Browsing by Author "Tholl, Dorothea"

Now showing 1 - 20 of 57
  • Thumbnail Image
    An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana
    Vaughan, Martha M.; Tholl, Dorothea; Tokuhisa, James G. (Biomed Central, 2011-03-10)
    Background Plant defense against herbivory has been studied primarily in aerial tissues. However, complex defense mechanisms have evolved in all parts of the plant to combat herbivore attack and these mechanisms are likely to differ in the aerial and subterranean environment. Research investigating defense responses belowground has been hindered by experimental difficulties associated with the accessibility and quality of root tissue and the lack of bioassays using model plants with altered defense profiles. Results We have developed an aeroponic culture system based on a calcined clay substrate that allows insect herbivores to feed on plant roots while providing easy recovery of the root tissue. The culture method was validated by a root-herbivore system developed for Arabidopsis thaliana and the herbivore Bradysia spp. (fungus gnat). Arabidopsis root mass obtained from aeroponically grown plants was comparable to that from other culture systems, and the plants were morphologically normal. Bradysia larvae caused considerable root damage resulting in reduced root biomass and water absorption. After feeding on the aeroponically grown root tissue, the larvae pupated and emerged as adults. Root damage of mature plants cultivated in aeroponic substrate was compared to that of Arabidopsis seedlings grown in potting mix. Seedlings were notably more susceptible to Bradysia feeding than mature plants and showed decreased overall growth and survival rates. Conclusions A root-herbivore system consisting of Arabidopsis thaliana and larvae of the opportunistic herbivore Bradysia spp. has been established that mimics herbivory in the rhizosphere. Bradysia infestation of Arabidopsis grown in this culture system significantly affects plant performance. The culture method will allow simple profiling and in vivo functional analysis of root defenses such as chemical defense metabolites that are released in response to belowground insect attack.
  • Thumbnail Image
    Aggregation Pheromone Biosynthesis and Engineering in Plants for Stinkbug Pest Management
    Lehner, Bryan W. (Virginia Tech, 2019-04-26)
    Stinkbugs (Pentatomidae) and other agricultural pests such as bark beetles and flea beetles are known to synthesize terpenoids as aggregation pheromones. Knowledge of the genes and enzymes involved in pheromone biosynthesis may allow engineering of pheromone biosynthetic pathways in plants to develop new forms of trap crops and agricultural practices for pest management. The harlequin bug, Murgantia histrionica, a specialist pest on crucifer crops, produces the sesquiterpene, murgantiol, as a male-specific aggregation pheromone. Similarly, the southern green stink bug, Nezara viridula, a generalist pest worldwide on soybean and other crops, releases sesquiterpene cis-/trans-(Z)-α-bisabolene epoxides as male-specific aggregation pheromone. In both species, enzymes called terpene synthases (TPSs) synthesize precursors of the aggregation pheromones, which are sesquipiperitol and (Z)-α-bisabolene as the precursor of murgantiol and cis-/trans-(Z)-α-bisabolene epoxide, respectively. We hypothesized that enzymes in the family of cytochrome P450 monooxygenases are involved in the conversion of these precursors to the final epoxide products. This study investigated the tissue specificity and sequence of these conversions by performing crude enzyme assays with protein extracts from male tissues. Furthermore, candidate P450 genes were selected by RNA-sequencing and co-expression analysis and the corresponding recombinant proteins tested for enzyme activity. To engineer the pheromone biosynthetic enzymes in plants, transient expression of the TPSs of both stink bugs was performed in Nicotiana benthamiana leaves. Both sesquipiperitol and (Z)-α-bisabolene were found to be produced and emitted from inoculated N. benthamiana leaves. Future work will implement stable transformation to engineer murgantiol biosynthesis in crucifer trap crops and develop similar approaches for pheromone engineering of other agricultural pests.
  • Thumbnail Image
    Ancient origin and conserved gene function in terpene pheromone and defense evolution of stink bugs and hemipteran insects
    Rebholz, Zarley; Lancaster, Jason; Larose, Hailey; Khrimian, Ashot; Luck, Katrin; Sparks, Michael E.; Gendreau, Kerry L.; Shewade, Leena; Koellner, Tobias G.; Weber, Donald C.; Gundersen-Rindal, Dawn E.; O'Maille, Paul; Morozov, Alexandre, V; Tholl, Dorothea (Pergamon-Elsevier Science, 2023-01)
    Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra-and inter -specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing-sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS -type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions.
  • Thumbnail Image
    Biochemical and Functional Characterization of Induced Terpene Formation in Arabidopsis Roots
    Sohrabi, Reza (Virginia Tech, 2013-08-13)
    Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis.
  • Thumbnail Image
    Biochemical, Molecular and Functional Analysis of Volatile Terpene Formation in Arabidopsis Roots
    Huh, Jung-Hyun (Virginia Tech, 2011-07-20)
    Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues.
  • Thumbnail Image
    Biosynthesis and Emission of Stress-Induced Volatile Terpenes in Roots and Leaves of Switchgrass (Panicum virgatum L.)
    Muchlinski, Andrew; Chen, Xinlu; Lovell, John T.; Köllner, Tobias G.; Pelot, Kyle A.; Zerbe, Philipp; Ruggiero, Meredith; Callaway, LeMar, III; Laliberte, Suzanne; Chen, Feng; Tholl, Dorothea (2019-09-19)
    Switchgrass (Panicum virgatum L.), a perennial C4 grass, represents an important species in natural and anthropogenic grasslands of North America. Its resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. However, little is known about the mechanisms of resistance of switchgrass against pathogens and herbivores. Volatile compounds such as terpenes have important activities in plant direct and indirect defense. Here, we show that switchgrass leaves emit blends of monoterpenes and sesquiterpenes upon feeding by the generalist insect herbivore Spodoptera frugiperda (fall armyworm) and in a systemic response to the treatment of roots with defense hormones. Belowground application of methyl jasmonate also induced the release of volatile terpenes from roots. To correlate the emission of terpenes with the expression and activity of their corresponding biosynthetic genes, we identified a gene family of 44 monoterpene and sesquiterpene synthases (mono-and sesqui-TPSs) of the type-a, type-b, type-g, and type-e subfamilies, of which 32 TPSs were found to be functionally active in vitro. The TPS genes are distributed over the K and N subgenomes with clusters occurring on several chromosomes. Synteny analysis revealed syntenic networks for approximately 30-40% of the switchgrass TPS genes in the genomes of Panicum hallii, Setaria italica, and Sorghum bicolor, suggesting shared TPS ancestry in the common progenitor of these grass lineages. Eighteen switchgrass TPS genes were substantially induced upon insect and hormone treatment and the enzymatic products of nine of these genes correlated with compounds of the induced volatile blends. In accordance with the emission of volatiles, TPS gene expression was induced systemically in response to belowground treatment, whereas this response was not observed upon aboveground feeding of S. frugiperda. Our results demonstrate complex above and belowground responses of induced volatile terpene metabolism in switchgrass and provide a framework for more detailed investigations of the function of terpenes in stress resistance in this monocot crop.
  • Thumbnail Image
    Biotic and abiotic mechanisms shaping multi-species interactions
    Maynard, Lauren Danielle (Virginia Tech, 2022-12-20)
    Interactions are important drivers of selection and community structure, which makes the study of multi-species interactions critical for understanding the ecology and evolution of organisms. This dissertation includes four data chapters that examine the biotic and abiotic mechanisms that shape multi-species interactions in both tropical and temperate ecosystems. The first three data chapters (Chapters 2–4) were completed within a Neotropical rainforest in Costa Rica and focus on one plant genus, Piper (Piperaceae). The final data chapter (Chapter 5) was conducted within a working landscape of soybean (Glycine max) fields in eastern Maryland, USA. In Chapter 2, I explore intra- and inter-specific dietary niche partitioning of Piper fruits among three frugivorous bats, illustrating the importance of fine-scale mechanisms that facilitate species coexistence and influence plant–animal interactions. In Chapter 3, I demonstrate how the chemical ecology of a Neotropical shrub, Piper sancti-felicis, shapes fruit interactions with antagonists (fruit fungi) and mutualists (frugivorous bats and birds), developing a foundation for understanding evolutionary ecology of plant chemical traits based on phytochemical investment patterns. In Chapter 4, I describe the direct and indirect impacts of elevated temperature and CO2 concentration on the plant traits and interactions in Piper generalense, improving our understanding of the effects of climate change on a Neotropical plant–herbivore system. In Chapter 5, I explore the biotic (herbivore-induced plant volatiles) and abiotic (fine-scale weather conditions) drivers affecting insectivorous bat foraging in soybean fields in eastern Maryland, providing a pathway to further investigate new strategies for integrated pest management. As a collective work, this dissertation disentangles the nuances of multi-species interactions, exploring foundational mechanisms underlying biodiversity maintenance as well as answering applied questions to address a changing climate and aid sustainable agriculture.
  • Thumbnail Image
    Cell-type specificity and herbivore-induced responses of primary and terpene secondary metabolism in Arabidopsis roots
    Zhang, Jingyu (Virginia Tech, 2013-09-02)
    Plants employ diverse defense mechanisms to combat attack by harmful organisms. For instance, plants produce constitutive physical barriers or use chemical compounds such as specialized secondary metabolites to resist herbivore or pathogen invasion. Considering the cost-efficiency and energy balance between defense, growth and reproduction, defense reactions in plants have to be regulated temporally and spatially. As more cost-efficient strategies, plants may induce their defense response only in the presence of the attacker or restrict constitutive defenses to specific tissues or cells. In this study, we investigated aspects of the spatial regulation and induced changes of primary and secondary metabolism in roots of Arabidopsis thaliana. Roots represent important organs for anchoring plants in the soil and taking up water and nutrients. Hence, it is assumed that roots are as well protected as aerial tissues by different defense mechanisms. The first part of this work is focused on the cell-type-specific biosynthesis of volatile terpenes in Arabidopsis roots. Terpenes are the most abundant specialized metabolites in plants and play an important role in plant defense against pathogens or herbivores. Terpene biosynthetic enzyme activities are often coordinated in specific tissues and cellular compartments. Fine-scale transcriptome maps of Arabidopsis roots have shown that terpene biosynthesis is restricted to particular cell types. However, the reasons and significance of this cell-type specificity are not well understood. We hypothesized that the formation of terpene metabolites is not restricted to specific cells but can be supported by different cell types. We, therefore, probed the plasticity of the cell-specific formation of terpenes by swapping the expression of the terpene synthase (TPS) genes, TPS08, TPS13 and TPS25, between different root cell types in the respective mutant background. To investigate the ectopic expression of TPSs at different levels, quantitative real-time PCR (qRT-PCR), confocal microscopy, and gas chromatography-mass spectrometry (GC-MS) were performed. We found that terpene synthase TPS08, which produces the diterpene rhizathalene and is normally expressed in the root vascular tissue, is functionally active when expressed in the epidermis or cortex, although at substantially lower levels compared to the wild type. We did not find an obvious correlation between the volatile emission level and gene transcript level of TPS08, which may be attributed to a reduced activity of the expressed TPS08-yellow fluorescent protein (YFP) fusion protein. When expression of TPS13 (producing the sesquiterpene (Z)-"-bisabolene) was directed from the cortex to the epidermis or stele, TPS13 gene expression and (Z)-"-bisabolene formation was supported by these cell types although to varying levels in comparison to wild type. TPS13-YFP fluorescent signal driven by the epidermal WER and GL3 promoters was primarily detected at the root tip. Terpene production was also observed for the (E)-"-farnesene sesquiterpene synthase TPS25 when its expression was switched from the endodermis and non-hair producing epidermal cells to hair producing epidermal cells although only a weak fluorescent signal was detected from the expressed TPS25-mGFP protein. Together, the results provide preliminary evidence for a relaxed cell specificity of terpene biosynthesis in Arabidopsis roots and suggest that tissue-specific terpene metabolite patterns could change depending on different selective pressures in rhizosphere. In the second part of this study, we performed global gene transcript profiling and primary metabolite analysis of Arabidopsis roots upon feeding by the generalist root herbivore, Bradysia (fungus gnat). In a microarray analysis, we identified 451 of 22,810 genes that were up-regulated more than 2-fold. Gene ontology (GO) analysis showed that 26% of those genes with predicted or known functions play a role in primary or secondary metabolism, while 24% are involved in cell signaling or in responses to stimulating factors, such as jasmonic acid (JA), ethylene, wounding, and oxidative stress. At the metabolite level, we observed only marginal changes of amino acid, sugar and carboxylic acid relative levels over a time course of 4 days of Bradysia feeding. There was a trend for increased levels of amino acids and the relative levels of sucrose were increased significantly ("=0.05) at the fourth day of feeding. In conclusion, the study provided evidence for the induction of genes related to primary and secondary metabolism and stress responses in Arabidopsis roots, but showed only marginal changes at the primary metabolite level. In addition, the work indicated that the formation of terpene-specialized metabolites in Arabidopsis roots is not restricted to specific cells, but can be supported by different cell types.
  • Thumbnail Image
    Characterization of the Sinorhizobum Meliloti Chotaxis System
    Castaneda Saldana, Rafael (Virginia Tech, 2019-12-19)
    Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds.
  • Thumbnail Image
    Chemical Manipulation of Honey Bee Behavior
    Larson, Nicholas R. (Virginia Tech, 2017-06-09)
    The loss of managed honey bee colonies, resulting from their unintentional exposure to pesticides, is a topic of concern for the agricultural and apicultural industry. Current methods for reducing pesticide exposure to bees involve the application of pesticides before crop bloom or in the evening when foraging bees are less likely to be exposed to these applications. There is an urgent need for additional protection procedures to reduce the annual losses of managed bee colonies. Another method for protecting these pollinators is the use of chemical deterrents to reduce the interaction times of foraging bees with pesticide-treated crops. Historically, insect repellents (IRs) have been used to prevent the spread of deadly human diseases by arthropod vectors. However, it has been shown that bees can be repelled from pesticide-treated crops using DEET and bee pheromonal compounds. Here, I report the toxicological and deterrent effects of bee pheromonal compounds, as well as the deterrent effects of heterocyclic amines (HCAs) on bees. The results of this study indicate that the bee pheromonal compounds, at 8, 20, 60 and 100% concentrations, are toxic to bees and inhibit the feeding of bees within a confined space. Additionally, the pheromonal compounds and the HCAs are as efficacious as DEET in deterring bees from treated food sources. The HCA piperidine was observed to effectively deter bee foragers from a sugar feeder in a high-tunnel experiment as well as from melon flowers and knapweed in field experiments. Electroantennogram recordings were conducted to verify an olfactory response of the bees to the tested compounds. Pheromonal compounds were readily detected by bee antennae; whereas, the HCAs did not elicit significant responses in the bee antennae. These data suggest that bee pheromonal compounds, as well as HCAs, may serve as candidates for the further investigation as repellents to protect bees from unintentional pesticide exposures.
  • Thumbnail Image
    Cool Temperature Effects on Productivity and Photosynthesis of Two Biomass Fuel Species: Switchgrass (Panicum virgatum) and  Miscanthus (Miscanthus x giganteus)
    Mitchell, Jackson Lee Bean (Virginia Tech, 2013-01-14)
    The world\'s highest yielding crops are C4 plants due to their higher water use efficiency, nitrogen use efficiency, and productivity compared with C3 plants.  With an increasing demand for renewable resources as a result of the decreasing global supplies of fossil fuels, we need to improve our understanding of the limitations of biomass fuel feedstock to improve yields and better satisfy energy requirements.  The ability to attain the goal feedstock production in the US is limited by available arable land and cool temperatures.  This study investigates the effects of cool temperatures on the productivity and photosynthesis of the two species with the highest potential for feedstock production in the US: switchgrass (Panicum virgatum) cv. Alamo and miscanthus (Miscanthus × giganteus).  At 14/12"C and a 14/10 hour light/dark photoperiod, switchgrass showed lower productivity and light saturated photosynthetic rates (Amax=10.3 "mol m-2s-1) compared with 28/25"C and the same photoperiod (Amax=18.8 "mol m-2s-1).  Miscanthus has demonstrated cold tolerance in previous studies, and here showed no significant decrease in the productivity or photosynthetic rates in cool, compared with warm, growing conditions (Amax=8.2 "mol m-2s-1 and 7.0 "mol m-2s-1 for warm and cool conditions, respectively). Also, this study examines the potential limitations of C4 photosynthesis by the enzyme pyruvate phosphate dikinase (PPDK) under the same cool conditions, transgenic switchgrass cv. Alamo were created with the insertion of the miscanthus PPDK gene. Productivity and photosynthetic responses of the transgenic plants were evaluated in cool and warm growth temperatures.  Of the two transgenic events tested here, line S(1) displayed cold tolerance, as seen in no loss of both carboxylation efficiency and the ratio of CO2 assimilation to electron transport (Asat/Jmax).  These results indicate that PPDK may pose a significant limitation to C4 photosynthesis in cool conditions and there is a possibility that cold season photosynthesis of switchgrass cv. Alamo could be improved.
  • Thumbnail Image
    The Curious Poisoned Weed: Poison Ivy Ecology and Physiology
    Dickinson, Christopher Cody (Virginia Tech, 2019-07-11)
    Poison ivy (Toxicodendron radicans (L.) Kuntze) is a native perennial liana widely recognized for the production of urushiol, and the associated contact dermatitis it causes in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of global change. Moreover, poison ivy is an important food source for avian species, and urushiol has numerous applications as a high-value engineering material. Thus, this curious weed has many avenues for future concern, and promise. Here, I address gaps in knowledge about poison ivy ecology and physiology so that we may better understand its weediness and utilize its benefits. I address three core areas: poison ivy establishment patterns; biotic interactions with multiple taxa; and the development of molecular tools for use in poison ivy. I found that the early life stage of seedling emergence is a critical linchpin in poison ivy establishment due largely to herbivore pressure from large grazers. I also describe the multifaceted relationship between poison ivy and avian frugivores that not only disperse the drupes of poison ivy but also aid in reduction of fungal endophytic phytopathogens. A survey of poison ivy urushiols yielded that while variation in urushiol congeners was high across individuals, relative congener levels were stable within individuals over a two month period. Lastly I demonstrate best practices for introducing and transiently expressing recombinant DNA in poison ivy as a step towards future reverse genetic procedures.
  • Thumbnail Image
    Diversity and function of terpene synthases in the production of carrot aroma and flavor compounds
    Muchlinski, Andrew; Ibdah, Mwafaq; Ellison, Shelby; Yahyaa, Mossab; Nawade, Bhagwat; Laliberte, Suzanne; Senalik, Douglas; Simon, Philipp; Whitehead, Susan R.; Tholl, Dorothea (2020-06-19)
    Carrot (Daucus carota L.) is an important root vegetable crop with high nutritional value, characteristic flavor, and benefits to human health. D. carota tissues produce an essential oil that is rich in volatile terpenes and plays a major role in carrot aroma and flavor. Although terpene composition represents a critical quality attribute of carrots, little is known about the biosynthesis of terpenes in this crop. Here, we functionally characterized 19 terpene synthase (TPS) genes in an orange carrot (genotype DH1) and compared tissue-specific expression profiles and in vitro products of their recombinant proteins with volatile terpene profiles from DH1 and four other colored carrot genotypes. In addition to the previously reported (E)-beta -caryophyllene synthase (DcTPS01), we biochemically characterized several TPS proteins with direct correlations to major compounds of carrot flavor and aroma including germacrene D (DcTPS7/11), gamma -terpinene (DcTPS30) and alpha -terpinolene (DcTPS03). Random forest analysis of volatiles from colored carrot cultivars identified nine terpenes that were clearly distinct among the cultivars and likely contribute to differences in sensory quality. Correlation of TPS gene expression and terpene metabolite profiles supported the function of DcTPS01 and DcTPS03 in these cultivars. Our findings provide a roadmap for future breeding efforts to enhance carrot flavor and aroma.
  • Thumbnail Image
    Ecology of Root-Feeding Insect Assemblages in Fire-Manipulated Longleaf Pine-Wiregrass Ecosystems
    Dittler, Matthew Jason (Virginia Tech, 2013-05-23)
    Root-feeding insects can have top-down influence on vegetative composition and ecosystem processes; however, they may respond to bottom-up factors such as soil resources, site productivity, and disturbance.  My research addressed the following questions: (1) Do disturbance (fire), vegetative composition, soil resources, and fine root standing mass influence the structure of root-feeding insect assemblages? (2) What types of roots do root-feeding insects eat, and do they forage selectively?  (3) Do root-feeding insects influence fine root productivity?  To address these questions, I studied root-feeding insect assemblages in longleaf pine wiregrass (Pinus palustris-Aristida stricta) ecosystems of southwestern Georgia, U.S.A.  On a random basis, study sites were burned at least every other year (B), or left unburned (UB) for about 9 years.  Fine root productivity and root-feeding insect abundances were sampled repeatedly across 54 random plots in UB and B sites.  In Chapter 2, I characterized spatial and temporal patterns of root-feeding insect abundance, understory plant composition, soil resource availability, and fine root standing mass within each plot.  Insect population densities were low overall, but abundance, patchiness, and diversity were greater in UB sites.  Abundance patterns were significantly related to vegetative composition.  In Chapter 3, I quantified the diet of root-feeding insects by measuring the natural abundance of carbon (C) and nitrogen (N) stable isotopes in insects and fine roots.  Using 13C abundance, I examined the contribution of warm season grass roots to insect diet, relative to the proportion of warm season grass roots within adjacent root standing crop samples; 15N abundance was used to detect omnivory.  Overall, insects appeared to be non-selective herbivores and omnivores that may alter foraging behavior to maintain a mixed diet (i.e. reducing or increasing warm season grass consumption when its abundance was high or low, respectively).  The extent of omnivory varied within and among taxa.  In Chapter 4, I estimated the top-down influence of root-feeding insects on fine root productivity by comparison of ingrowth cores with or without an insecticide treatment.  I detected a weak positive effect of herbivores on the productivity of non-grass fine roots (< 10% of fine root productivity).    
  • Thumbnail Image
    Elucidation of the Specificity of S. meliloti Chemoreceptors for Host Derived Attractants
    Webb, Benjamin A. (Virginia Tech, 2016-08-24)
    The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields.
  • Thumbnail Image
    Emergence of terpene chemical communication in insects: Evolutionary recruitment of isoprenoid metabolism
    Rebholz, Zarley; Shewade, Leena; Kaler, Kylie; Larose, Hailey; Schubot, Florian; Tholl, Dorothea; Morozov, Alexandre V.; O'Maille, Paul E. (Wiley, 2023-05)
    Insects have evolved a chemical communication system using terpenoids, a structurally diverse class of specialized metabolites, previously thought to be exclusively produced by plants and microbes. Gene discovery, bioinformatics, and biochemical characterization of multiple insect terpene synthases (TPSs) revealed that isopentenyl diphosphate synthases (IDS), enzymes from primary isoprenoid metabolism, are their likely evolutionary progenitors. However, the mutations underlying the emergence of the TPS function remain a mystery. To address this gap, we present the first structural and mechanistic model for the evolutionary emergence of TPS function in insects. Through identifying key mechanistic differences between IDS and TPS enzymes, we hypothesize that the loss of isopentenyl diphosphate (IPP) binding motifs strongly correlates with the gain of the TPS function. Based on this premise, we have elaborated the first explicit structural definition of isopentenyl diphosphate-binding motifs (IBMs) and used the IBM definitions to examine previously characterized insect IDSs and TPSs and to predict the functions of as yet uncharacterized insect IDSs. Consistent with our hypothesis, we observed a clear pattern of disruptive substitutions to IBMs in characterized insect TPSs. In contrast, insect IDSs maintain essential consensus residues for binding IPP. Extending our analysis, we constructed the most comprehensive phylogeny of insect IDS sequences (430 full length sequences from eight insect orders) and used IBMs to predict the function of TPSs. Based on our analysis, we infer multiple, independent TPS emergence events across the class of insects, paving the way for future gene discovery efforts.
  • Thumbnail Image
    Environmental and behavioral factors associated with the infestation of vineyards by larvae of grape root borer
    Rijal, Jhalendra P. (Virginia Tech, 2014-04-03)
    Grape root borer, Vitacea polistiformis (Harris), is an oligophagous pest of grapevines in the eastern USA. Neonates must burrow into the soil to find grape roots. In Virginia, larvae feed on roots for ~2 years, then pupate just beneath the soil surface. Emerging adults leave an empty pupal exuviae at the soil surface around the vine base. There was no relationship between weekly captures in pheromone traps and pupal exuviae counts, indicating that exuviae sampling is most appropriate to assess infestations. Exuviae sampling in Virginia vineyards revealed infestations that ranged from light to very heavy. Eighteen biotic and abiotic variables were measured and used in analyses that assessed their relative contributions to differences in exuviae density. Water holding capacity and clay/sand ratio were most strongly associated with pupal exuviae density; these variables were used to develop a model for predicting the extent of infestation of individual vineyards. The spatial distribution of pupal exuviae was characterized using non-spatial and geospatial techniques. Although the non-spatial method (Taylor's Power Law) indicated that exuviae showed an aggregated distribution in all blocks, spatial methods (variograms, SADIE) revealed aggregated distributions only in blocks with ≥ 0.5 pupal exuviae per vine. Independent pupal exuviae samples for population assessment in vineyards can be achieved using sampling points separated by >8.8 m. Combined results from geospatial analyses and the temporal distribution of pupal exuviae within years enabled the development of a practical and quantitative sampling protocol. Bioassays used to measure the behavioral response of larvae to host stimuli revealed that neonates were attracted to grape root volatiles. In soil column bioassays, larvae moved vertically and horizontally over distances of up to 120 cm and apparently perceived the presence of grape roots from a distance of 5 cm in soil. Results are discussed in relation to their potential implications for monitoring and managing grape root borer.
  • Thumbnail Image
    Exciting times in plant biotic interactions
    Innes, Roger W.; Gu, Yangnan; Kliebenstein, Dan; Tholl, Dorothea (Oxford University Press, 2022-04-26)
  • Thumbnail Image
    Exploring protein interactions and intracellular localization in regulating flavonoid metabolism
    Bowerman, Peter A. (Virginia Tech, 2010-08-02)
    The organization of biological processes via protein-protein interactions and the subcellular localization of enzymes is believed to be fundamental to many aspects of metabolism. Although this organization has been demonstrated in several systems, the mechanisms by which it is established and regulated are still not well understood. The flavonoid biosynthetic pathway offers a unique system in which to study several important aspects of metabolism. Here we describe a novel toolset of mutant alleles within the flavonoid biosynthetic pathway. In addition, we discuss the use of several of these alleles together with a number of emerging technologies to probe the role of subcellular localization of chalcone synthase, the first committed flavonoid biosynthetic enzyme, on metabolic flux, and to characterize a novel chalcone synthase-interacting protein. The over-expression of this interacting protein induces novel phenotypes that are likely associated with the production or distribution of auxin. Further, interaction analyses between recombinant flavonoid biosynthetic enzymes point to the possibility that post-translational modifications play an important role in promoting interactions.
  • Thumbnail Image
    Exploring the genetic basis of germination specificity in the parasitic plants Orobanche cernua and O. cumana
    Larose, Hailey Lee Ann (Virginia Tech, 2018-04-17)
    Seeds of the root parasitic plants of the genus Orobanche germinate specifically in response to host-derived germination signals, which enables parasites to detect and attack preferred hosts. The best characterized class of germination stimulants is the strigolactones (SLs), although some species respond to non-SL compounds, such as dehydrocostus lactone (DCL). Recent work indicates that SLs are perceived by members of the KARRIKIN-INSENSITIVE2 (KAI2) gene family, and suggests that within parasitic Orobanchaceae the KAI2 genes have undergone duplication and specialization. The "diverged" clade of these genes, termed KAI2d, has been shown to bind SL germination stimulants in model system assays, but the precise role for KAI2d in regulating germination specificity in a parasitic plant has not been demonstrated. To address this issue, we used genetic and genomic approaches involving two closely related species, Orobanche cernua and O. cumana, which differ primarily in host range and stimulant preference. Orobanche cernua parasitizes tomato (and other Solanaceous crops) and responds to orobanchol, the major SL from tomato roots, whereas O. cumana specifically parasitizes sunflower and responds to DCL. Crosses between O. cernua and O. cumana produced hybrid populations that segregate for stimulant specificity, creating a tractable genetic system. Orobanche cernua contains four KAI2d genes (numbered OrceKAI2d1-4), while O. cumana contains six genes (OrcuKAI2d1-6). The DNA from 94 F2 hybrids was genotyped to identify the KAI2d gene composition and these were correlated with germination phenotype. The pattern of segregation indicated that the KAI2d genes are linked, but pointed to OrceKAI2d2 as a likely orobanchol receptor. Response to DCL was associated with inheritance of all O. cumana KAI2d genes together. Each KAI2d gene was expressed in the Arabidopsis thaliana kai2 mutant background and tested for ability to recover the mutant phenotype when exposed to SLs (including orobanchol, 5-deoxystrigol and GR24) or DCL. One O. cernua gene, OrceKAI2d2, responded to all SLs, but not DCL in this system. No DCL-specific KAI2 genes were identified. In summary, we have identified the likely SL receptor in O. cernua, and show evidence that the DCL receptor is either not a KAI2d protein, or uses KAI2d in combination with other signaling pathway components.