levels of IGDs to
pinpoint candidate defensive metabolites. To demonstrate the toxicity of individual IGD candidates, they
are orally delivered to mites. Once the Arabidopsis defences are established, the metabolomic analysis of
mite extracts upon the application of an IGD is used to determine the pattern of compound modification in
mite gut and to elucidate which detoxification gene family(ies) contributes to TSSM resistance. The
function of putative detoxification enzymes in TSSM is further investigated by the utilization of
environmental RNAi interference to knock down the candidate’s gene expression. In conclusion, the
overall investigation of plant-pest interactions aims to identify plant defensive compounds and
mechanisms used by the TSSM to overcome their toxicity. This will not only enrich the understanding of
ecological dynamics in plant-pest interaction but will also translate into tangible benefits for agriculture,
including the development of resilient crop varieties and innovative pest management.

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