Author ORCID Identifier

https://orcid.org/0000-0003-4487-1986

Date Available

6-1-2024

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Entomology

First Advisor

Dr. Lynne Rieske-Kinney

Abstract

Unprecedented weather events associated with climate change, coupled with extensive anthropogenic stresses, have reduced the effectiveness of traditional forest pest management approaches, which can no longer keep pace with invasive species overwhelming naïve landscapes and native pests experiencing eruptive and expansive outbreaks. Double-stranded RNA-(dsRNA) mediated gene silencing, a type of RNA interference (RNAi), has been coopted for use as a biopesticide against a multitude of agricultural and horticultural pests and could serve as a powerful tool for woody plant protection. While effective initiation of gene silencing and subsequent mortality have been demonstrated in multiple tree pests, effective and efficient methods of delivery and plant compatibility must first be understood.

To better understand how dsRNA applied as a root soak is taken up by woody plants, I investigated the translocation and persistence of different dsRNAs in both coniferous and deciduous seedlings. For Chapters 2 and 3, I submerged the roots of deciduous white oak (Quercus alba, L.) and coniferous loblolly pine (Pinus taeda, L.) in a water and dsRNA solution and used end-point RT-PCR to detect the presence of intact, exogenous dsRNA in various tissue types over the course of 7 days. While both seedling types showed rapid uptake and whole-plant dissemination, dsRNA recovery quickly waned over time in loblolly pine while detection decreased only slightly in white oak. This work shows that naked dsRNA can rapidly disseminate throughout woody plants and sheds light on possible differences between coniferous and deciduous trees.

Since plants possess intrinsic RNAi machinery that can respond to and defend against invading dsRNA, I annotated RNAi-related proteins (RRP) in loblolly pine and investigated the expression response of their corresponding mRNAs in the presence of dsRNA for Chapter 4. Annotation of RRP in loblolly pine, as well as selected proteins in five Pinus spp., established new sequences for these proteins and highlight the diversity and possible redundancy in some of these vital pathways. Differential expression analysis identified only two isoforms of RRP that were upregulated in dsRNA treated seedlings, demonstrating an overall lack of RNAi response detected. Despite this, pathway enrichment revealed upregulation of autophagy and plant hormone signal transduction pathways, which may be responding to the dsRNA challenge. Knowing how, and to what degree, plants may recognize and respond to insect-specific dsRNA will help to increase compatibility with plant uptake and may reduce plant processing of these insect biopesticides.

Finally, in Chapter 5, I began the work to establish transgenic Arabidopsis thaliana (L.) Heynh. for the production of insect specific dsRNA. Despite the successful production of entry clones, I documented unique and erroneous recombination during the LR reaction when attempting to generate the expression clone. This issue with pHELLSGATE12 recombination is previously undocumented but now replicated with multiple entry clones. However, restriction digest procedures may circumvent this issue while also allowing for flexibility in future intron containing constructs.

Overcoming the barriers associated with dsRNA deployment, specifically that of practical delivery and efficient production, will help move this technology closer to commercial use. Overall, my data demonstrate the applicability of root delivered dsRNA in both deciduous and coniferous seedlings and its compatibility within woody plant tissue.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.205

Funding Information

This work was supported by funds provided by the United States Department of Agriculture, Animal and Plant Health Inspection Service (Grant no. AP20PPQS & T00C061), the University of Kentucky, and the Kentucky Agricultural Experiment Station under McIntire-Stennis 2351197000.

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