Myzus persicae secretes an auxin-synthesizing enzyme while feeding

The green peach aphid (Myzus persicae) is a generalist herbivore, feeding on a variety of species in several different plant families. Many of these plants are valuable agricultural crops such as cabbage, potato, tomato, and corn. Investigating the feeding mechanisms of M. persicae, and the effects of herbivory on plants is vital to developing aphid-resistant crops. In previous research, auxin biosynthesis from tryptophan was identified through LC-MS of aphid-fed artificial diet. Other studies indicated increased expression of DAO1, the gene encoding the enzyme DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) in Arabidopsis thaliana (Arabidopsis), 48 hours after the start of aphid feeding. It is hypothesized that whilst aphids are secreting an auxin-synthesizing enzyme, plants respond to aphid feeding by upregulating the expression of DAO1. Utilizing mutant and transgenic Arabidopsis lines (DR5:GUS and dao1 DR5:GUS), we aimed to visualize increased levels of auxin at the aphid feeding site through GUS staining. Results of GUS staining were inconclusive. Interestingly, aphid-fed dao1 DR5:GUS Arabidopsis leaves did not show more GUS activity than aphid-fed DR5:GUS Arabidopsis leaves. Although further replications of this study are needed, these results may suggest the upregulation of auxin inactivation through a mechanism other than DAO1. Further investigation into mechanisms of auxin catabolism within dao1 Arabidopsis may help elucidate these results. In addition, 24-hour aphid choice assays with wildtype and dao1 Arabidopsis were performed to determine if M. persicae preferred feeding on Arabidopsis plants that were unable to oxidize auxin. Statistical analysis of aphid choice assays did not reveal any significant differences, which is distinct from prior no-choice assays where aphids produced more progeny on dao1 Arabidopsis.

Being a part of the REU program at the Boyce Thompson Institute has been an invaluable first research experience for me. In addition to learning new lab techniques and protocols, I gained insight into what a career in scientific research entails. The weekly seminars also allowed me to learn about various areas of plant biology and connect with passionate faculty and staff at BTI and Cornell. I was additionally able to participate in scientific outreach in the local community, which taught me how to effectively convey scientific research to the general public. I am especially grateful to my PI and mentor, Georg Jander, who never emphasized my mistakes and instead encouraged my learning. These past ten weeks have truly been unforgettable for me, and I will carry the skills I have learned here into each and every new step I take in the future.

Grafting and micrografting of Nicotiana benthamiana to study the phenotypic role of nicotine

Grafting and micrografting in the model species Nicotiana benthamiana allows the study of the systemic movement of molecules and their phenotypic effects. One example involves the alkaloid nicotine, that is produced in the roots but accumulates in the leaves of this species. Mutant lines were developed in Georg Jander’s lab that lack enzymes involved in nicotine biosynthesis (the reductase A622 and Putrescine methyltransferase PMT), with the A622-deficient line (a622) showing lower nicotine levels and a cold-sensitive, dwarf phenotype leading to the hypothesis that nicotine may be related to plant growth, and therefore, that a nicotine-producing rootstock may rescue the dwarf phenotype. Grafting trials were done using wildtype, nicotine-producing and low-producing plants (a622 and pmt mutants), for which sterile seedlings growing in culture media were cut to produce scions and rootstocks that were joined in recovery plates. Later on, additions to the micrograft protocol included the use of entomological needles to fix cotyledons, scion and rootstock. A successful micrograft was obtained that was transfered to substrate and may be evaluated in the future. On the other hand, grafts were done between 4-week-old plants of the nicotine-producing and mutant lines (a622, pmt and the a622 pmt double mutant) using the transverse cut and butt alignment and the wedge-cleft methods. It is suggested to keep making grafting and micrografting trials using these and different protocols to ensure successful results that will make possible to evaluate growth of this intraspecific N. benthamiana chimeras.

I am grateful with BTI and Georg Jander for this opportunity of gaining hands-on experience in pure research with Nicotiana benthamiana, especially in evaluating grafting protocols. The feedback and weekly lab meetings within Georg’s group were essential to improve my scientific-communication skills and to know what his other students were doing. And when it comes to BTI, I thank the weekly seminars, networking events and the incredible panel of Graduate Studies that took place as unique opportunitites to know what the BTI and CALS faculty were working on. This summer was full of tremendous experiences: I felt fortunate to be surrounded by a tremendous, state-of-the-art research environment and by friends, food and fun, which will be a memory for all my life. I leave this internship with some scientific literature that I found during the development of my project, that I hope to use for my undergrad thesis in Colombia, but I also leave with an even deeper motivation to continue working on Plant-Science research.

Evaluation of herbivorous insect resistance amongst Card4, Card6, Card8 and DAO-1 mutant lines of Erysimum cheiranthoides

The biosynthetic pathway of cardiac glycosides has not yet been fully deciphered. The chemical diversity of these compounds is large, and little is known about the biological function of the different stereochemistries. In this project, Erysimum cheiranthoides CRISPR/Cas9 mediated knockout mutants were used to investigate insect preference toward particular genotypes in the field. Moreover, genotypes’ resistance against Brevicoryne brassicae (cabbage aphids; adapted to glucosinolates but not cardiac glycosides). This was conducted by performing choice assays and no choice with cabbage aphids, in addition to a seven-week long field study of visiting insects in a natural setting. The results showed that there is a difference in insect preference in laboratory settings and in natural settings. In laboratory settings, the cabbage aphids generally show preference for mutant lines compared to wildtype. They also show preference in Card4 and Card6 mutant lines in laboratory settings. No significant difference could be shown in preference between Card4 and the Card9 mutant lines in laboratory choice assays. Some slight statistical difference can be shown between genotypes and flea beetle resistance. Overall, no profound differences of insect preference between the different mutant lines are shown in the field study.

I am so grateful that I received the opportunity to come all the way from Sweden to spend the summer here at BTI and in the U.S. Working with Professor Jander and with all of the members of the Jander lab, I have learned more than I thought was possible during only ten weeks. This has been my very first research experience, and I am so glad that I got to experience working in academia, getting hands-on experience with bioassays, field work, GC-MS and how to construct and analyze experiments. This experience has really opened up my eyes to plant science and research in general and made me curious for more of what academia has to offer. In addition to the research, I’m so grateful to have learned more about science communication and for the opportunity to write my own outreach article through this excellent REU program.

Revealing candidate genes for maize resistance against Spodoptera exigua (beet armyworm) via GWAS

Maize crops worldwide suffer substantial losses every year due to damage caused by pests, such as the armyworm. Secondary metabolites, specialized complex molecules synthesized by plants, can act as a defense mechanism against insects. The production of secondary metabolites can vary widely between maize populations, providing an opportunity to study the genes encoding for these compounds by comparing metabolic profiles across various maize inbred lines with their genetic differences. HPLC and mass spectroscopy can be used to generate lists of mass features present in each sample, and these features can be correlated with the relative caterpillar resistance between the different maize lines. We performed genome-wide association studies (GWAS) on a diverse panel of maize plants using both caterpillar feeding performance and metabolomic profiles as traits to identify candidate genes for resistance against armyworms. This study reveals several potential genes associated with armyworm resistance in maize, providing a path forward for further research. Once potential genes are found, mutant lines can be generated to investigate their functions. Eventually, genetically modified maize plants may be developed with increased resistance against pests to reduce agricultural losses.

Through my 10 weeks at the BTI I gained invaluable skills and knowledge about plant science, metabolomics, and conducting research as a whole. Under the guidance of my mentor Guillermo Jimenez I learned a variety of new skills such as preparing and running samples through HPLC, processing of mass spectroscopy data and statistical analysis, as well as effective experimental design. The insights of those in the Jander lab as well as many others, the informative seminars, and overall immersion into plant science I experienced gave me a much clearer understanding of the positive impact that can be made with scientific research. My experiences here have influenced my decision to pursue a PhD after my undergraduate studies and hopefully secure a career in research.

Adaptation of a tissue culture-independent transformation method and genomic editing tools for Coffea arabica

Coffea arabica represents about 60% of global coffee production. This cultivar is highly sensitive to climate change and pathogens such as coffee leaf rust (Hemileia vastatrix) and coffee berry disease (Colletotrichum kahawae). Conventional methods for genetically modifying plants involve biolistic or Agrobacterium tumefaciens-mediated transformation and subsequent tissue culture, which is a long and tedious process with coffee plants. However, new techniques for tissue culture-independent transformation, which have been developed recently for several plant species, can be adapted to genetically engineer coffee. Another recent advance is the implementation of RUBY, a reporter gene that encodes the betalain biosynthesis pathway as a visual screening marker for the successful integration of exogenous genes in plants. The main objective of this project is to combine tissue culture-independent plant transformation approaches and the RUBY reporter system for CRISPR/Cas9 mutagenesis and stable transgene expression in C. arabica. As part of the project, different A. tumefaciens strains and inducible promoter systems for the RUBY gene are being used to optimize coffee transformation.

This internship has been so far the best experience in my life. At the beginning of the summer, I had expectations of getting more involved in the general workflow of researching and talking with professors about opportunities for graduate school. Being surrounded by international scientists in the Jander lab made me feel proud of being a Latina in STEM and motivated me to keep going in this long path of academic and personal formation. By working on this project, I not only learned new techniques related to plant science, but I also had the opportunity to put into practice the knowledge that I have acquired in my home university. I want to highlight the help of my mentor, Boaz Negin, for allowing me to apply troubleshooting by my own before asking him because it challenged me, increased my confidence, and made me more independent in the lab.

Identification of voruscharin biosynthetic genes through differential gene expression analysis of Asclepias species

Cardiac glycosides are secondary metabolites produced in plants as toxic defenses against herbivorous insects. They have evolved independently at least twelve times in different plant families, and function by inhibiting the NA+/K+ ATPase enzyme in animal cells, making the plants that produce them toxic. Despite their ecological importance, many steps of the biosynthetic pathways of cardiac glycosides remain unknown. Milkweed species in the genus Asclepias are optimal candidates to investigate cardiac glycoside biosynthesis due to tremendous variation among species. Different species not only synthesize different cardiac glycosides but also vary greatly in cardiac glycoside concentrations. There are a small number of cardiac glycosides containing nitrogen and sulfur, making their biosynthetic pathways ideal for studying. Voruscharin is one such cardiac glycoside; it is present in Asclepias curassavica (tropical milkweed). Thus, the A. curassavica genome may be compared to that of a closely related species, Asclepias incarnata (swamp milkweed), that does not produce voruscharin. Asclepias syriaca (common milkweed) also produces a nitrogen and sulfur-containing cardiac glycoside that is not present in a close relative species, Asclepias tuberosa (butterfly weed). A. curassavica and A. syriaca are expected to use similar enzymes in this process. I performed the preparations for the assembly of the A. tuberosa and A. incarnata transcriptomes in order to compare differential expression under stress in species pairs. Genes that are upregulated in both A. curassavica and A. incarnata or both A. syriaca and A. tuberosa as a defense response are not likely to be involved in voruscharin biosynthesis.

My summer in the Jander lab gave me insight into what it would be like to perform research full-time. I was able to learn new lab protocols and techniques as well as how to manage my time spent doing it. Since I only had a mentor for the first few weeks, I became more confident in asking various lab members and BTI staff for help. As a result, I was also able to develop closer relationships with the people I was working alongside. Through the weekly seminar series, I not only learned new science but also how to communicate science. I am incredibly grateful to have had the opportunity to participate in the BTI REU and look forward to continuing research in plant science.

The Effects of Species and Diet on Metabolites Present in Aphid Saliva

Despite their minute physical size, aphids are an important, complex family of insects (Aphididae: Hemiptera) that includes many agronomically important pests. Most aphids feed on the phloem of plants using their specialized mouthpart called a stylet. While feeding, aphids are also injecting their saliva, rich in proteins and other components known to impact the plant-aphid relationship. Many of the proteins present in aphid saliva allow the aphid to continue feeding by dampening the host plant’s defensive reaction. It is understood that host plant and aphid species impact the composition of saliva. However, most other components of aphid saliva, like metabolites, are not understood well. A range of aphid species and host plants were compared to see if diet or aphid species influences the metabolites present in saliva. Saliva samples were processed using Liquid Chromatography and Mass Spectrometry in a non-targeted metabolomics approach. Results demonstrate that the host plant on which the generalist aphid species, Myzus persicae, is feeding does impact adult aphid size and survival. While diet did seem to impact the aphid saliva composition, surviving aphids did not always produce abundant saliva with unique compounds. Additionally, saliva from five aphid species with various ecological preferences was compared, revealing that while they share a portion of unique mass features, species also influences composition of metabolic features in the saliva. Further analysis of this data set needs to be performed to understand the trends seen in metabolite composition across species and host plants. These conclusions demonstrate the complexity of aphid saliva and warrant further investigation into the metabolome of aphid saliva to focus on metabolites that could be assisting the aphid while feeding.

This summer internship at Boyce Thompson Institute was not only fun but also highly educational and eye-opening. Getting the hands-on experience of working in a professional lab as an undergraduate student has helped me to gain new skills and confidence. Having my mentor to guide me through the project gave me stability but also granted me a new connection and friend in the scientific community. The community that was created by the REU students was very encouraging. This internship reinforced my decision that I want to continue in my educational journey and pursue a graduate degree in plant biochemistry. These experiences and the people I met have helped me to know more of what to expect as a grad student and answered my questions honestly. This internship has cultivated my interests in plant biology and has helped me to feel more prepared for my future in research.

Voruscharin in cardiac glycosides

Cardiac glycosides (CGs) are a class of organic compounds that are used as medicines to slow the heart rate and strengthen the muscle. These organic compounds can be found in milkweed plants and make the plant very toxic to most animals and insects. However, monarch butterfly caterpillars are well adapted to most CGs, efficiently sequestering them as a defense mechanism against predators. One exception is Voruscharin, a thiazolidine-ring containing cardiac glycosides that are produced in trophic milkweed (Asclepias Curassavica) that strongly inhibits the butterfly’s sodium potassium ATPase. Requiring costly detoxification to be converted into a less toxic CG. It’s of interest to know its biosynthesis, especially how its thiazolidine ring was formed on the sugar residue. Recently, the Jander lab found that cysteine might serve as a donor of the thiazolidine group in voruscharin. As cysteine is actively involved in glutathione biosynthesis, I would like to see whether cysteine incorporation into voruscharin is influenced when glutathione biosynthesis pathway is inhibited by buthionine sulfoximine (BSO). BSO is a well known inhibitor of glutamate cysteine ligase (GCL), the enzyme that controls the glutathione biosynthesis. In this study buds and seedlings of trophic milkweed were collected and soaked in water solution or growth medium. The solutions were supplemented with isotope-labeled cysteine and tested with and without BSO. The comparison on isotope labeled voruscharin and glutathione in the treatments might also help determine whether GCL is the enzyme catalyzing the cysteine incorporation into voruscharin.

The PGRP program has taught me how much patience, precision and knowledge goes into lab work. I‘ve also learned that when mistakes happen with research, it’s how you deal with them that’s important. While working in the lab I made mistakes and was encouraged to try again and keep practicing. Working with my mentor, Fumin Wang, brought a new perspective on what goes into research and the significance each experiment has on the overall project. I am thankful that my experience has been eye opening to my passion in environmental work.

Localizing Catechol-Glucoside Synthesis Genes May Provide New Insight Into The Functions of Maize Benzoxazinoids

Benzoxazinoids are a type of grass species metabolite that are essential to herbivory and pathogen defense. Prior research has indicated their potential to carry information across the cell, regulating non-benzoxazinoid metabolite networks. Operating off of this theory, a metabolomics analysis determined that benzoxazinoids involved in callose production in maize regulate Acetylcatechol -Glucoside and Catechol-Glucoside, two non-benzoxazinoid metabolites. My research localized the enzymes involved in the synthesis of these compounds in order to understand their function and provide evidence for benzoxazinoids’ purpose as a signal molecule. We transferred three candidate genes into EGFP vectors and infiltrated N.Benthamiana, a model plant for maize. Only the acetyltransferase gene was localized, and flourescencing occurred in the chloroplasts and cell membrane. This suggest that the compound could be involved in photosynthesis or conversion of molecules to different forms.

My research experience at BTI gave me insight into how labs are funded and what daily life in a lab is like. I gained exposure to a variety of techniques for DNA and structural analysis, including protoplast isolation, PCR, proton spectrum emissions (MSQC and COSY), and plasmid isolation. After the initial learning process, the lab environment became very conducive to growing my independence, and the ceiling for learning remained exceedingly high. It was a very special opportunity to work with people from diverse cultural and academic backgrounds, and I am grateful to my mentor and everyone in the Jander Lab.

“Subcellular localization and role in pollen development of the jasmonic acid receptor (COI) protein family in Zea mays“

Project Summary:

The jasmonic acid (JA) signaling pathway regulates plant development and environmental responses. In the pathway, the Coronatine Insensitive (COI) protein binds JA-Ile and initiates degradation of the Jasmonate-Zim Domain (JAZ) repressors, which inhibit activity of JA gene transcription factors. Six COI orthologs (four COI1s and two COI2s) are present in maize as a result of gene duplication events. Preliminary experimental results inspired two independent experiments: (1) subcellular localization of the COI1 proteins, and (2) COI2 pollen viability assays. For Experiment 1, previous COI1 and COI2 subcellular localization experiments in N. benthamiana showed that COI2s were localized primarily in the nucleus, while COI1s were localized in cytosolic organelles. Additionally, bimolecular fluorescence complementation in N. benthamiana leaves showed that, while COI1s only weakly interacted with JAZ proteins in the nucleus, COI2s showed strong interactions. We hypothesized that, if COI1 has a lower affinity for JAZ than COI2, expression of COI1 in maize protoplasts should restore nuclear localization due to the presence of the host JAZ. When maize protoplasts were transformed with COI-GFP plasmids, COI1 appeared to localize in the nucleus. However, the results were difficult to interpret since the cells ruptured and the expressing nuclei were free-floating. For Experiment 2, previous work suggested that the homozygous coi2 double mutations are lethal. To determine when the coi2a coi2b double mutant pollen dies, pollen viability and germination assays were performed. COI2a/coi2a coi2b/coi2b and coi2a/coi2a COI2b/coi2b plants had significantly higher amounts of non-viable pollen and exhibited lower pollen germination rates than their single-mutant siblings.

My Experience:

Surrounded by a diverse group of plant scientists, I have learned far more than lab techniques and greenhouse protocols during my time at BTI. The most valuable lesson I learned from this experience is the importance of thoughtful discourse and collaboration in science and research. I realized that one of the most essential skills to have as a scientist is the ability to clearly communicate complex topics in a way that is not intimidating or confusing for scientists or non-scientists. Additionally, my lab has demonstrated that open dialogue amongst fellow scientists is also crucial in the pursuit of refining and uncovering scientific conclusions. My project would not have been successful without the guidance and support of my peers, lab mates, and mentor. BTI’s emphasis on collaboration has amplified my appreciation for working with a team and constantly considering new ideas, questions, and perspectives that help me develop as a scientist.

“Investigating water relations in OST1-silenced Nicotiana benthamiana plants with AquaDust”

Project Summary:

Capturing the effects of drought on plant water relations in real-time is challenging. However, a nanotechnology approach involving a dyed hydrogel known as AquaDust can be used to monitor the local water potential (ψ) within the apoplastic spaces of leaves. AquaDust, in combination with a gas exchange machine, can be used to measure the vapor pressure deficit (VPD), rate of transpiration (E), and rate of carbon assimilation (A) as metrics for plant growth and water use efficiency. Our goal was to explore the local water potential and hydraulic conductance in the xylem and mesophyll cells of mutated tobacco leaves. Virus-induced gene silencing (VIGS) protocols were implemented in Nicotiana benthamiana to knock down the expression of OST1, which encodes a protein that regulates stomatal closure. Four-week-old  N. benthamiana plants were inoculated with tobacco rattle virus (TRV) to suppress the gene expression. The treatment group in this experiment consisted of the TRV genome with DNA fragments of our gene of interest (TRV-OST1). Three controls were used to determine whether the phenotype was affected by this treatment. One control, which was nonspecific to the genes in the N. benthamiana, was known as (TRV-GFP). The negative control was the (TRV-PQ-11-empty vector). The positive control (TRV-PDS) was used to determine how long it took for the inoculation to silence N. benthamiana’s gene expression by producing a bleaching phenotype. We studied how silencing OST1 gene expression prevented plants from closing their stomata. When the stomata remained open, we determined that the gas exchange mechanisms affected would trigger changes in the VPD and local water potential, which could then be investigated with AquaDust.

My Experience:

I am fortunate to have had the opportunity to learn and work in both Abe Stroock and Georg Jander’s labs, as part of the NSF-funded CROPPS project. I came to the Boyce Thompson Institute with minimal bioinformatics and molecular biology knowledge. However, because of the strong support network I encountered here, my critical thinking skills and problem-solving abilities grew to new heights. My mentors, Honglin Feng and Sabyasachi Sen, helped me gain a dynamic understanding of the molecular cloning processes, bioinformatics, and programmable plant systems used to carry out the experimental design and functional assays of our project. This ranged from learning virus-induced gene silencing protocols to generate mutated N. benthamiana tobacco plants with altered phenotypes to utilizing the AquaDust nanogel to take measurements at the molecular level within the leaves of living plants using a fiber-optic spectrometer. From this research experience, I gained the confidence to learn new things, ask questions, and envision opportunities as a plant researcher.

“Structural diversity in cardenolides: Gaining insights into the biosynthetic pathway”

Project Summary:

Thanks to its phytochemical defense diversity, the Erysimum genus is better equipped to avoid herbivory than other plants in the Brassicaceae family. Erysimum produces two classes of toxic compounds, glucosinolates and cardenolides, a type of cardiac glycosides. Cardenolides act as inhibitors of the Na+, K+- ATPase ion channel, an essential and conserved membrane protein in animal cells that helps maintain the electrochemical gradient involved in many cell and organ functions. In human medicine, cardenolides are used as a treatment for heart arrhythmias and congestive heart failure. Despite their medical and ecological importance, the cardenolide biosynthetic pathway remains unknown. To address this knowledge gap, the Jander lab has established Erysimum cheiranthoides (wormseed wallflower) as a model system for studying cardenolide biosynthesis. Along with this, the lab applied mutagenesis and discovered plants with altered phenotypes, including the absence of derived cardenolides such as cannogenol and strophanthidin, presumably because of a mutation in a cytochrome P450 that hydroxylates digitoxigenin. This mutation is useful for studying the evolution of the biosynthetic pathway, as the Erysimum cytochrome P450 is homologous to a known Arabidopsis thaliana cytochrome P450 that hydroxylates steroid-like compounds. Transient overexpression of the P450 enzyme via Agrobacterium tumefaciens infiltration, implemented in E. cheiranthoides mutants that are deficient in derived cardenolides, successfully complemented the mutant phenotype. The cytochrome p450 was also transiently expressed in Nicotiana benthamiana plants via A. tumefaciens infiltration and addition of the substrate digitoxigenin. Yeast transformation was used to express the p450 in Saccharomyces cerevisiae, with addition of digitoxigenin to determine whether the derived compounds are produced.

My Experience:

This summer research internship has been one of the best experiences of my life, and I know it will mark an important milestone in my professional career. I remember that at the beginning of the internship I had expectations of getting an insight into academic life and the commitments that come associated with working in research, as well as acquiring new skills that would serve me in a possible future career as a research scientist myself. But today, reflecting and admiring the experience from the other side, I am certain that I got many other things out of this summer. The challenges of moving to an English-speaking country for two months, in the role of a short-term researcher, have been considerable. I have had opportunities to prove to myself that I have a strong sense of responsibility and resilience, and I have gained a lot of confidence in myself and my skills. I highly value the experience of working in a high-level research laboratory, and doing science has made me realize that it has to be collaborative. It was invaluable working with my mentor, Gordon Younkin, who was there to guide and support me but let me have the driver’s seat in developing my own project, to which I could contribute my knowledge.

“Investigating the activation/deactivation mechanisms of jasmonate signaling in maize (Zea mays)”

 Project Summary:

The jasmonate signaling pathway controls many plant processes, including growth, development, and defense. One key component in this pathway is the fatty acid derivative jasmonic acid (JA). JA is induced by both abiotic and biotic stresses such as mechanical wounding and herbivory. Upon induction, JA is converted into its bioactive form JA-isoleucine (JA-Ile), which is perceived by its F-box COI1 receptor. JA-Ile perception results in a wide transcriptional reprogramming activating the immune system of the plant. This process is costly; therefore, plants need to maintain a fine-tuned homeostasis to optimize fitness and guarantee survival. In Arabidopsis thaliana, JA conjugation/deconjugation to Ile is achieved by amido-synthases/-hydrolases of the GH3 superfamily, respectively. A phylogenetic analysis of GH3 proteins revealed two candidate genes for conjugation and deconjugation of JA to Ile in maize. The objective of the work was to understand the role of the identified enzymes, JIH1 and IAA-AS, in regulating the jasmonate signaling pathway in Zea mays (maize). To achieve this goal, two experiments were conducted, (i) quantification jasmonate content on mutant lines, and (ii) a caterpillar performance assay on the same set of plants. The data showed the absence of JIH1 did not have a clear impact on maize defense against the generalist insect Spodoptera exigua. Results obtained with IAA-AS were inconsistent and further studies are needed to assess the role of this enzyme in maize. Future studies should be conducted on the activity of IAA-AS by employing different mutant lines than those analyzed in this study. Additionally, it may be helpful to find other metabolites that are impacted by these enzymes through an untargeted metabolomic analysis.

My Experience:

The past few weeks in the Jander lab have been an eye-opening experience. Being immersed in an environment with passionate researchers has exposed me to the vastness of plant biology research, a truly humbling experience. Working with my mentor, Dr. Jimenez-Aleman, I was able to get a glimpse of how to approach certain problems, not only with the available technology but also in the most efficient way. Caterpillar performance assays, phytohormone extraction, and running and analyzing samples via UPLC-MS/MS are just some of the techniques that I learned for my project. Along with learning new approaches and lab techniques, I was able to gain confidence with previously learned lab techniques, which has helped me to develop into a better scientific researcher. I will take all of these acquired techniques, approaches, and skills to my home institution, where I will continue to investigate key enzymes in the jasmonic acid signaling pathway in maize.

“Transportation of cardiac glycosides across tissues in Milkweeds (Asclepias)”

Project Summary:

Plants in the genus Asclepias, milkweeds, have been a popular study subject for ecological, biochemical, and medicinal purposes. In natural ecosystems, milkweeds, as the only host plants, are essential for ecologically important insect species like the monarch butterflies. Milkweeds accumulate cardiac glycosides (CGs), which have been used to treat heart failure and have been intensively studied for their medical importance. In addition, CGs are defensive chemicals that protect milkweeds from insect herbivores. Characterizing CGs and their biosynthesis will provide further knowledge regarding their functions and promote CG usage for medical and agricultural purposes.

My project is to investigate the possibility of CG transport between different tissues through grafting experiments using two milkweed species, A. curassavica and A. incarnata, which have differing CG profiles. In the grafting experiments, the shoot and root of A. incarnata was cross-grafted with the root and shoot of A. curassavica. In parallel, each species was self-grafted with its roots and shoots as controls. Once the grating junction had healed, we determined the abundance of CGs by using liquid chromatography and Orbitrap Q-Exactive mass spectrometry (LC-MS). Then, CGs were identified and annotated using Thermo Xcalibur Qual Browsers. Three CGs that are specific to A. curassavica, voruscharin, calotropin, and calactin, were analyzed. According to our results, we did not observe transport of these CGs from A. curassavica to grafted A. incarnata tissues, in either the shoot-root or the root-shoot direction. Through this project, we conclude that, without stress, A. curassavica does not transport voruscharin, calotropin, and calactin between tissue types.  

My Experience:

My experience this summer at Boyce Thompson Institute has extended my knowledge in the lab setting and scientific research. Working with my mentor has given me the opportunity to learn an abundance of new techniques and procedures in the lab. The weekly lectures, seminars, and lab meetings have expanded my understanding of the vast array of research topics and subfields of biology. It has been extremely meaningful to discuss with undergraduates, post-docs, and professors who are passionate about their work and study, both within and outside their field of biology. The collaboration and positive work environment at BTI has helped guide my interests and inspired me to pursue science in college and in my future career.

“Overexpression of candidate insect herbivory resistance genes in Setaria viridis and the effects on Spodoptera frugiperda herbivory”

Project Summary:

This project investigates how insect herbivory of Setaria viridis (hereafter Setaria) is affected by overexpression of three candidate genes encoding chitinase, thionin, and pathogenesis-related protein Bet VI family. The transgenic candidate genes sequences were confirmed to ensure the correct genes were inserted into the Setaria genome. Subsequently, the overexpression of the three candidate genes in respective plants was confirmed using quantitative Real-Time Polymerase Chain Reaction. Selecting for the Setaria lines with the greatest overexpression of the respective candidate genes at the transcript level, comparative herbivory experiments using Spodoptera frugiperda (fall armyworm) were and will continue to be conducted.

My Experience:

This project investigates how insect herbivory of Setaria viridis (hereafter Setaria) is affected by overexpression of three candidate genes encoding chitinase, thionin, and pathogenesis-related protein Bet VI family. The transgenic candidate genes sequences were confirmed to ensure the correct genes were inserted into the Setaria genome. Subsequently, the overexpression of the three candidate genes in respective plants was confirmed using quantitative Real-Time Polymerase Chain Reaction. Selecting for the Setaria lines with the greatest overexpression of the respective candidate genes at the transcript level, comparative herbivory experiments using Spodoptera frugiperda (fall armyworm) were and will continue to be conducted.

“Evaluation of Insect Preference in Erysiumum cheiranthoides Mutant and Wildtype Plants”

Project Summary

Cardiac glycosides are a class of secondary metabolites involved in plant defense against herbivores. One class of cardiac glycosides, cardenolides, are found in multiple plant families, and evolved in the genus Erysimum of the Brassicaceae family between one and three million years ago, providing defense against glucosinolate (a different class of defensive metabolite)-resistant insects. Research into cardiac glycosides could be beneficial in creating more pest-resistant crops, possibly increasing crop yields and decreasing harmful pesticide use. Additionally, cardiac glycosides have a medical application, being used as a heart medication.

Using mutant-line 635 Erysimum cheiranthoides (wormseed wallflower) plants with a mutation in a gene associated with cardenolide production, as well as wildtype Erysimum, I performed bioassays to investigate the effect of the differing cardenolide content present in the plants of the F2 generation on insects. I used Myzus persicae (peach aphid), as well as Plutella Xylostella (diamondback moth) and Spodoptera exigua (beet armyworm) caterpillars for choice assays, observing the preference of insect feeding between a mutant and wildtype leaf. I also compared M. persicae reproduction on whole mutant and wildtype plants. The results show that cardenolide differences in the 635 mutant line do affect insect preference, although that preference may differ between herbivores, possibly due to physiological difference or an additional unknown difference between the mutant and wildtype plants.

My Experience:

My first week or two in the Jander lab were filled with learning and re-learning basic lab skills. Following a year and a half of the coronavirus, my science classes in school had been severely lacking in lab experience, and there were plenty of new tools and techniques to master, such as micropipetting, creating overnight bacterial cultures, and streaking plates to create bacterial colonies. While my mentor, Marty Alani, was always there to help and supervise me, I was able to gradually become more proficient at doing things myself over the last 6 weeks. Things quickly became less overwhelming and confusing in the lab, and I was able to set up the bioassays independently. Later on, I also took part in more advanced and exciting procedures like gene transformations of Erysimum using agrobacteria dipping. Overall, this internship has been a very engaging and valuable experience.

Exogenous Hormone Application Modulates Cardiac Glycosides in the Non-Model Brassica Wormseed Wallflower

Cardiac glycosides, also called cardenolides, are defensive compounds produced in twelve different plant families that act as a broad-spectrum herbivore defense. In binding to and inactivating Na+, K+-ATPase, a crucial and highly conserved ion channel in animal cell membranes, cardiac glycosides provide a durable and effective deterrent to feeding. Specifically, cardiac glycosides hamper muscle function, eventually slowing and stopping the heart. The cardiac glycoside chemical structure contains a steroid moiety, a lactone ring, and sugars. The structural similarity to steroid hormones leads us to hypothesize that biosynthesis of cardiac glycosides in plants might closely mirror that of other phytosteroids and that the two pathways may share intermediates. My project focused on modulating hormone levels in plants through exogenous applications and artificial growth media to see whether there were subsequent effects on cardenolide content. I performed two hormone application experiments, one involving growth of Erysimum cheiranthoides (wormseed wallflower) seedlings on agar growth medium supplemented with the plant hormones auxin and brassinolide, and the other direct leaf application of these two hormones. I then ran UPLC-MS analyses of metabolite extracts in order to compare cardiac glycoside content between my control and hormone groups. I found that seedlings growing on plates supplemented with brassinolide had increased cardiac glycoside levels, but that the treatment either had no effect, or decreased cardiac glycosides in adult leaves. I found that indole acetic acid (auxin) had no effect on cardiac glycosides in either experiment, and that 1-naphthaleneacetic acid, a synthetic auxin analog, also had no effect when present in seedling growth medium. In future experiments, I would like to perform a transient overexpression of genes early in the brassinosteroid pathway to determine whether this would also increase cardiac glycosides, implying that these two phytosterols share pathway intermediates. I also would like to grow plants on medium supplemented with brassinosteroid analogs to see if cardiac glycosides similarly increase, thereby determining whether the increase is due to the direct effects of the hormone on the plant or due to a shunting of pathway intermediates to cardiac glycosides, which might result from a surplus of brassinosteroid hormones.

My Experience:

Having worked in the Jander lab a previous summer, I was expecting an experience like what I already knew, of carrying out experiments and letting my mentor decide what I would do for his and my projects. That was in high school for me, and having finished my first year in college, I found that I could apply my basic coursework to my research and get a lot more out of it. Compared to my previous experience, I’m much more in the driver’s seat and am making my own decisions about my project. My mentor taught me many new techniques and now lets me be relatively independent. I’ve been having lots of trouble with one of my experiments, yet all my effort researching troubleshooting for that technique, asking other lab members, and trial and error has given me a deeper understanding about the technique and how it works. I’m a Cornell student, and my experiences this summer encouraged me to see this project through to the finish; I’ll continue to do research in the Jander Lab during the school year. I’d just like to thank my mentor Cynthia, Dr. Jander, and all the other Jander Lab members for encouraging me and for making such a positive learning experience!

“Exploring Novel Strategies for Aphid Pest Control”

Aphids, comprised of 4,000 species, are serious agricultural and forestry pests. Aphids can damage plants by directly sucking plant phloem sap, vectoring plant viruses, and providing a hotbed for growth of sooty mold by their honeydew. Current pest control methods involve spraying varying amounts of chemical pesticides which have introduced foreign chemicals that pollute the environment and infected plants that people eventually consume. My project aims to explore safer techniques that includes two RNA silencing methods: 1) plant-mediated virus-induced gene silencing (VIGS) and 2) morpholino-mediated gene silencing. Plant-mediated VIGS involves using Agrobacterium transformation to infect plants with Tobacco rattle virus (TRV) that carries double-stranded RNAs targeting an aphid gene. The goal is for aphids to ingest this double-stranded RNA through natural feeding on VIGS plants, in turn blocking the target gene expression via RNAi, and ultimately decreasing aphid population. We are still in the phase of screening target gene candidates that can effectively reduce aphid survivorship via the plant mediated VIGS method. Morpholinos, also known as phosphorodiamidate morpholino oligomers (PMOs), have been used as gene silencing reagents. We aim to explore morpholinos for their potential to control aphids. PMOs are synthetic oligonucleotides that contain a backbone of morpholine rings connected by phosphorodiamidate linkages. PMOs function by complementary binding to the target mRNA sequence, thereby blocking mRNA translation or modifying pre-mRNA splicing. We designed a PMOs to targeting an exon-intron junction of aphid gene white, aiming to alter its exon splicing. Alteration to white by this PMO will result either dysfunction or reduced expression. Thus, we expect aphids who ingest this PMO to demonstrate discolored eyes because white has been shown to be responsible for eye color in other insects. We have designed experiments that will allow for optimal uptake of PMO, using techniques such as topical delivery, gene gun bombardment, artificial diet, and microinjections. After such experiments, we have observed lighter eye phenotypes but have since complemented with biological replicate experiments and qPCR/PCR experiments to confirm the altering of the gene white. These results have looked promising, but we are still far away from eliminating all possible conflicting variables and confirming the efficacy of morpholinos for controlling aphid pests.

My Experience

Not only has the Boyce Thompson Institute experience expanded my scope of scientific knowledge, but it has also sharpened my abilities to regard the minuscule details of lab work and experiment management. I now know what it means to design sufficient control experiments and limit sources of potential cross contamination, the little things that lead the experiment in the right direction. It is easy in class to get carried away in the unimportant details of science like memorizing the steps to the scientific method or memorizing the amino acids. But with the help of my mentor, this program provided the perfect space to apply the concepts I have learned in introductory science course to experiments in real life. Working with my mentor has shown me what it means to actually pursue research because in the work there are so many ups and downs and variables that are not in your control. But it’s all about how you use what you have to your advantage to try and contribute to the scientific world. I definitely got to experience first-hand some of the concepts I read about in my AP Biology textbooks. Most importantly, this program has raised my confidence level in pursuing research science to the point where I’m excited to start my own journey into scientific research fields.

Virus-mediated overexpression of peptides in Golden Bantam maize to reduce fall armyworm larval growth

To protect themselves against herbivorous insects and microorganisms, plant species have evolved physical and chemical weapons. The recently discovered PROPEP1 gene, which encodes the precursor of the Pep-1 peptide in maize (Zea mays), has been found to play a crucial role in activating and amplifying the innate immune responses of the plant. Since we know that the peptide regulates the biosynthesis of benzoxazinoids, defense proteins, and jasmonic acid, which are all provide protection against pests such as fall armyworm (Spodoptera frugiperda), we expect that Pep-1 over expression will reduce larval growth. We set out to determine whether the overexpression of Pep-1 with Sugarcane mosaic virus in the maize would increase the plants’ resistance to pests. In order to overexpress the peptide in the maize variety Golden Bantam, a series of cloning processes were performed along with different methods of injections in order to get a high level of viral vector infection. Because there was insufficient time for a bioassay with the cloned Pep-1 gene, the bioassay was performed with plants expressing UyCT3 and LqhIT3 (scorpion venom proteins that are toxic to insects). In this bioassay we found that caterpillars growing on the UyCT3- and LqhIT3-expressing plants experienced significantly reduced growth compared to GFP-expressing control plants. From this result we can deduce that overexpression of cloned peptides in a virus vector can be used to reduce the growth of S. frugiperda.

My Experience

Working in the Jander Lab at Boyce Thompson Institute this summer has been an invaluable experience, introducing me to the plant world. Each lab member’s passion for science not only made the Jander Lab a welcoming place to work, but a safe place to ask hundreds of questions. Entering the program with little knowledge from high school biology, my mentors Seung Ho and Mahdiyeh took the time to explain every protocol with detail and taught me the importance of laughing at unexpected results. Arming me with new scientific skills and teaching me to use high tech machinery, my mentors challenged my high school thinking styles, pushing me to come up with other possibilities when procedures failed. The PGRP intern experience has offered me a real world setting to test my curiosities in plant defense mechanisms and has allowed me to realistically apply my results to better sustain the health of our future crops and plants.

Coi Receptors in Maize: Beyond Jasmonic Acid Perception

Jasmonic acid (JA) is a lipid-based phytohormone that plays an important role in plant defense. It is released in response to tissue wounding and induces plants to undergo specific transcriptional changes that protect them from herbivores. JA induction leads to the production of secondary metabolites that aid plants in fighting invasion and the release of volatile substances that attract parasitoids. In Arabidopsis, JA is known to be perceived by a protein called Coi1 (Coronatine Insensitive 1). In maize (Zea mays), however, evolutionary gene duplications have resulted in the presence of at least four Coi genes: Coi1a, Coi1b1, Coi1b2, and Coi2. These genes are either evolutionarily conserved, or have undergone neofunctionalization to detect other signaling molecules. The scientific community has yet to determine which combination of Coi1 proteins are the true receptors of JA, and if any of the Coi proteins are responsible for perceiving other distinct ligands.

In this experiment, we exposed single mutants of maize (Coi transposon insertion mutants) to caterpillar feeding and observed a potential difference in caterpillar weight, which would give us insight into JA perception, and potentially be indicative of another defense pathway. We first genotyped each plant by DNA extraction, amplification with polymerase chain reaction (PRC), and gel electrophoresis. In our bioassays we subjected plants that were homozygous mutant or wild type to feeding by fall armyworms (Spodoptera frugiperda), and made progress toward creating an efficient method of data collection. We also extracted RNA from five of the plants that we genotyped to be homozygous and performed reverse transcription to make cDNA, which will serve as proof that the mutant plants are incapable of expressing the Coi gene.

The future objectives of this project are replication of the bioassay to ensure the accuracy of results, as well as follow-up experiments using Coi double mutants, and later triple mutants.

My Experience

Through the BTI internship, I have been able to apply the knowledge gained in school to a real-life laboratory setting. By working alongside motivated scientists, I was able to learn new skills and methods, such as polymerase chain reaction (PCR), DNA extraction, and reverse transcription. I also acquired an in-depth understanding about my project. Not only did this experience teach me scientific techniques, but it also allowed me to work on my scientific fluency and oratory skills. The weekly seminars, lab meetings, and scientific papers read enhanced my ability to understand scientific content, and the poster presentation at the end of the internship allowed me to practice conveying my thoughts in an articulate manner. This experience has allowed me to think about my future in science; I have been inspired to pursue a biology related field.

“A QTL Containing Maize ETHYLENE INSENSITIVE 2 Regulates Two Antifungal Metabolites and Resistance Against a Common Fungal Pathogen, Fusarium Graminearum”

Project Summary

Resistance to fungal pathogens is an important plant defensive and crucial for their survival and reproduction. Ethylene takes part in plant responses to necrotrophic pathogens, such as Fusarium graminearum. One way in which ethylene contributes to fungal resistance is by regulating the production of defensive plant metabolites. Previously, a quantitative trait locus (QTL),was discovered by genetic mapping with maize (Zea mays) B73 x Mo17 recombinant inbred lines, was shown to control both expression of ETHYLENE INSENSITIVE 2 (EIN2) and accumulation of the defensive metabolites smiliside A and Smigliside C. The main objective of this project was to validate this genetic mapping result with a collection of near isogenic lines from the same two parental maize inbred lines, Mo17 and B73. Specifically we found that the smiliside A/smigliside C ratio can only be increased by 1-aminocyclopropanecarboxylic (1-ACC, an ethylene biosynthetic precursor) in near isogenic lines that carry the Mo17 allele at this locus. These near isogenic lines also exhibited higher resistance against F. graminearum. This result demonstrates that ethylene regulation of smiliside A/smigliside C metabolism is dependent on the identified genetic locus and that it contributes to resistance against F. graminearum.

My Experience

My experience in the Jander lab at the Boyce Thompson Institute has been very beneficial for me, both academically and personally. With the help of my lab mates and specifically my mentor I have learned a wide array of new techniques including, DNA extraction, qPCR, colony PCR, and RNA extraction. My mentor and all the members of my lab were very supportive and welcomed me, encouraging me to ask questions and be involved. Overall, the experience allowed to get a firsthand look of what working in a lab is really like and better understand potential majors and careers I would be interested in exploring in the future.

“Aphid Produced Auxin in the Response of Arabidopsis thaliana to Aphids”

Project Summary:

Aphids are one of the most abundant order of sap sucking pests interacting with agricultural crops. My mentor discovered that one of the components in aphid saliva is the auxin indole-3-acetic acid (IAA), a naturally occurring phytohormone in plants. The goal of this research is to determine the role of aphid made auxin in aphid-plant interactions. We looked for plant genes involved in either auxin signaling, regulation, transportation, or storage, that are also expressed differently after aphid treatment. Two auxin related genes DFL1 and Pin5 were found to be induced during aphid treatment. DFL1 is a Gretchen Hagen 3 gene that is involved IAA amino acid conjugation. Amino acid conjugates are known to be involved in auxin inactivation, regulation, and storage. Pin5 is a gene that codes for an auxin transporter which might regulate intracellular auxin homeostasis and metabolism. The cause of the induction of Pin5 and DF1 during aphid treatment is unclear. My research project focused on determining if DFL1 and Pin5 have roles in aphid reproduction. To test this, Arabidopsis thaliana with a mutation in either the DFL1 or the Pin5 gene were selected and at maturity, a leaf on every mutant and wild type plant were infected with an aphid (Myzus persicae) nymph.  Infected leaves were then isolated from the rest on the plant for a week and then the number of aphids per leaf was counted. Results will shed light on the role of auxin in aphid interactions with plants.

My Experience:

The Boyce Thompson Institute Summer High School Internship Program provided me a meaningful experience which validated my desire to pursue collegiate studies in biology and genetics. Entering the program, I knew that I wanted to continue to study science, but was unsure on research biology as a path.  I am now confident in my decision to study cellular biology or genetics. My mentor Leila was awesome and I’m excited by the new skills and techniques I learned, such as how to extract RNA and genotype plants. I would have never had this opportunity in my high school. I’ve grown tremendously in my ability to work others through my interactions with my mentor and other scientists. I will always be grateful for my time here at BTI!

“Functional Analysis of a Putative UDP-Glycosyltransferase Involved in the Biosynthesis of Cardiac Glucosides in Erysimum cheiranthoides”

Project summary:

Cardiac glycosides are a class of defense-related plant metabolites that inhibit animal Na+, K+-ATPase membrane ion transporters, and thereby deter certain insect species from feeding or ovipositing on the plants that produce them. Despite their potential medical and agricultural applications, the biosynthetic pathway of cardiac glycosides remains incomplete to this day. Candidate genes were selected based on comparisons of 3′-RNA-seq and LC-MS analyses, performed before and after elicitation of defenses in Erysimum cheiranthoides (wormseed wallflower), a Brassicaceae species that is phylogenetically close to the model plant Arabidopsis thaliana. This identified a gene coding for a predicted UDP-glycosyltransferase that could catalyze the last step of the production of erycordine, a cardiac glycoside that was previously identified in E. cheiranthoides. Through cloning in plasmid vectors for over- or under-expression, then transformation via Agrobacterium tumefaciens and A. rhizogenes, the UDP-glycosyltransferase candidate gene was expressed transiently in the leaves and roots of E. cheiranthoides. LC-MS analysis of cardiac glycosides was performed to confirm whether or not there are effects on this metabolic pathway. In addition to obtaining the complete pathway of cardiac glucoside biosynthesis, the discovery of new molecules and the improvement of the methods for obtaining these compounds could be a significant asset for medical research. Moreover, cardiac glycoside biosynthesis could be used to protect crops and thus help reduce the use of pesticides.

My Experience:

Studying in the first year of a master’s degree in Plant Integrative Biology and Breeding in France, I had to do a two-month internship to complete my school year. By coming to the BTI, in addition to acquiring a first real professional experience in research laboratory, I took this opportunity in the United States to improve my English and gained many new laboratory techniques. I was also able to put to other techniques to use that I knew but I had never realized in application for. As the weeks went by, I gained more and more confidence in the manipulations and I began to really appreciate the research work, even with the few moments of frustration when the results were not what I expected. Apart from my research topic, participating in the PGRP also allowed me to discover a range of new plant biology research topics by attending the various seminars and becoming friends with the other students in the program. The bioinformatics courses offered have also improved my understanding of the methods used to obtain candidate genes. Before spending my summer in Ithaca, I wasn’t very motivated to continue my studies and obtain a PhD, but it is now a career path that I am considering more seriously.

“Physiological Mechanism of Fusarium graminearum-induced Aphid Vulnerability on Maize Seedlings”

Project Summary

Plants are subject to attack by many types of pests. While there has been significant research on plant interactions with these pests separately, less is known about how plants respond to multiple pests in separate parts of the plant. In my project, we investigated the interaction of corn leaf aphids (R. maidis), a leaf phloem feeder, and F. graminearum, a fungus that infects corn roots.  A previous experiment has shown a significant increase in corn leaf aphid reproduction on B73 corn seedlings infected with F. graminearum. This could be explained by a decrease in benzoxazinoids, a major plant defense chemical, in the leaves after F. graminearum root infection.  To test if the F. graminearum-induced R. maidis growth is dependent on the induced benzoxazinoid reduction, we used two benzoxazinoid deficient mutants, bx1 and bx2, as well as their shared wild type progenitor T43, to compare aphid reproduction on fungus- and mock-inoculated plants.  We found that the increase in aphid fecundity present in fungal infected plants is not conserved in the T43 genetic background, because we found no significant difference in aphid reproduction on fungal-infected and mock plants.  Understanding this type of interaction could inform farmers about more resistant corn varieties, or could just provide further elucidation into the way that plants defend themselves against pests.

My Experience:

Overall, I’ve had a very educational and constructive experience in my lab.  My mentor, and everyone else in my lab, are very supportive of me and my work.  If I ever am unclear on anything, whether it is some sort of extraction protocol or just how to use a machine, I can count on any person to help me out, and sometimes, people count on me to help them out in the same way.  I’ve been able to learn many laboratory techniques such as DNA extraction, PCR, and gel electrophoresis, all of which I wouldn’t have the chance to do outside of a lab environment. When my mentor shows me a new technique, he also explains what the technique does, and why the results are important to my experiment.  I feel like I really understand what I’m doing in the lab and why it matters, which is the most important part of this internship for me.

“Variation in Trichome Density, Latex Inducibility, and Resistance Against a Specialist Insect Herbivore in Natural Populations of Common Milkweed”

Project Summary:

The common milkweed, Asclepias syriaca, a native weedy plant that occurs throughout eastern North America, is the major food source for migrating monarch butterflies (Danaus plexippus) and their larvae. When damaged, milkweed plants immediately exude a sticky, gelatinous, milky-white substance known as latex that serves as a physical barrier to herbivores. This latex, along with all other plant parts, contains cardenolides of varying polarity and effectiveness against herbivores. The growth rate of Danaus plexippus is related to the amount of latex produced from abrasions, leaf cardenolide concentrations, and the presence of leaf trichomes. However, the causes of variation in these traits among natural populations of A. syriaca are poorly known.

Here we measured two defensive traits, latex inducibility and density of leaf trichomes, as well as Monarch larva performance on ~200 A.syriaca accessions. We found that there were high variations of latex response among different accessions post larva damage. Also, the greater the amount of leaf trichomes present, the more latex the caterpillars ingested, and the greater risk of death. Studying the phenotypic variation amongst natural accessions of milkweed allows us to identify the genetic and molecular basis of A. syriaca in future studies.

My Experience:

Over the past 10 weeks, I’ve improved several of my lab skills, made impactful connections, and have gained more knowledge than my brain can hold. My mentor, and friend, Jing Wei, has taught me different lab techniques, showed me how to be an independent researcher, and has challenged me to become a better problem solver. I am now excited and prepared more than ever to conduct my own research in the future. I have discovered my love for plant genomics research, and can fully appreciate all of the hard work and failure that goes into research.

Project Summary

Plants are constantly exposed to pathogen attack, yet they manage to thrive and fight off these threats.  Our food supply is dependent on crops’ ability to fend off these pathogens, making their biochemical defense systems a point of research interest.  From a previous comparative metabolomics study, we identified two feruloyl acetylsucroses in maize seedling roots that could be strongly induced upon infection by Fusarium graminearum, a common fungal pathogen.  We hypothesized these metabolites could play a role in maize biochemical defense against this fungal pathogen. Further genetic study on this metabolite led us to hypothesize that ethylene signaling is a positive regulator of these metabolites.  Taking an integrated genetic and physiological approach, we treated seedlings of B73 wildtype and aminocyclopropane-1-carboxylic acid synthase (ACS) 2/6 double mutant, a maize genotype which lacks an enzyme in the ethylene biosynthetic pathway and therefore produce less ethylene, with aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene.  We then used mass spectrometry to analyze harvested root extract to measure the concentration of the two feruloyl acetylsucroses.  In results, we found that the ACS 2/6 double mutant seedlings had constitutive lower content of these two metabolites, and that the ACC treatment induced accumulation of these two metabolites in both B73 wildtype and the ACS 2/6 mutant seedling roots. Interestingly, exogenous ACC treatment rescued the ACS 2/6 mutant seedlings from their metabolite deficiency. These results strongly support our hypothesis that ethylene signaling is a positive regulator of the two feruloyl acetylsucroses. In the future, we hope to see if other maize phytohormones induce these metabolites by studying other known signaling pathways involved in maize biochemical defense.

My Experience

This summer PGRP intern experience has been extremely rewarding.  I take away from this internship not only research experience in a professional lab environment, but also lifelong problem-solving skills I gained and great memories working on my project and with my mentor.  This summer I refined basic lab techniques from running PCR to creating different stock solutions we use in lab daily.   These are incredibly useful skills for whatever lab I find myself in in my future endeavors.  I learned to think like a scientist; when I encounter a problem, I can find ways to fix the issue myself as opposed to relying on my mentor to search for an answer.  Learning to become a more independent thinker and worker has allowed me to better understand what I am doing in lab with the bigger picture, as opposed to solely focusing on day-to-day tasks.  This has in turn made me more confident in my abilities as a scientist.  I would like to thank all the PGRP staff and all those in the Jander Lab for making my summer experience so positive and rewarding.

“Determining the functions of herbivore-inducible maize genes on defense against Spodoptera exigua“

Project Summary

The survival and fitness of plants is affected by a wide variety of biotic and abiotic stresses; hence plants must defend themselves. Jasmonic Acid (JA) is a plant hormone induced by insect feeding and helps plants defend themselves by producing chemicals that harm the attacking insects or attract their predators.

However, there is much less research on the JA signaling pathway in monocots, such as maize, as compared to dicots. Our project, therefore, is focused on testing the functions of two herbivore-inducible genes in maize that may have roles in affecting JA flux. These genes are highly induced in maize after herbivory, pointing to their importance in plant defense. Even though the homolog of the first gene (Allene Oxide Cyclase2, AOC2) is reported to have a role in JA biosynthesis in dicots, its role in monocots has not been described.

The second gene (IAA-Amidase), despite its high induction, has not been described to affect herbivore resistance in any other plant species. In both projects, we used the reverse genetics approach to reduce the transcript levels of the genes and test the effect of gene silencing on the herbivore-induced jasmonate and metabolite accumulation and on caterpillar performance. Our results suggest that these genes affect the herbivore-induced metabolome of maize plants and the performance of the caterpillars, increasing knowledge on the biosynthesis and signaling pathways of JA, which will not only be important for developing insect-resistant lines of maize, but will elucidate the ancient history of the split between monocots and dicots.

My Experience

This internship gave me the hands-on research opportunity I had been wanting. Because my mentor taught me each step and the science behind the step for every procedure I learned, and then let me do the procedure on my own, I feel confident in my ability to work in any biology lab, regardless of concentration.

Another major take-away from this internship is an appreciation for the immense amount of work that goes behind each scientific publication. For example, prior to running an experiment, one must make sure each of the plants are the right genotype by conducting PCR, gel electrophoresis, and DNA sequencing. Then, during the experiment, researchers troubleshoot any errors that came up. Scientific research takes a lot of detail and patience, but it makes the result all the more rewarding.

“Indole-3-Glycerol Phosphate Synthase Characterization in Maize Plants”

Project Summary

In plants, indole-3-glycerol phosphate synthase (IGPS) proteins control the biosynthesis of precursors for the production of important metabolites, including tryptophan, benzoxazinoids, and volatile indole. The maize genome contains three predicted IGPS genes. In our research, we aimed to characterize these genes by investigating their activity in the production of defense compounds, as well as their localization within maize cells.

To determine the functional activity of IGPS genes, we prepared cDNA constructs from each gene and transformed them into E.coli trpC9800, which has a tryptophan auxotrophy due to lack of IGPS. Thereby, a tryptophan complementation assay was used to confirm the activity of each maize IGPS gene. To examine IGPS activity in planta, we obtained maize knockout lines with mutations in each individual IGPS gene, and measured benzoxazinoid and tryptophan content by liquid chromatography-mass spectrometry, as well as volatile indole production by gas chromatography-mass spectrometry.

To localize the IGPS proteins in maize cells we used the Gateway cloning technique to fuse each gene with a yellow fluorescent protein (YFP) marker. IGPS-YFP in the plant cells was visualized by fluorescence microscopy to identify the subcellular location of the fluorescence, which is indicative of the location of the IGPS protein. This research is important for understanding how plants defend against herbivory. By understanding the function of IGPS genes in maize plants, scientists will be able to obtain a more complete picture of the plant, contributing important knowledge and technology for maize production with improved food safety, high quality, and yield.

My Experience:

My summer at BTI has been invaluable. Over the past six weeks, I have learned what it is like to propose, design and perform a research project. I have learned a variety of lab techniques ranging from PCR and RNA extraction to plasmid preparation and Gateway gene cloning. One of the most valuable skills that I have learned through this program is how to think critically and how to face setbacks when experiments do not work. This internship taught me that science is not one dimensional, and that I should approach questions and analyze results with an open mind. I know that these skills will definitely follow me in my future research endeavors. I would like to thank my mentor Annett Richter, Georg Jander, the Jander Lab, and BTI for providing me with this incredible opportunity.

Transcriptomic and metabolomic analyses reveal the herbivore-induced natural variability in jasmonate-mediated plant defense responses of two inbred maize lines

Project Summary

In maize plants, Jasmonates are a class of signaling molecules responsible for responses to biotic stresses. Specifically, jasmonates play important roles during plant defense against insect herbivores and necrotrophic pathogens. In our research we explored the genetic and transcriptomic differences between the defenses of the B73 and NC350 maize lines. In measuring the expression of jasmonate encoding genes after herbivory, we found an increasing trend in the level of expression. In general, the B73 line exhibited the most induction which suggests that the defensive response is greater than that of the NC350 line. In addition to gene expression analysis, we conducted a caterpillar performance assay to test the defensive ability of both plant lines. Our results revealed that the NC350 line is slightly more defensive than the B73 line since the caterpillars favored feeding from the B73 line. In our metabolomic testing, we measured the abundance of four defensive compounds, DIMBOA-Glc, DIMBOA, HDIMBOA-Glc, and HDIMBOA. We found that the level of caterpillar performance is inversely correlated to the amount of HDIMBOA present and that the NC350 line produces a greater amount of HDIMBOA making it increasingly defensive.

My Experience

Over the past six weeks at BTI I learned an incredible amount of skills to aid in my future endeavors. I came into the program hoping to get a head start in the field I intend to pursue in college, yet I have received much more than a head start. The fact that someone can come into a program and be involved so quickly and so completely is unbelievable. This was my first intern research experience, and I am not sure that any future internship could top the enjoyment I’ve had throughout this one.

Exploring the Role of CYP72A Genes in Maize Stress Response

Project Summary

Cytochrome P450 (CYP) genes are conserved in nearly all genomes and comprise approximately 1% of plant genomes. CYP proteins perform a wide range of biochemical functions, and are organized into subfamilies based on structural and functional similarities. CYPs are critical for the essential and species-specific biochemical pathways comprising plant primary and secondary metabolism. Secondary metabolites are required for plant stress responses and interactions with other organisms. A subfamily of CYPs thought to be involved with plant secondary metabolism is CYP72A, as these genes are expressed in response to various plant stress conditions. Little is known about the biochemical functions of this CYP subfamily. This study aims to elucidate the role of CYP72A genes, primarily CYP72A26, in stress response in Zea mays. Quantitative reverse-transcriptase PCR was performed to investigate gene expression of CYP72A26 in three varieties of corn under normal and stress conditions. Additionally, metabolite analysis was performed to explore the biochemical role of CYP72A26 in wildtype and mutant plants that were exposed to caterpillar herbivory. Results suggest that CYP72A26 expression levels vary among different varieties of corn, but this gene does not appear to be significantly induced by caterpillar feeding. However, we detected metabolic differences between mutant and wildtype plants. Further investigation is necessary to explain the role of CYP72A26 in maize stress responses. Determining the role CYP enzymes play in plant stress response is critical for the successful selection of stress resistant plants. In the face of climate change and overpopulation, pesticide-free stable and stress resistant crops are essential.

My Experience

My experience as a research intern in the Jander lab has been exceptionally rewarding. Leeann Thornton has been an amazing mentor, both professionally and personally. This summer has taught me how to transition from an undergraduate student to a full-time researcher. I have also greatly enjoyed working with pioneers of scientific discovery, seeing up-close the work of extraordinary scientists who are changing the world for the better. Most importantly, this summer has allowed me to cohesively connect the various epistemologies I have gained during my undergraduate career. As a senior, I am transitioning from college to the professional world. I have learned how to integrate my skills, knowledges, and passions, and hopefully I will continue to do so as I strive to pursue a career in medicine.  I am excited to enter the next frontier of my education and I thank BTI tremendously for further preparing me for

Plant polyamine alternative pathways

Project Summary

Polyamines are multifunctional biological molecules found in all living organisms. In plants, polyamines respond to biotic and abiotic stressors, so understanding their regulatory and biosynthetic pathways provide insight to the plant defense system. An Arabidopsis thaliana acetyltransferase, NATA1, has been shown to have important regulatory functions on plant polyamines. The goal of this project was to further explicate the function of NATA1 and two decarboxylases, ADC1 and ADC2, in a potential polyamine/acetylpolyamine biosynthetic pathway and their part in plant biotic stress response.

Genotyped nata1 mutant Arabidopsis leaf samples were analyzed on LC-MS after 3 days of Nδ-acetylornithine treatment. The detection of acetylputrescine suggests the existence of a yet-unidentified alternative pathway to polyamine formation. Given the somewhat similar structure of arginine and Nδ-acetylornithine, we tested whether ADC1 and ADC2 also function as Nδ-acetylornithine-decarboxylase. Arabidopsis ADC1 and ADC2 were cloned under cauliflower mosaic virus (CaMV) 35S promoter and overexpressed in Nicotiana benthamiana before being purified. Acetylputrescine accumulation was detected when purified ADC1 and ADC2 were co-incubated withNδ-acetylornithine in an in vitro assay.  To investigate the biological function of the alternative pathway, Pseudomonas syringae was infiltrated into Col-0, nata1, adc1, adc2, nata1 adc1 and nata1 adc2 mutant Arabidopsis plants. Polyamine level was analyzed using HPLC and transcript level of defense-related marker genes and genes on polyamine biosynthesis pathway (AIH, CPA, ADC1, ADC2, and NATA1) were quantified using qPCR. My results suggest the existence of an alternative polyamine biosynthesis pathway and further investigation is warranted to assess how these acetylated polyamines function in the plant defense system.

My Experience

My summer as a PGRP intern has been both challenging and extremely rewarding. I came into this program knowing little to nothing about molecular biology and genetics. Thankfully my mentor, Yann-Ru Lou, was an amazing teacher and role model. She has taught me many lab skills that will be useful well into graduate school. She has also taught me the essential skill of how to be patient and perseverant when experiments or machines do not work. In the future I hope I am lucky enough to be a part of a lab as brilliant and helpful as the Jander Lab. I am truly thankful for my summer at BTI and all the people that have contributed to my growth as an aspiring plant scientist. I have never been more excited to attend graduate school and continue conducting plant science research.

Aboveground Resistance in Solanum tuberosum in Response to Belowground Herbivory

Project Summary

Resistance responses to herbivory are crucial for plants to survive and adapt to the threats that surround them. The responses can be categorized into alterations in the primary and secondary metabolism of the plant. Generally, when studying resistance responses, only one insect species and a specific area of a plant are taken into consideration. However, in nature, more than one insect could attack different parts of a plant simultaneously. In the current project, potato (Solanum tuberosum) and two different types of insects were used in different organs of the plant. Tecia solanivora was used to infect the tubers and Spodoptera furgiperda was later used to feed from the leaves. The approach we took with this experiment was to try to decipher the defense interaction between the above and belowground parts of the plant. Specifically, we wanted to know how belowground herbivory by T. solanivora affects S. furgiperda feeding on the leaves, and what defense mechanisms are triggered by belowground herbivory. The experimental techniques that were used include quantitative PCR (qPCR), liquid chromatography-mass spectroscopy (LC-MS), and spectrophotometry. Previously, we found that higher lipoxygenase 3 transcripts were accumulated in the aboveground leaves. We also observed thatS. frugiperda  larval mass in the aboveground section of the plants in which belowground herbivory was present was lower than the mass of the controls, indicating that a resistance response was elicited by belowground herbivory. This contributes to our understanding on how different plant organs respond to insect herbivory and how they interact with one another.

My Experience

Working in the Jander lab gave me an idea of what I would like to pursue as a career in the future, and it was a great honor to be part of the PGRP internship program. I learned many basic molecular biology and biochemistry techniques such as qPCR, soluble sugars and starch quantification using spectrophotometry, and LC-MS. Before this internship I didn’t have much knowledge about what working in a lab was like. Something important that I learned was to plan ahead when doing an experiment, because it is always good to have a backup plan, and knowing that if you make a mistake, you can fix it without having to put the whole experiment in danger. I really enjoyed working alongside my mentors Pavan Kumar and Erandi Vargas Ortiz who helped me achieve a better understanding of everything I did at the Boyce Thompson Institute.

Identification and characterization of cardiac glycoside-related genes in milkweed

Project Summary

Cardiac glycosides have been used for human medicinal purposes, historically combatting against many cardiac illnesses. However, if one overdoses on cardiac glycoside, the excess content in the body can very easily lead to other diseases and potential death. This project will help us reduce these risks by determining the genes that affect the production of cardiac glycosides and their biosynthetic pathways. We are focusing on cardiac glycoside production in different subspecies of milkweed, specifically Asclepias incarnata pulchra and Asclepias incarnata incarnata because of their difference in cardiac glycoside levels, and on making milkweed a genetic and genomic model plant system. Now, the primary focus is determining which genes are crucial in cardiac glycoside production. Recent transcriptomics have identified candidate genes for testing. To determine their supposed roles in cardiac glycoside biosynthesis in milkweed, these genes of interest will be targets for silencing usingAgrobacterium-mediated transformation harboring RNAi constructs containing our gene(s) of interest. After (hopefully) altering the expression of candidate genes, we expect to find changes in cardiac glycosides levels, thereby identifying genes that are present in the biosynthetic pathway. Through this research, we can eventually map out the pathway and produce safer and more effective pharmaceuticals.

My Experience

Before the PGRP summer internship, I had no experience in a laboratory setting besides my science classes. This program gave me valuable exposure to the scientific environment and allowed me to learn about plant science from great researchers. The opportunity to work in a lab and do work that could have real-world applications is extremely valuable. I have gained a deeper respect for the research process and the work that goes behind a publication. The amount of troubleshooting and all the different ways to approach a problem can be frustrating, but I feel that I have gained the most from this internship through these experiences. They forced me to go back and reevaluate what I’ve done and think about what I should’ve done differently, leaving me with a better understanding of the scientific process. This research experience has been very rewarding and will definitely influence my future career decisions. I am very grateful for Felix Fernandez-Penny, my mentor, Georg Jander, Nicole Waters Fisher, and Tiffany Fleming for giving me this opportunity and making it as enjoyable and educational as possible.

Natural variability in jasmonate expression among parental recombinant inbred lines in maize

Project Summary

On a day-to-day basis, plants are exposed to a vast range of environmental stresses. Despite unfavorable conditions, they must maintain Darwinian fitness, setting up defense systems that do not compromise their growth and development. The phytohormone jasmonic acid (JA) and its receptor-active derivative, jasmonic acid isoleucine (JA-Ile), are key components in induced immunity against a variety of stresses such as attacks by herbivores and pathogens as well as other forms of tissue damage. Our research had two parts: firstly, we used mass spectrometry to profile the parental recombinant inbred lines of maize for variation in levels of JA and its conjugates, collectively known as jasmonates. We also used genotyping techniques with the intention of elucidating the functions of specific genes in the JA biosynthesis and response pathways, and comparing their roles in maize to those previously discovered in model plants, such as Arabidopsis. We hypothesized that there is significant natural variation in jasmonate levels among maize lines, and while the functions of jasmonate signaling genes will be largely conserved in maize, there will be divergence specific to the defensive and developmental needs of the maize plant. The study of jasmonic acid and other forms of maize defense is vital in its potential to increase crop yield while decreasing use of chemicals such as pesticides and insecticides, positively contributing to both the world economy and the environment.

My Experience

Although my weeks at BTI included several exciting successes, a large portion of my time was spent troubleshooting, frequently backtracking to pinpoint the cause of my disappointing results. Not only was this valuable to my understanding of the experimental process, my frustration gave me a new appreciation for the timeframe of research; each discovery summarized in a paper or magazine article represents months, if not years, of the concerted effort of many scientists. Working closely with a mentor showed me the value of collaboration and especially mentorship in the laboratory environment.  Coming in with little to no prior experience in plant science, my mentor was invaluable in getting me up to speed in no time. The positive experience I’ve had in my brief time at BTI has shown me the potential for research, regardless of field, to be both fulfilling and inspiring.

The influence of a polyamine oxidase inhibitor on the hydrogen peroxide defense system of Arabidopsis thaliana against Pseudomonas syringae

Bacterial infection by Pseudomonas syringae can cause leaf-spot and blast diseases that defoliate plants, leading to high loss in agricultural production. The aim of this project is to investigate the influence of polyamines and polyamine metabolism in Arabidopsis thaliana when subjected to Pseudomonas syringae infection. Because plants are sessile organisms they need to provide efficient defense against biotic and abiotic stresses. In order to protect themselves against pathogens plants have evolved several defense systems. Polyamines, such as spermidine and spermine, have multiple functions in plants, including growth, development, and defense against various environmental stressors, and these molecules play an important role during hypersensitive response triggered by pathogen attacks. During pathogen infection plants are able to degrade polyamines with the help of polyamine oxidases releasing hydrogen peroxide (H2O2) in order to destroy pathogenic microorganisms, such as Pseudomonas syringae. Plants were treated with guazatine in order to inhibit polyamine oxidases, and infiltrated with Pseudomonas syringae strain DC3000 afterwards. Bacterial growth is measured in guazatine treated and untreated groups to determine differences of plant defense. Furthermore, polyamine oxidase activity, H2O2 content, polyamine levels and gene(s) expression profiles were analyzed. In further research resulting from this project, different sections of possible H2O2 production pathways could be more narrowly defined.

My Experience

I had a great time exploring both the daily work of a researcher at BTI and the American culture. I met a lot of people from all over the world, improved my laboratory working skills and attended great events such as the annual BTI summer picnic. I also really enjoyed the weekly seminars with talks given from researchers of different scientific fields concerning plant research, and I was able to make new contacts which will be very helpful for my further career. I really appreciate the chance to participate in a summer internship in this beautiful town and all the priceless memories it gave to me!

Understanding the role of Turnip Mosaic Virus (TuMV) in plant-insect interactions

Plants are susceptible to insect and pathogen attacks, including viral infections that spread from one plant to another through insect vectors, causing severe crop loss each year. Previous research has shown that green peach aphids, Myzus persicae, feeding on Turnip Mosaic Virus (TuMV)-infected plants produce a significantly increased number of progeny compared to aphids feeding on mock-infected plants. A few important virus proteins may account for higher reproduction of aphids due to increase nutrient content in the plants. Further experiments were performed with Agrobacterium-infiltrated Nicotiana benthamiana plants transiently expressing isolated TuMV protein compared to empty vector control plants. N. benthamiana with ten aphids were observed for three hours in the dark to assess aphid preference. Additional analyses of significant viral proteins were conducted by using high performance liquid chromatography (HPLC) to measure free amino acid contents in the plants. Investigating these plant-insect-virus interactions will enhance our understanding of the pathogenesis of the Turnip Mosaic Virus as well as the spread of the virus by insects. Eventually, this will allow us to improve plant development, endurance, and insect resistance, thereby increasing plant defenses, lessening the use of insecticides in the agriculture industry, and increasing global food crop.

My Experience

The Plant Genome Research Program (PGRP) internship was an amazing opportunity for me, opening doors for me to conduct research in a professional scientific environment at a vibrant active campus. By working with colleagues in the Jander lab, I gained a wide variety of ideas and techniques, including RNA extraction, cDNA synthesis, polymerase chain reaction (PCR), agarose gel electrophoresis, plant infiltration, and HPLC. Being mentored by an expert in the field, I have learned to think scientifically, deal with troubleshooting, work more efficiently, propose my own experiments, and improve my scientific writing ability. The summer spent at BTI has exposed me to a broad range of research areas in plant sciences via the PGRP seminar series. This internship has definitely prepared me for graduate school and a scientific research career. I am truly thankful to have been a part of the PGRP program.

Maize Plant-Insect Interactions

Meiotic recombination is the basis of evolutionary change over time. While recombination has been extensively studied, still little is known of the mechanisms behind this process. Many studies have suggested that recombination is in part needed for pairing of homologous chromosomes. The mutantdsyCS, which is defective in homologous chromosome pairing during meiosis, was used to further study the mechanisms of how the recombination pathway is coordinated with pairing. Previous work done in the Pawlowski lab shows that there are a substantially reduced number of RAD51 foci in thedsyCS mutant leading to a complete elimination of homologous chromosome pairing. However, it is still unknown at what stage the mutation is occurring. Immunolocalization coupled with three dimensional deconvolution microscopy was used to observe the number of foci of γ-H2AX and MLH3 proteins in wild type and mutant maize. A reduced number of γ-H2AX foci as well as a reduced number of MLH3 were detected at all the prophase stages that were observed, drawing the conclusion that indsyCS mutants the formation of double strand breaks were impaired. The more knowledge about the mechanisms behind meiosis can have many applications, including plant breeding, cancer genetics, and birth defects.

My Experience

As a PGRP intern with BTI, I was able to answer a lot of the questions I had about research, graduate school, and research careers. I knew that I wanted to do research, but wasn’t sure which area I wanted to do research in. Pawlowski’s lab is focused on meiosis, which is very complex but applicable to all organisms that undergo meiosis. I had done research in a laboratory setting before and I felt like the new techniques I learned here perfectly complemented my previous research experience. I plan on going to graduate school in the plant sciences, but didn’t know what being a graduate student really entailed. This experience has been invaluable in helping me see what being a graduate student in this field would be like. I felt like an integral part of the lab here and I am very grateful that I had the opportunity to be a part of the Pawlowski lab and the BTI program.

Knocking Out O-methyltransferase Targets on Chromosome 9

By researching maize, scientists have been able to manipulate plant genes to fight diseases and repel insects. In order to discover more about the corn leaf aphid resistance pathway involving benzoxazinoid biosynthesis, I tried to isolate mutant knockout alleles of the o-methyltransferase paralogs on Chromosome 9 using the transposable element Dissociation (Ds). This would help to determine the importance of each of these genes in insect resistance. Ac and Ds are transposable elements that move during DNA replication. If Ds inserts into a gene such as the o-methyltransferases, their expression can be disrupted and more can be learned about them. To carry out this project, I planted nearly one hundred plants from a genetically closely linked Ds testcross population and sampled tissue from each seedling in pools from which DNA was extracted. Then through multiple PCR reactions surveying the o-methyltranferase target genes and four others in the region, I was able to amplify the genes and search for Ds insertions. With the results of this experiment, more can be known about the genetic makeup of maize. We are one step closer to identifying the role that these genes play in benzoxazinoid biosynthesis.

My Experience

Through this internship, I learned a lot about plant genetics and molecular biology and I acquired many new skills and techniques. I learned how to do technical lab work, such as DNA extractions and PCR reactions. My co-workers in the lab were absolutely amazing to work with and couldn’t have been more supportive. My mentor Kevin Ahern, gave me a significant amount of background information before I started my project, and helped me with any question I had along the way. I am now confident in successfully completing a DNA extraction as well as running PCR reactions. Because of this internship, I now know that I definitely want to major in biology, and pursue the subject as a future career.

Deciphering the genetic architecture of aphid-induced callose deposition in Zea mays L.

Previous experiments showed that corn leaf aphids (Rhopalosiphum maidis Fitch) feeding on maize (Zea mays L.) inbred line CML322 produce significantly more offspring than aphids feeding on inbred line B73. A quantitative trait locus (QTL) analysis done on a B73 x CML322 recombinant inbred population highlighted a locus on maize chromosome 1 that explains the majority of the demonstrated variance. Callose deposition, a plant defense against aphids, may account for this difference in aphid growth. We preformed further experiments with B73 and CML322 and saw that less offspring were produced on B73, which also had significantly greater callose deposition, as opposed to CML322 which showed more offspring production and less callose formation. This demonstrated the hypothesized negative correlation between callose deposition and reproductive fitness of the aphids (more babies). Analysis of B73 x CML322 recombinant inbred lines is being conducted to determine whether aphid reproduction and callose formation QTL co-localize. This will enable us to decipher better the genetic architecture of callose formation in maize.

My Experience

As a high school student, the BTI research internship gave me an amazing opportunity to experience a professional scientific setting. I greatly expanded my knowledge of modern biology, not only through working in the lab but also through the many seminars that were given by the other labs in the building. This internship has only increased my interest in the sciences, and definitely has impacted my decision on a course of studies for college. I’m truly happy to have been a part of such a great program.

Identifying Quantitative Trait Loci for Spodoptera exigua Resistance in Maize

In some geographic regions of the world the relatively high abundance of the Spodoptera exigua, beet armyworm, causes severe crop losses. This leads to the heavy application of insecticides, which can have potential deleterious effects on the environment and could result in the outbreak of other pests. Our research is aimed at reducing the plant injury that is brought on by beat armyworms. Based on preliminary experiments with 26 maize inbred lines, we identified three maize lines (P39, B73, and Oh7B) that show significant differences in their resistance to the beet armyworm. To map genes influencing beet armyworm resistance, we measured caterpillar growth on B73 x Oh7B and B73 x P39 recombinant inbred populations. In the P39 recombinant inbred population, quantitative trait loci were identified on chromosomes one and four. Candidate genes were selected for further analysis and once identified would aid in marker assisted selection for beet armyworm resistance in maize.

My Experience

As a biology major, I have always had a desire to understand exactly what takes place in a research setting. Spending my summer here at BTI has not only fulfilled that inner desire, but has also given me the tools and knowledge to further excel along my career path. Two years ago, before my first year in college, I would have never imagined that someday the opportunity would present itself for me to engage with experts in the field of plant biology. While my background does come from growing up in underprivileged area, I must say that participating in this internship on such a prestigious campus has been extremely wonderful and I am truly grateful for being given the chance to experience two full months of hands-on research. Though I was not experienced in the field, my mentor and other members of the Jander lab were helpful and encouraging in every possible manner. Within my short span of time here, I have gained high hopes for a scientific research career.

Developing a bioassay to study Arabidopsis thaliana defense response to aphid salivary components

This summer, I studied the interactions between Arabidopsis thaliana and green peach aphids (Myzus persicae). Aphids ingest sap from the sieve elements of plants using a stylet appendage, a long hollow mouthpiece. Saliva plays an essential role in aphid feeding- both sheath saliva, which gels upon secretion to form a barrier around the stylet, and watery saliva, which is continuously spit into the phloem upon feeding. Watery saliva has been shown to contain specialized proteins that work to deter and disable the plant’s normal defense mechanisms in response to feeding. The Jander lab recently discovered that aphid saliva infiltrated onto a leaf can elicit many of the same responses as actual aphid feeding on the plant- an important step forward in this area of research and the background for my project.

My Experience

My research was focused on creating a bio assay to facilitate the study of aphid-plant interactions. My assay was designed to identify possible plant immune response changes in reaction to salivary proteins collected from aphid diet. Responses that I measured include Ca+2 release, increased reactive oxygen species concentration, pH change, and callose deposition. Techniques that I employed in these experiments include diaminobenzidine and aquiline blue staining, RNA extraction, plant infiltration, PCR and qPCR, and Gateway cloning. The time I spent in the Jander lab opened my eyes to the wide variety of opportunities and research areas in plant science, I now have a better idea of the course of study I want to pursue in college and graduate school. I am sincerely grateful I was able to participate in the PGRP program.

Nδ-acetylornithine is a defensive non protein amino acid in Arabidopsis thaliana

In response to insect feeding or treatment with the plant defense elicitor methyl jasmonate, the small crucifer, Arabidopsis thaliana (Arabidopsis) produces the non-protein amino acid N-delta-acetylornithine. My project was focused on determining whether this amino acid has deleterious effects on aphids feeding from the plants. As part of my project, I conducted artificial diet assays with aphids, measured plant amino acids by high performance liquid chromatography, and conducted plant growth experiments. The results from my summer project are included in this publication:

Adio, A.M., Casteel, C.L., De Vos, M., Kim, J.H., Joshi, V., Li, B., Juery, C., Daron, J., Kliebenstein, D.J. and Jander, G. 2011. Biosynthesis and defensive function of N-delta-acetylornithine, a jasmonate-induced Arabidopsis metabolite. Plant Cell 23: 3303-3318

Expressing Aphid Salivary Genes in Nicotiana tabacum to Determine Protein Function

The proteins found in aphid saliva are known to mediate plant-aphid interactions. In the Jander Lab, I cloned aphid salivary genes and then transiently expressed them in N. tabacum, performing aphid bioassays to determine if the encoded proteins induced plant resistance or facilitated aphid feeding. For this project, I learned RNA and DNA work, gene cloning, and tobacco infiltration.

During my PGRP internship, I learned a variety of techniques commonly used by molecular biologists, as well as some that are more specific to research in plant-aphid interactions. The internship also reinforced my desire to pursue a career as a biological research scientist.

My Experience

The proteins found in aphid saliva are known to mediate plant-aphid interactions. In the Jander Lab, I cloned aphid salivary genes and then transiently expressed them in N. tabacum, performing aphid bioassays to determine if the encoded proteins induced plant resistance or facilitated aphid feeding. For this project, I learned RNA and DNA work, gene cloning, and tobacco infiltration.

Production of protease inhibitors in Arabidopsis

Many plants produce inhibitors of insect gut proteases as a defense against herbivory.  The Jander lab had previously isolated mutants of the genetic model plant Arabidopsis thaliana with altered protease inhibitor production. During my summer internship I assayed the protease inhibitor activity in mutant and wildtype plants and conducted experiments to measure the effects on insect growth.

Defense against Herbivores: The role of Glucosinolates in Arabidopsis

My project investigates the effects on glucosinolates on caterpillars. I used tu Arabidopsis mutants impaired in glucosinolate production.

Regulated amino acid accumulation under drought stress in Arabidopsis and tomato

Amino acids produced under drought-stress serve as osmolytes to protect plant cells against dehydration. Abscisic acid (ABA) regulates many osmotic stress responses, including production of many osmolytes. We have used mutants of tomato and Arabidopsis defective in ABA biosynthesis, to study drought-induced amino acid accumulation. In tomato mutants defective in ABA biosynthesis, we found that the accumulation of branched chain amino acids in drought-stressed leaves was significantly reduced compared to wild-type. However, these effects were rescued in ‘sit’ mutants by exogenous ABA supplementation, suggesting a direct role of ABA in amino acid biosynthesis. Analogous results were obtained in Arabidopsis ABA-defective mutants that also produced reduced amino acids under stress, in reproductive tissues. To understand the transcriptional regulation of drought-induced amino acid synthesis, we identified and used mutants of transcription factors that are strongly induced under drought-stress. Three transcription factor mutants accumulated significantly reduced amino acid content under stress, implying their function in regulating amino acid biosynthetic genes.

Functional Analysis of Aphid Salivary Proteins

Aphids rely on salivary proteins to infest plants. We have identified ~20 candidate genes encoding salivary proteins in Myzus persicae. I have started cloning and expressing 3 aphid salivary proteins. In planta expression allows for functional characterization of these proteins.

Winnie is currently a junior at Cornell university. During the summer, Winnie worked under her mentor, post-doctoral associate, Martin DeVos on effect of (E)-beta farnesene, a chemical found in aphids’ alarm pheromone, on green peach aphids’ behavior. While in the Jander lab, Winnie learned several molecular biology techniques including PCR, DNA extraction, and bacterial transformation.

The results from my summer project are included in this publication:

de Vos, M., Cheng, W.Y., Summers, H.E., Raguso, R.A. and Jander, G. 2010. Alarm pheromone habituation in Myzus persicae has fitness consequences and causes extensive gene expression changes. Proc Natl Acad Sci U S A 107: 14673-14678

Ksenia is a student at local Ithaca High School. In the Jander lab, Ksenia worked on a project investigating Ksenia was working to identify egg laying stimulants for Plutella xylostella (diamondback moth) and Pieris rapae (white cabbage butterfly). For this project, Ksenia under the supervision of post-doc Martin DeVos learned RNA/DNA work and gel electrophoresis.

The results from my summer project are included in this publication:

De Vos, M., Kriksunov, K.L. and Jander, G. 2008. Indole-3-acetonitrile production from indole glucosinolates deters oviposition by Pieris rapae. Plant Physiology 146: 916-926

Ludmilla is currently a student at Cornell University. She worked in the Jander lab with Dr. Jander on a project working to identify the biochemical pathway of meta-tyrosine synthesis in fescue. For this project, Ludmilla learned HPLC, GC-MS, and plant maintenance.

The results from my summer project are included in this publication:

Huang T., Rehak L., and Jander G. 2012. meta-Tyrosine in Festuca rubra ssp. commutata (Chewings fescue) is synthesized by hydroxylation of phenylalanine. Phytochemistry 75: 60-66

Tatiana is a student at the University of Puerto Rico, Mayaguez campus. She worked in the Jander lab under the guidance of post-doc Minsang Lee looking for an SMM molecule that could transport between leaves and seeds. For this project, Tatiana learned HPLC, PCR, and how to graft Arabidopsis plants.

The results from my summer project are included in this publication:

Lee, M.S., Huang, T.F., Toro-Ramos, T., Fraga, M., Last, R.L. and Jander, G. 2008. Reduced activity of Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed methionine content. Plant Journal 54: 310-320

Bryan is currently a student at local Ithaca High School. In the Jander lab, Bryan worked with post-doc Jae Hak Kim on a project studying glucosinolate induction by Myzus persicae, or the green peach aphid, using artificial diets. Glucosinolates and their breakdown products are important deterrents to aphid feeding. Bryan’s poster presentation at the Symposium earned him first prize and the Colonel’s Plaque for the Jander lab!

The results from my summer project are included in this publication:

Ellerbrock, B.L.J., J.H. Kim, G. Jander. 2007. Contribution of glucosinolate transport to Arabidopsis defense responses. Plant Signaling and Behavior  2: 282-283

After taking a year off to work at Monsanto, Karen will be returning to Heidelberg College (OH) after her summer in the Jander lab. Under the guidance of post-doc Vijay Joshi, Karen researched the presence of threonine aldolase as expressed on chromosome 3 of Arabidopsis. This summer, Karen’s presentation at the Summer Student Symposium earned her first prize and the Colonel’s Cup for the lab!

The results from my summer project are included in this publication:

Joshi, V., K. M. Laubengayer, N. Schauer, A. R. Fernie, G. Jander. 2006. Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool. Plant Cell 18: 3564-3575

Karen enjoys playing beach volleyball and power boating, and really liked the ice cream socials at BTI!

Teresa is from Dryden, NY and currently attends Ithaca High School. This summer, Theresa is working with Dr. Georg Jander at Boyce Thompson Institute. Her project studies two aspects of aphid biology. First, she is working toward identifying chemicals that plants, such as Arabidopsis, produce in order to defend themselves from aphids. Secondly, she is comparing the reproduction of different strains of aphids on Arabidopsis.

In her free time, Teresa enjoys reading, playing tennis, spending time with friends and going to the cinema.

Nδ-acetylornithine is a new defensive non protein amino acid induced by methyl jasmonate inArabidopsis thaliana

During my internship in the Jander lab at the Boyce Thompson Institute for Plant Research (BTI), I investigated the role of Nδ-acetylornithine, a non-protein amino acid that is induced by methyl jasmonate and is probably involved in the defense against aphids. In order to further determine the function of this compound in Arabidopsis thaliana, I used an aphid artificial diet approach to determine its direct impact on Myzus persicae reproduction. I also learned how to use the HPLC to analyze amino-acid profiles linked to the biosynthesis of Nδ-acetylornithine. Furthermore, I worked with Pseudomonas syringae and a nata1 A. thaliana mutant, which does not produced Nδ-acetylornithine, to see if this compound is linked to the polyamine biosynthetic pathway.

My Experience

As a French student, this research experience was a very good opportunity for me. It enabled me to discover how research works in a foreign country, in this very effervescent and active campus. I was very well welcomed by my lab team and I really enjoyed learning how to become a future scientist with my PI and my mentor.

The results from my summer project are included in this publication:

Adio, A.M., Casteel, C.L., De Vos, M., Kim, J.H., Joshi, V., Li, B., Juery, C., Daron, J., Kliebenstein, D.J. and Jander, G. 2011. Biosynthesis and defensive function of N-delta-acetylornithine, a jasmonate-induced Arabidopsis metabolite. Plant Cell 23: 3303-3318

Methods for transient expression in milkweed

Project Summary

Cardiac glycosides are a diverse family of plant secondary metabolites that have historically been used for a multitude of human medicinal purposes. Their applications include uses in medications warding against heart disease and more recently research has been conducted into their ability to act as cancer suppressants. Current studies aim to learn more about the genetic pathways involved in cardenolide (cardiac glycosides in their aglycone form) biosynthesis. Unlike other model plant systems such as maize, wheat, and rice, Asclepias syriaca (common milkweed)lacks a reference genomic platform. Research partnered between the Agrawal, Mueller and Jander labs aims “to advance A. syriaca from an ecological model system to a genetic and genomic model system that can be used to investigate the biosynthesis of medicinally relevant plant metabolites.” This work will provide a reference platform for further investigation of A. syriaca at genomic and genetic levels and will supply information that holds with it the potential for future innovations in medicine using cardenolides. This summer, my research project will begin developing a set of reliable methods for genetic transformation of Asclepias species,A. pulchra, A. incaranta,and A. syriaca, chosen on the basis of their varying levels of different cardiac glycosides. These include methods for gene silencing, and gene overexpression. To begin, we will try implementing already established transformation protocols for other species in milkweed. This will include the infiltration of plants with Agrobacterium constructs for over expression as well as Virus-Induced Gene Silencing (VIGS). VIGS will target the phytoene desaturase (PDS) gene, which will induce an intense photobleaching phenotype if silenced. Furthermore, the PDS sequence should remain relatively constrained between plant species (as it exists in all varieties) allowing us to draw from experiments done in other model systems.

My Experience

This summer marks the beginning of my second year as in intern in the Jander Lab here at BTI. Having begun as a high school intern in the summer of 2013, I look forward to the new opportunities presented with participation in the undergraduate program. It’s been such a joy and incredible experience working alongside the members of the Jander Lab over the past year and I can’t wait to begin a new chapter as I embark on a new project this summer. Working with my mentor, Postdoctoral Scientist, Tengfang Huang has been a rewarding experience so far and I look forward to what is to come as the summer continues. The chance to work with new people allows insight into new methods, perspectives, and analyses of the work being done. Tengfang has already taught me so much and I am incalculably grateful for his guidance and patience. I’d like to extend a special thanks to Tiffany Fleming, Georg Jander, Tengfang Huang and all of the Jander Lab, fellow interns, and the wonderful staff at BTI for making my time here so memorable and such a delight to return to each day.