Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 60% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 40% |
Citrus greening disease is the most important disease of citrus. It has devastated the industry in many citrus growing regions, causing the loss of over 8,000 jobs and billions of dollars of revenue in Florida alone. Citrus greening disease is associated with a bacterium called "Candidatus Liberibacter asiaticus," (CLas) which is spread from tree-to-tree by an insect vector called the Asian citrus psyllid. Vector spread of this bacterium has allowed this disease to reach epidemic proportions. Transmission begins when the insect feeds on citrus plants, and a protective sheath forms around its mouth-parts. The bacterium moves into the insect body and must cross and replicate in insect tissues to be transmitted to the next host plant. We propose to develop molecules that block the formation of the protective sheath or block the movement of CLas through insect tissues. These two strategies are expected to produce new tools to block transmission of this serious bacterial pathogen.
The major goal of this project is to develop novel strategies to control the vector spread of citrus greening disease or huanglongbing, the most serious disease of citrus. First described in China over 200 years ago, it is now found in many citrus-growing regions around the world, including the United States. Since the initial detection of the disease in Florida in 2004, the Florida citrus industry has lost over $9 billion in revenue and over 8,000 jobs. HLB infection in citrus is associated with the Gram-negative bacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative, propagative manner. As D. citri individuals feed, they deposit salivary sheaths around their piercing-sucking mouthparts that are made of protein and carbohydrates and are required for effective feeding. D. citri spreads CLas throughout the grove during a long latency period where the trees remain asymptomatic and the pathogen cannot be reliably detected by molecular methods. HLB research is difficult because CLas is an obligate biotroph and cannot be grown in culture. CLas does not require systemic infection in the host plant for transmission. Horizontal transmission of CLas to psyllid nymphs via infected flush tissues, or the youngest plant tissues, facilitates a rapid spread that is unprecedented for insect vector-borne plant pathogens. The D. citri gut is the first site of interaction between CLas and D. citri. A quantitative, comparative transcriptomic study of CLas-infected and uninfected D. citri showed that D. citri gut cells express long non-coding RNAs in response to CLas infection. The function of these long non-coding RNAs in D. citri and CLas infection in the insect is now known. This project focuses on the development of RNA-based molecular therapies to disrupt interactions between the psyllid vector and CLas, referred to hereafter as interdiction molecules. There are a variety of strategies under development for expression of exogenous RNA molecules in citrus and psyllids to control citrus greening. I propose to develop interdiction molecules that interfere with function and CLas interactions in the insect gut and salivary sheath. I will pursue two specific aims in parallel that are complementary in their goals. The success of one is independent of the other.
The goal of Aim 1 is to explore the role of long non-coding RNAs discovered in the gut of the insect. These long non-coding RNA transcripts are expressed in response to CLas-infection. Several RNA-based strategies will be employed to interfere with the function of these molecules, and the effect on acquisition and transmission of CLas will be explored. Five long non-coding RNAs were selected for investigation. Two of these were among the most abundant RNA transcripts in the gut of D. citri, two were up-regulated, and one was down-regulated when D. citri fed on CLas-infected plants. Function of these long non-coding RNAs will be disrupted using antisense RNA molecules delivered to insects via artificial diet. The overarching goal of this project is to develop long non-coding RNAs as a novel target to control pathogen transmission. To do this, I will first characterize their expression in the insect in different conditions, and will examine the effect of reducing their expression on CLas acquisition and transmission.
The goal of Aim 2 is to develop RNA aptamers that disrupt D. citri feeding. These molecules will target the most abundant proteins in the insect stylet sheath, a structure that polymerizes around the insect's mouthparts. The sheath lends structural stability to the stylet during feeding, and is hypothesized to help the insect avoid the plant's immune system. If the sheath's polymerization is blocked, the insect cannot feed and therefore cannot initiate the cycle of pathogen transmission.RNA aptamers are single stranded RNAs that bind to a particular target with specificity. Aptamers can be selected to virtually any target using a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamers can be compared to antibodies in that they bind with affinity to a specific target. However, they are more advantageous than antibodies because they penetrate tissues more easily and can be synthesized in vitro. RNA aptamers are currently being used to treat human diseases, such as macular degeneration and cancer, but have not yet been applied to any plant diseases. Aptamers will be developed that bind to the most abundant proteins in the stylet sheath. These aptamers will be evaluated for their ability to block sheath polymerization and therefore act as feeding inhibitors.
Aim 1: To develop interdiction molecules that target and block the function of long non-coding RNAs in the psyllid gut.I have already used RNA modeling software [14] to predict the structure of these lncRNAs, and synthesized antisense constructs against regions predicted to be important for the structural integrity of each lncRNA. These antisense RNAs will be administered to both nymph and adult ACP via artificial diets enclosed in parafilm membrane sachets and via detached leaves soaked in a solution with antisense RNAs. The antisense constructs are expected to function by steric hindrance, interfering with the proper folding and molecular interactions of lncRNAs in the gut. CLas acquisition assays will be performed first to test the efficacy of the antisense molecules in blocking the acquisition, replication, or translocation in the psyllid. Antisense molecules that show a negative impact on CLas acquisition will be further tested for blocking transmission of CLas by the insect. Experiments will focus on lchanges in the abundance and localization in the gut associated with CLas exposure.Aim 2: To develop interdiction molecules that disrupt the psyllid salivary sheath, an essential feeding structure.I have unambiguously identified three proteins in the salivary sheath proteome with multiple unique peptide assignments using tandem mass spectrometry. Portions of these three proteins predicted to be soluble will be expressed using affinity purification tags. Aptamer selection will be carried out using a library of 2.5x1015 RNA aptamers, each with a unique central region giving it a specific secondary structure, and PCR primer binding sites on either side. This library will be bound to a column containing purified protein. The column will be washed, and bound aptamers will be eluted. This process will be repeated for at least five cycles or until specific aptamers can be enriched from the aptamer libraries. Columns containing only the affinity tag will be used as a control to select aptamers that bind to the column itself or the affinity tag. The final library with be amplified using the PCR primer sites after five rounds of enrichment, and sequenced using Illumina. Electrophoretic Mobility Shift Assays will be used to validate that aptamers identified bind to the intended protein, and to establish a concentration curve for aptamer binding. I will use a simple bioassay on an agar plate to measure sheath inhibition. I will use this method to screen aptamers for efficacy, and it will allow me to easily and rapidly screen a large number of aptamers. In a typical selection, about 100 aptamers will be enriched and less than ten will be highly abundant. The ten most abundant aptamers will be screened individually, and the remaining 100 aptamers will be screened in pools of ten.
Target Audience
The importance and progress of the research proposed here was communicated directly to citrus growers at the Citrus Greening Grand Challenge Meeting in Fort Pierce, FL. In addition, the lead scientist performed outreach to female students in grades six through eight with the goal of engaging young women in the STEM fields. This work was also communicated to underrepresented minorities during the Diversity Preview Weekend at Cornell University, and during the Boyce Thompson Institute Live Stream event which reaches hundreds of remote audience members. To engage the scientific community, this work was presented in invited seminars at the National Institute of Health,Albert Einstein College of Medicine, and Vanderbilt University. Finally, much of this work has been published in peer-reviewed scientific journals to engage the broader scientific community (see Products section).
Changes / Problems
Nothing Reported
Training & Professional Development
This project is highly interdisciplinary, and allowed me to establish strong collaborations with leading researchers at Cornell, the USDA, and the University of Washington. Each of these collaborations expanded my technical skillset, as I learned cutting-edge methods such as protein expression and purification, insect bioassays, and RNA aptamer selection from experts. By presenting the work at various scientific conferences, I improved my scientific communication skills and expanded my professional network. This ultimately led me to secure apostdoctoral position at Vanderbilt University.
Dissemination Streams
This work has been communicated to fellow scientists at three conferences this past year, as well as three invited seminars. In addition, this work has been directly communicated to citrus growers during a grand challenge meeting. Several peer-reviewed publications make this work available to the broader community. Outreach efforts to young women, underrepresented minorities, and virtual communities expanded awareness of the work beyond scientific researchers.
Next Reporting Steps
Nothing Reported
Target Audience
Scientific findings were orally communicated to a group consisting of citrus growers and scientists during the Citrus Greening Grand Challenge meeting, and feedback was received from both growers and scientists. Findings were also presented to fellow scientists and industry representatives during the United States Human Proteomics Meeting, the Gordon Conference on Tropical Infectious Disease, and the Cornell Virology and Vector Biology meeting. I also communicated about my work to a general audience via a podcast appearance and an interview during a Boyce Thompson Institute live stream event. Findings were also communicated to citrus growers via an article in the grower-facing magazine Citrograph.Finally, I led a workshop about genetics for a group of girls in grades 6-8 as part of the Expanding Your Horizons (EYH) program.
Changes / Problems
No problems were encountered.
Training & Professional Development
This project has allowed A. Kruse to establish collaborations with labs in different departments within Cornell as well as different institutions, to apply cutting-edge technology to a very important disease problem. These collaborations helped her learn to manage multiple professional relationships and deadlines. A. Kruse also presented her work at professional academic meetings, to groups of growers, and to other institutions during invited seminars (see target audience and product sections). She had the opportunity to actively participate in the Boyce Thompson Institute (BTI) Post-Graduate Society (PGS). Through the PGS, she participated in an alumni mentorship program which allowed her to learn about diverse careers in science. She also organized an informational workshop to connect international scientists with an immigration lawyer to learn about the types of Visas available for scientists. This helped her learn how to organize events and communicate effectively with non-scientists. She also served as the president of the Plant Pathology Graduate Student Association (GSA) which improved my leadership and communication skills. In this capacity, she organized a peer-to-peer mentorship program to improve the graduate community. Finally, she mentored an undergraduate, Anna Yaschenko, and assisted her in completing an independent research project. A. Yaschenko won the Best Poster Award at the BTI undergraduate symposium for this work.
Dissemination Streams
This work was presented by Angela Kruse viatwo invited seminars at the NIH and at the Albert Einstein College of Medicine. Shealso presented at the United States Human Proteomics meeting, theGordon Tropical Diseases Conference, the Cornell Virology and Vector Biology Meeting, and the Cornell School of Integrated Plant Sciences Symposium. In addition, a section of this work is published in a paper in the peer-reviewed Journal of Proteome Research. A. Kruse co-authored an article in the citrus grower-facing magazine Citrograph. Shealso communicated this work to non-scientists with the help of the BTI communications department. She appeared as a guest on the BTI Science Bomb podcast, and the BTI Giving Tuesday Livestream, and was interviewed for a press release about my work entitled "Blood, Sweat, and Tears: All in a day's work fighting citrus greening disease." See the Products and Target Audience sections for more information.
Next Reporting Steps
Specific Aim 1: To develop interdiction molecules that target and block the function of long non-coding RNAs in the psyllid gut The major remaining goal in this aim is to deliver antisense RNAs to silence the lncRNAs of interest identified by bioinformatics and expression analysis. These will be delivered via artificial diets, and insect mortality and CLas acquisition will be measured. Specific Aim 2: To develop interdiction molecules that disrupt the psyllid salivary sheath, an essential feeding structure A sheath-binding aptamer shows promise in inhibiting sheath activity of the ACP. This aptamer will be further tested in artificial diets. Next, the RNA will be tagged with a fluorescent molecule to allow us to visualize where it goes within the insect using microscopy. The aptamer will also be synthesized with fluorinated nucleotides to test whether this improves the activity of the aptamer compared to a control aptamer with no binding activity. The aptamer will also be delivered to plants via detached leaves to measure how far it can move within the leaf, then whether it inhibits insect feeding and pathogen transmission. Now that key sheath proteins have been identified and expressed in insect and bacterial cells, they will be purified as targets for aptamer selection. Resulting aptamers can be used in combination among themselves and with the existing sheath aptamer to establish more broad-spectrum control of insect feeding. <br><br>
<br>What was accomplished under these goals? Impact Statement: Citrus greening disease is currently the most serious threat to the citrus industry. It is caused by a bacterium hereafter called CLas. CLas can be spread between plants by a sap-sucking insect called the Asian citrus psyllid (hereafter called ACP). The ACP ingests CLas while feeling on plants, and CLas moves into and replicates within the insect's gut. CLas then moves into the hemolymph, or insect blood, to circulate until it can reach the salivary glands and be injected into the next plant host. This process, which is called circulative transmission, requires highly specific molecular interactions between the bacteria and insect. Disrupting this process would help to prevent the spread of CLas and minimize the associated environmental and economic consequences that result from the disease. A. Kruse worked with her supervisor, Michelle Heck, and collaborators Michael MacCoss from the University of Washington and Robert Shatters from the USDA-ARS in Fort Pierce Florida to use proteomics and transcriptomics to learn about the effects of CLas on the ACP gut. This provided protein and RNA targets that may be important for transmission. The work below builds upon these data by targeting proteins and long non-coding RNAs (lncRNAs) in the insect with the goal of developing novel strategies to control transmission of CLas. Specific Aim 1: To develop interdiction molecules that target and block the function of long non-coding RNAs in the psyllid gut. Angela Kruse worked with undergraduate Anna Yaschenko and Surya Saha to develop a bioinformatics pipeline to better curate long non-coding RNAs (lncRNAs) from the ACP. A. Kruse and A. Yaschenko did expression analysis of several lncRNAs and found that their expression and length is affected by host plant, infection status, and lifestage. They also determined that lncRNAs from the ACP have many repetitive regions that are likely the result of transposon activity. Importantly, A. Yaschenko was able to learn how to do basic molecular biology and troubleshoot experiments under the mentorship of A. Kruse. A. Kruse designed antisense constructs to silence abundant lncRNAs from the insect. She also established protocols to synthesize RNAs and test their size and activity against insects using artificial diets. Experiments to test the antisense constructs are underway. Key Outcomes: Improved bioinformatics tools allow us to better find lncRNAs and make sure they are really lncRNAs. Expression analysis gives us a profile of which lncRNAs are expressed under specific conditions, including CLas infection. Specific Aim 2: To develop interdiction molecules that disrupt the psyllid salivary sheath, an essential feeding structure. A. Kruse worked with Drs. Robert Shatters and Michael MacCoss to use proteomics to identify three major proteins from the ACP salivary sheath. These have strikingly similar functions to one another, making them promising targets for inhibition of insect feeding. A. Kruse expressed these in both bacterial and insect cells, and found that one of them binds copper directly to form a protein complex. This process is also an essential step towards purifying these proteins for aptamer selection and testing their activity. To increase the number of proteins that can serve as targets for aptamer selection, and to learn more about the biology of transmission, A. Kruse developed a technique to extract small quantities of hemolymph (insect blood) from the ACP using microcapillaries. She worked with fellow Heck lab member, Dr. John Ramsey, to extract large amounts of hemolymph and conducted a proteomics experiment to learn how the hemolymph responds to CLas. This experiment revealed several immune proteins that respond to CLas and showed changes in the insect's metabolism indicative of a higher level of hunger in the insect. The immune proteins identified can serve as aptamer targets in the future. In parallel to the protein work above, A. Kruse did an aptamer selection against whole stylet sheaths and found one enriched sequence. When delivered in artificial diets, this aptamer inhibits the formation of the sheath. This aptamer can be further tested as a treatment to prevent ACP feeding. Key Outcomes: Three sheath proteins were discovered in the salivary sheath of the insect vector associated with citrus greening disease (the ACP). An RNA aptamer selected against whole sheaths inhibits the formation of sheaths in artificial diets, and is now being evaluated as a treatment to prevent ACP feeding. <br><br><b>Publications</b><br>