Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Grbic, Vojislava

Abstract

The arms race between plants and herbivores has resulted in a great diversity of plant compounds to act as defences against attackers. It has concurrently resulted in herbivorous pest adaptations to host defences, including plant-host defence suppression through the action of secreted effectors, and detoxification of phytochemicals ingested during feeding. While these two mechanisms of herbivore adaptation are relatively well studied, they have not been tested for use at the same time. This study uses the model plant species Solanum lycopersicum (tomato), and the model arthropod species Tetranychus urticae (two-spotted spider mite), to characterize the utilization of the above-mentioned mechanisms in an experimental adaptation set-up. Two spider mite strains, non-adapted (ancestral) and tomato-adapted, were used to infest tomato under different experimental conditions to interrogate the adaptation process. Tomato adaptation was validated through plant damage and mite performance assays. Transcriptional analysis of differentially expressed genes demonstrated an attenuation of the response to non-adapted mites by adapted ones, indicating the defence response to be deficient in induced defence programs, such as jasmonic acid biosynthesis and protease inhibitor biosynthesis. This was supported with marker gene and hormone quantification. However, inhibition activity was found to be differentially induced in different tomato cultivars, being highly induced in Moneymaker and attenuated in Heinz samples fed on by adapted mites, suggesting mites still encounter protease inhibitors as a plant defence in certain tomato cultivars despite being adapted to tomato in general. A mite co-infestation experiment was used to demonstrate that any benefit to host-plant modulation occurs only at the feeding site. Characterization of mite protease activity and fecundity post-inhibition by a synthetic inhibitor, E-64, suggest that mites increase their protease activity to overcome tomato protease inhibitors. Detoxification was also found to be involved in tomato adaptation, whereby inhibiting different classes of enzymes (cytochrome P450s, esterases, or glutathione-S-transferases) resulted in decreased fecundity on tomato.

Summary for Lay Audience

Insight into the molecular mechanisms of plant host adaptation by herbivores can inform future agricultural practices and technologies to ensure continued food production in a sustainable, ecologically friendly way. This research investigates two such adaptation mechanisms. First, suppression of plant defences. Using this mechanism, a herbivore can utilize a host plant by suppressing the plant response to herbivory, decreasing the amount of defences a plant produces in response to attack, and making the plant a more hospitable host. Detoxification of plant compounds is the second mechanism of adaptation studied here. Detoxification of toxic plant compounds can also make a host plant suitable for development and reproduction. Detoxification does not decrease the amount of plant defences produced, but it renders toxic metabolites that are ingested during feeding to be non- or less-functional against the herbivore. I use the two-spotted spider mite as a model herbivore that has been documented to use these two mechanisms of suppression to feed on tomato plants, and investigate whether these two mechanisms can be used simultaneously. Previous research has only studied these two mechanisms independently, but I hypothesize they can be used simultaneously. I used a variety of techniques to characterize the adaptation status of a tomato adapted mite population by comparing it to a non-adapted mite population sharing genetic ancestry. Quantification of gene expression and plant hormone accumulation indicated that the adapted mite population can attenuate the tomato response to mite feeding, compared to the non-adapted strain. A co-infestation experiment revealed that any physiological benefit to adapted mites must occur at the feeding site and is not transmitted systemically throughout the plant. I also characterized tomato protease inhibitor activity and mite protease activity to ascertain how mites were overcoming tomato protease inhibitors (an anti-digestive plant defence). Results suggests that mites have high protease activity to overcome tomato protease inhibitors and may not be relying on suppression of this plant defence class. Finally, I characterized the involvement of three prominent detoxification enzyme classes, namely carboxyl/choline esterases, glutathione-S-transferase, and cytochrome P450, using synthetic inhibitors of these classes. Results from detoxification inhibitor experiments supports adapted mites also using detoxification as a mechanism to overcome tomato toxin metabolites. Overall, this research supports the conclusion that spider mites, and probably herbivores generally, can use multiple mechanisms of adaptation concurrently.

Figure_3.1_Mite_damage_on_Moneymaker.html (965 kB)
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Figure_3.2_Mite_damage_on_multiple_tomato_cultivars.html (1080 kB)
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Figure_3.3_Mite_dispersal_on_tomato.html (970 kB)
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Figure_3.4_Mite_fecundity_on_tomato.html (967 kB)
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Figure_3.5_Microarray_QC.html (160 kB)
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Figure_3.6_Suppression_assay.html (970 kB)
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Figure_3.7_Moneymaker_hormone_analysis.html (1211 kB)
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Figure_3.8_Protease_inhibitor_marker_gene_analysis.html (1031 kB)
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Figure_3.9A_Percent_inhibition_of_commercial_cathepsin_L.html (974 kB)
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Figure_3.9B_Percent_inhibition_of_commercial_cathepsin_L.html (974 kB)
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Figure_3.9C_Protease_inhibitor_marker_gene_analysis.html (1033 kB)
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Figure_3.10_E-64_inhibition_of_cathepsin_L_activity.html (1172 kB)
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Figure_3.11_E-64_fecundity_assay.html (1187 kB)
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Figure_3.12_Mite_cysteine_protease_activity_on_rearing_and_experimental_hosts.html (1162 kB)
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Figure_3.13A_Esterase_activity_following_inhibition.html (971 kB)
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Figure_3.13B_GST_activity_following_inhibition.html (1042 kB)
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Figure_3.13C_CYP_activity_following_inhibition.html (1016 kB)
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Figure_3.14_Detoxification_inhibitor_assay_Moneymaker.html (1104 kB)
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Figure_3.15_Detoxification_inhibitor_assay_Castlemart_and_def-1.html (1292 kB)
Statistical analysis

Available for download on Tuesday, September 01, 2020

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