Heather E. Pence

Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

Systemic RNAi in western corn rootworm, Diabrotica virgifera virgifera, does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.

Interaction of organic solvents with the epicuticular wax layer of wheat leaves.

After foliar application, compounds that are not absorbed into leaves can be removed from the leaf surface by dipping or rinsing in dilutions of organic solvents in water. However, interactions between solvent mixtures and the epicuticular wax layer have received little attention, and information on potential physical and chemical intactness of the plant surface following application of solvents is limited. In this study, wheat leaves were dipped in organic solvents at different dilutions with water, and the major component of the leaf epicuticular wax layer, 1-octacosanol, was analyzed to assess damage to the wax layer. Dipping leaves in dilutions of organic solvent higher than 60% by volume resulted in only negligible or low levels of 1-octacosanol extraction, while no 1-octacosanol was detected in any mixtures containing less than 40% organic solvent. Furthermore, analysis of leaf surfaces by scanning electron microscopy showed structural intactness of the epicuticular wax layer when organic solvent mixtures were used. Therefore, our results demonstrate that the epicuticular wax layer of wheat leaves is not altered physically or chemically by organic solvent solutions up to 40% by volume. These findings validate the use of solvent washing procedures to assess unabsorbed compounds on wheat leaf surfaces.

Improved insect-proofing: expressing double-stranded RNA in chloroplasts

Abstract RNA interference (RNAi) was discovered almost 20 years ago and has been exploited worldwide to silence genes in plants and animals. A decade later, it was found that transforming plants with an RNAi construct targeting an insect gene could protect the plant against feeding by that insect. Production of double‐stranded RNA (dsRNA) in a plant to affect the viability of a herbivorous animal is termed trans‐kingdom RNAi (TK‐RNAi). Since this pioneering work, there have been many further examples of successful TK‐RNAi, but also reports of failed attempts and unrepeatable experiments. Recently, three laboratories have shown that producing dsRNA in a plants chloroplast, rather than in its cellular cytoplasm, is a very effective way of delivering TK‐RNAi. Our review examines this potentially game‐changing approach and compares it with other transgenic insect‐proofing schemes.

Histopathological Effects of Bt and TcdA Insecticidal Proteins on the Midgut Epithelium of Western Corn Rootworm Larvae (Diabrotica virgifera virgifera)

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is a major corn pest in the United States, causing annual losses of over

RNAi targeting of rootworm Troponin I transcripts confers root protection in maize

1 billion. One approach to protect against crop loss by this insect is the use of transgenic corn hybrids expressing one or more crystal (Cry) proteins derived from Bacillus thuringiensis. Cry34Ab1 and Cry35Ab1 together comprise a binary insecticidal toxin with specific activity against WCR. These proteins have been developed as insect resistance traits in commercialized corn hybrids resistant to WCR feeding damage. Cry34/35Ab1 is a pore forming toxin, but the specific effects of Cry34/35Ab1 on WCR cells and tissues have not been well characterized microscopically, and the overall histopathology is poorly understood. Using high-resolution resin-based histopathology methods, the effects of Cry34/35Ab1 as well as Cry3Aa1, Cry6Aa1, and the Photorhabdus toxin complex protein TcdA have been directly visualized and documented. Clear symptoms of intoxication were observed for all insecticidal proteins tested, including swelling and sloughing of enterocytes, constriction of midgut circular muscles, stem cell activation, and obstruction of the midgut lumen. These data demonstrate the effects of these insecticidal proteins on WCR midgut cells, and the collective response of the midgut to intoxication. Taken together, these results advance our understanding of the insect cell biology and pathology of these insecticidal proteins, which should further the field of insect resistance traits and corn rootworm management.

Correlative Confocal and Environmental Scanning Electron Microscopy for Investigating the Fungal Invasion of Plant Surfaces in Their Native State

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Effects of Bacillus Thuringiensis Cry Proteins On the Morphology of Western Corn Rootworm (Diabrotica virgifera virgifera) Midgut Cells

Zymoseptoria tritici (wheat leaf blotch) can cause up to 50% yield losses in untreated wheat in Europe. Fungicide applications can significantly reduce yield losses, but fungicide resistance has been documented [1]. Hence, new fungicides are necessary for long-term management of this disease. Most fungicidally active molecules inhibit fungal spore germination and/or germ tube growth. The ability to visualize the impact of active molecules on fungal structures growing on wheat leaves would greatly facilitate the development of new fungicides. However, imaging fungal structures in situ is non-trivial due to the fixation, dehydration, and critical point drying required for conventional high-vacuum SEM. Development of an imaging method that requires less sample preparation but still allows visualization of fungal infections would improve our understanding of this disease and aid development of future treatments and disease management strategies.

Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm ( Diabrotica virgifera virgifera ) and Neotropical brown stink bug ( Euschistus heros )

Western Corn Rootworm (WCR) is a major pest in the United States corn industry, causing annual losses of over one billion dollars [1]. WCR feed on corn roots, which reduces nutrient and water uptake and can cause the plants to lodge, and thus considerably reduces crop yield and quality. One way to combat this pest is the incorporation of genes encoding Bacillus thuringiensis (Bt) crystal (Cry) proteins into the corn genome. Cry proteins interact with the midgut epithelial cells of WCR and form pores in their plasma membranes, disrupting the delicate brush border microvilli, and ultimately causing lysis and/or apoptosis of these cells [2]. Cry-damaged cells induce healing responses in nearby stem cells to repair the midgut in an attempt to prevent death of the insect [3, 4]. Herein, we characterize the impact of Cry34/Cry35Ab1 toxins on the anterior midgut morphology of WCR, thereby adding valuable data to our knowledge of the mode-of-action (MOA) for Cry34/Cry35Ab1.