Source: NATIONAL AGRICULTURAL LIBRARY submitted to
HARNESSING NATURAL VARIATION IN TRANSMISSION OF LIBERIBACTER BY THE ASIAN CITRUS PSYLLID TO DEVELOP NOVEL HLB CONTROL STRATEGIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1009024
Grant No.
2016-70016-24779
Project No.
MD.W-2015-10475
Proposal No.
2015-10475
Multistate No.
(N/A)
Program Code
CDRE
Project Start Date
Feb 1, 2016
Project End Date
Jan 31, 2020
Grant Year
2016
Project Director
Cilia, M.
Recipient Organization
NATIONAL AGRICULTURAL LIBRARY
ABRAHAM LINCOLN BUILDING
BELTSVILLE,MD 20705
Performing Department
US Department of Agriculture
Non Technical Summary
Huanlongbing (HLB) is the most serious disease of citrus. Candidatus Liberibacter asiaticus (CLas), the pathogen associated with HLB, is spread around the grove by the Asian citrus psyllid, a sap-sucking insect vector. Controlling the insect spread of CLas represents the most promising way to control the disease. However, there is a paucity of information on how CLas is spread by the psyllid and currently no tools for a grower to use to detect CLas in the insect. Current detection methods in insects and in plants rely on qPCR and microscopy, and both methods require a laboratory, a skilled researcher, expensive equipment and costly consumables. Our team will pursue four research objectives and one educational objective to better understand how the psyllid transmits CLas in an effort to develop new transmission blocking tools. Drawing upon a new cutting edge scientific field called synthetic biology, we will also develop a low-tech biosensor that a grower can use to detect CLas in insects in the field, one of the goals of the SCRI program. Undergraduate researchers will be involved in all aspects of the synthetic biology project. Although the research plans within each objective are synergistic, the success of one objective does not depend on the others. We realize our research plans are ambitious but our team is up to the challenge.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120999113050%
2113110104050%
Goals / Objectives
Huanglongbing (HLB) is a tritrophic disease complex involving citrus host trees, the Asian citrus psyllid (ACP) insect and a phloem-restricted, bacterial pathogen Candidatus Liberibacter asiaticus (CLas). HLB is considered to be the most devastating of all citrus diseases, and there is currently no adequate control strategy. In Florida, an estimated 40-70% of all citrus trees are infected, and HLB effects include production declines (10-20% per year), diminished fruit quality and increased production costs. Some growers have already been forced into bankruptcy. California and Texas have the ACP and isolated reports of HLB, where the spread of HLB is imminent without discovery and implementation of new management practices. Control of ACP-mediated CLas transmission represents a promising, new avenue for HLB control, but there is a paucity of tools available for growers to control HLB from this angle.Our team will pursue four research objectives and one educational objective. Although the research plans within each objective are synergistic, the success of one objective does not depend on the others. We will:1. Discover genetic populations of ACP that segregate for CLas acquisition and transmission competency.2. Functionally characterize the ACP endosymbiont toxin diaphorin and establish whether the relationship between the ACP endosymbiont Profftella and the ACP is a viable target for ACP control.3. Functionally characterize ACP proteins involved in CLas transmission.4. Develop an ACP-CLas yeast biosensor that can be used by growers5. Enhance cross-disciplinary undergraduate education and research at Cornell through participation in the International Genetically Engineered Machines (iGEM) Competition.The overall, long-term goal of our project is to develop new tools that can be used by citrus growers for CLas-infected ACP monitoring and management, as detailed in Objectives 3 and 4. Two short-term goals of our project that we will achieve during the lifetime of our grant include: to characterize populations of the ACP that vary in their ability to transmit CLas and to produce a yeast biosensor that can readily detect CLas-infected ACP. The ACP populations will be immediately useful for challenging citrus germplasm against HLB and ACP inoculation and feeding. A long-term goal is the distribution of the biosensor technology to growers. A second long-term goal of our proposal is to leverage the knowledge of the molecular basis of CLas transmission by the ACP to produce a genetically modified citrus that will prevent the spread of CLas within a grove. All research will be performed with constant engagement of the stakeholders to provide support and guidance on technology advancement through to commercialization.
Project Methods
Objective 1: Colonies of potentially good or poor vectors will be established using an individual adult female ACP paired with a single male collected from various locations. Insects will be determined to be CLas-free to start and CLas transmission efficiencies will be calculated using transmission assays. Efficient and poor transmitting lines will be crossed and their progeny randomly mated to develop populations segregating in CLas transmission ability.Objective 2: Mass spectrometry-based proteomics will be used to identify proteins interacting with the toxin diaphorin. Potential receptors will be functionally validated surface plasmon resonance and other methods. CTV technology will be used for silencing the diaphorin receptor in the ACP.Objective 3: The CTV technology has been established by the Dawson lab previously. Construction of CTV vectors that target ACP proteins involved in valine catabolism, immune system regulation and bacterial entry is currently in progress in the Cilia and Dawson labs, greenhouse tests to follow.Objective 4: Ongoing research in the Cornish?Laboratory has demonstrated the ability to take?advantage of an endogenous G protein-coupled?receptor (GPCR) signaling pathway present in Saccharomyces cerevisiae to drive a lycopene?biosynthetic pathway and directed evolution. These methods may be implemented to engineer?a GPCR to bind a specific peptide biomarker of CLas in infected ACP. These methods will be used to engineer yeast to turn red when mixed with insects or plant tissue harboring CLas, enabling a rapid and clear determination of CLas status by a grower in the field.Objective 5: iGEM undergraduate research teams will be formed at Cornell and Columbia Universities and will assist with achieving objective 4.

Progress 02/01/16 to 01/31/20

Outputs
Target Audience:The target audience for the work completed over the course of this grant is citrus growers and producers in areas where Huanglongbing is endemic (Florida) as well as areas at risk of infection (Texas, California). In addition, most of the personnel involved in this work have presented their research at the International Research Conference on Huanglongbing in March, 2017 and 2019, which is well attended by researchers, stakeholders, and regulatory agencies. We have also focused efforts on engaging undergraduates and underrepresented minorities in experiential learning opportunities through the iGEM program. Changes/Problems:One modification that we have made to our experimental workflow is that a magnetic bead based solution hybridization has been selected to replace a solid support resin affinity chromatography approach for identification of proteins in the ACP which interact with diaphorin. This change was made because whereas the chemical strategies to connect diaphorin to a solid support resin relied on linkage to a particular site on diaphorin, the coating on the magnetic bead allows diaphorin to bind in a wide range of orientations, ensuring that no specific epitopes are blocked, which may be critical for protein-protein interactions. Towards the search for peptide biomarkers of CLas infection (objective 4), we were unsuccessful in finding any small peptides that were unique to CLas (+) psyllids. Initial screens found over 1000 candidates, but using more sensitive methods, all candidates were detected in both CLas (+) and CLas (-) psyllids. This presented a significant challenge, as the crux of the biosensor aim was identifying robust and consistent biomarkers that are predictive of the infection state of psyllids. However, this finding initiated new lines of research that demonstrated that the yeast biosensor technology is amenable for the detection of larger protein ligands, which broadens the pool of candidates to include multiple existing datasets on the complete proteome of the psyllid. What opportunities for training and professional development has the project provided?Most of the personnel involved in this work, including graduate students and post-doctoral researchers, have presented their research at scientific conferences, seminars, and in local symposia. These are valuable professional development opportunities that can improve scientific communication skills. Work on this grant has also supported the training of a post-doctoral researcher, a visiting graduate student, and an undergraduate student in the Cilia lab. In addition to scientific training in the fields of proteomics and peptidomics, the post-doc has been significantly involved in managing the grant and the team. Through the iGEM competition, four undergraduates worked as a team to develop the plant pathogen biosensor described above. This project presented many opportunities for professional development, including effective communication, teamwork, and presentation skills. In addition, these students were trained in specific concepts related to their project and biological engineering. Towards the aim of opening the diversity "pipeline" to R1 research institutions, our iGEM team included a biotechnology major from the University of Puerto Rico-Mayaguez. This student, Mariela A. Rivera De Jesús, enjoyed her experience at Cornell so much that she subsequently applied and was admitted to the PhD program in Biomedical Engineering. It is our vision that the experience of this student will motivate other bright, young undergraduates to look beyond the typical path and to reach their full potential as researchers and scholars, and we will continue to use the iGEM project as a key recruiting tool for students interested in bioengineering/synthetic biology. How have the results been disseminated to communities of interest?Over the past three years, we have published four papers with several others submitted or in the final stages of preparation. In addition, we have presented this work to the scientific community at conferences (International Research Conference on Huanglongbing 2017, 2019; American Phytopathological Society 2018, 2019) and for smaller working groups (Cornell Virology and Vector Biology interest group, lab symposia, etc.). These opportunities to disseminate information also brought forth discussions and valuable feedback, helping guide our efforts towards understanding and managing the pathogen. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? With funding support from USDA NIFA, we have taken a multi-pronged approach to unravel the interactions between 'Candidatus Liberibacter asiaticus' (CLas), which causes the citrus disease Huanglongbing (HLB), and the Asian citrus psyllid (ACP), the insect that transmits HLB. Over the course of this grant, we have identified lines of ACP with diverging ability to acquire and transmit CLas. We also progressed towards understanding the role of diaphorin, a toxin produced by the ACP endosymbiont 'Candidatus Profftella armatura', by solving the crystal structure and characterizing analogs that are responsive to CLas infection. Our work on developing a yeast biosensor for disease diagnosis has run into a roadblock, as we were unable to find reliable biomarkers, but efforts by undergraduates has led to the development of a promising alternative biosensor. Data from this research has also led to the identification of candidate ACP neuropeptides and antimicrobial peptides, which hold potential for ACP management and disease eradication. Finally, we have demonstrated that interfering with the expression of ACP genes can affect CLas titer in psyllids, and is a promising strategy for the control of HLB. Progress is detailed below. Objective 1. Discover genetic populations of ACP that segregate for CLas acquisition and transmission competency. In an effort to understand the genetic basis of vector competency, we have established and tested the ability of 15 ACP isofemale linesto acquire and transmit CLas. These lines originated from psyllids collected from different locations to maximize genetic diversity, and there was a significant difference found in acquisition rates among the lines. Further examination demonstrated that acquisition and transmission rates are positively correlated, and that CLas titer in the psyllid is positively correlated with acquisition and transmission rates (Ammar et al. 2018 PLoS One 13:e0195804). We have previously found that hemocyanin, a copper binding protein, is upregulated in CLas-exposed insects. We hypothesized that this protein may affect vectoring ability due to its known role in the immune response. To test this, we quantified the mRNA levels of hemocyanin in Laurel 8 (good vectoring ability) and Laurel 16 (poor vectoring ability), and found that the expression was significantly higher in Laurel 16 (Ammar et al. 2018 PLoS One 13:e0195804). The isofemale lines are a valuable research tool resulting from this project. Crosses between isofemale lines with divergent vectoring ability are underway, and genetic analysis of progeny is expected to lead to the identification of ACP genes/pathways involved in CLas acquisition and transmission. Objective 2. Functionally characterize the ACP endosymbiont toxin diaphorin and establish whether the relationship between the ACP endosymbiont Profftella and the ACP is a viable target for ACP control. The psyllid endosymbiont 'Candidatus Profftella armatura' produces the cytotoxin diaphorin, but the function is unknown. To better understand this toxin, we solved the crystal structure of diaphorin-methanol monosolvate (Szebenyi et al. 2018). We identified the minor diaphorin analogue and confirmed that it occurs at a higher proportion relative to diaphorin in CLas (-) ACP than CLas (+) ACP. This finding is consistent with previously published evidence of alterations in diaphorin biosynthesis and degradation in CLas (+) ACP (Ramsey et al. 2015). We developed an HPLC-MS method that is sensitive to diaphorin at concentrations as low as 20 picomolar. This method enables the analysis of diaphorin in ACP honeydew, bacteriomes, citrus leaves, etc., which will enhance our understanding of the distribution of diaphorin in ACP and its function in plant-insect-pathogen interactions. Finally, we have demonstrated a negative effect of CLas exposure on Profftella titer in male ACP bateriomes and a positive effect in female ACP ovaries, which suggests a functional and sex-specific role of Profftella, and possibly diaphorin, on CLas propagation in ACP (Hosseinzadeh et al. 2019 Microbial Ecol. 78:206-222). Objective 3. Functionally characterize ACP proteins involved in CLas transmission. We have pursued two approaches to characterize proteins likely to impact CLas transmission: delivery of silencing constructs via artificial diets and microinjection, and development of citrus plants infected with Citrus tristeza virus (CTV) vectors producing dsRNA and siRNA designed to silence target genes. Using the first approach, we delivered dsRNA targeting four ACP proteins, and successfully silenced two of the targets. Silencing of the hemocyanin 1 resulted in 2-fold reduction of the titer of CLas (Hosseinzadeh et al. 2019 PLoS One 14:e0216599). Towards the second approach, we have received citrus plants infected with CTV constructs targeting five genes predicted to be involved in ACP immunity and metabolism. Infected plant material has been propagated with a subset being co-infected with CLas. CTV infection and insert stability was verified. Using the newest iteration of the ACP assembled genome, we have validated the genetic organization of the targets and developed a qPCR assay to monitor their expression in psyllids. Assays testing the effects of silencing of these ACP targets on psyllid mortality and CLas acquisition/transmission are on going. Objective 4. Develop an ACP-CLas yeast biosensor that can be used by growers We employed validated directed evolution strategies to engineer a yeast biosensor that specifically recognizes small protein indicators of infection in ACP, which we hypothesize would accumulate long before the bacteria have exceeded the threshold for detection with current methods. Using optimized methods for the extraction of peptides, we identified >1000 candidate biomarkers. Attempts at validation were not successful, and we were unable to proceed as outlined originally. However, the psyllid peptidome is a rich dataset that yielded a number of candidate antimicrobial peptides and neuropeptides, and a manuscript has been published describing these results (Fleites et al. 2020 J. Proteome Res. 19:1392-1408). A series of candidate antimicrobial peptides has been synthesized and testing of these peptides against the closest culturable CLas relative Liberibacter crescens is underway. In addition, this work has led to a new line of funding via the HLB MAC initiative to study ACP neuropeptides identified. To date, we have screened eight biostable neuropeptide analogs, one of which has shown insecticidal activity against ACP. Objective 5. Enhance cross-disciplinary undergraduate education and research at Cornell through participation in the International Genetically Engineered Machines (iGEM) Competition. The undergraduates that participated in the International Genetically Engineered Machines (iGEM) Competition sought to develop a cell-free immunoassay that could be cheaply produced, widely distributed, and implemented directly by non-experts in the field and inspection centers at international borders. Their results demonstrated that the readouts are rapid, reliable, and can be made with little to no specialized equipment. In addition, we used the iGEM framework to develop new modes of recruitment that we anticipate will bring a fresh approach to opening the diversity "pipeline". We have begun to build an active network that connects undergraduates to graduate students who will particularly resonate with them and, expose undergraduates to sustained research and faculty mentoring experiences at an R1 research institution. We are also devising professional development programs to train our graduate students to become effective mentors and engaging both Cornell faculty and visiting faculty mentors from minority-rich undergraduate institutions to form a long-lasting mentoring network of role models.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fleites, L. A., Johnson, R., Kruse, A. R., Nachman, R. J., Hall, D. G., MacCoss, M., and M. L. Heck. 2020. Peptidomics approaches for the identification of bioactive molecules from Diaphorina citri. J. Proteome Res. 19:1392-1408.


Progress 02/01/18 to 01/31/19

Outputs
Target Audience:The target audience for the work completed over the course of this grant is citrus growers and producers in areas where Huanglongbing is endemic (Florida) as well as areas at risk of infection (Texas, California). In addition, most of the personnel involved in this work have presented their research at the International Research Conference on Huanglongbing in March, 2017 and 2019, which is well attended by researchers, stakeholders, and regulatory agencies. We have also focused efforts on engaging undergraduates and underrepresented minorities in experiential learning opportunities through the iGEM program. Changes/Problems:One modification that we have made to our experimental workflow is that a magnetic bead based solution hybridization has been selected to replace a solid support resin affinity chromatography approach for identification of proteins in the ACP which interact with diaphorin. This change was made because whereas the chemical strategies to connect diaphorin to a solid support resin relied on linkage to a particular site on diaphorin, the coating on the magnetic bead allows diaphorin to bind in a wide range of orientations, ensuring that no specific epitopes are blocked, which may be critical for protein-protein interactions. Towards the search for peptide biomarkers of CLas infection (objective 4), we were unsuccessful in finding any small peptides that were unique to CLas (+) psyllids. Initial screens found over 1000 candidates, but using more sensitive methods, all candidates were detected in both CLas (+) and CLas (-) psyllids. This presented a significant challenge, as the crux of the biosensor aim was identifying robust and consistent biomarkers that are predictive of the infection state of psyllids. However, this finding initiated new lines of research that demonstrated that the yeast biosensor technology is amenable for the detection of larger protein ligands, which broadens the pool of candidates to include multiple existing datasets on the complete proteome of the psyllid. What opportunities for training and professional development has the project provided?Most of the personnel involved in this work, including graduate students and post-doctoral researchers, have presented their research at scientific conferences, seminars, and in local symposia. These are valuable professional development opportunities that can improve scientific communication skills. Work on this grant has also supported the training of a post-doctoral researcher and a visiting graduate student in the Cilia lab. In addition to scientific training in the fields of proteomics and peptidomics, the post-doc has been significantly involved in managing the grant and the team. Through the iGEM competition, four undergraduates worked as a team to develop the plant pathogen biosensor described above. This project presented many opportunities for professional development, including effective communication, teamwork, and presentation skills. In addition, these students were trained in specific concepts related to their project and biological engineering. Towards the aim of opening the diversity "pipeline" to R1 research institutions, our iGEM team included a biotechnology major from the University of Puerto Rico-Mayaguez. This student, Mariela A. Rivera De Jesús, enjoyed her experience at Cornell so much that she subsequently applied and was admitted to the PhD program in Biomedical Engineering. It is our vision that the experience of this student will motivate other bright, young undergraduates to look beyond the typical path and to reach their full potential as researchers and scholars, and we will continue to use the iGEM project as a key recruiting tool for students interested in bioengineering/synthetic biology. How have the results been disseminated to communities of interest?Over the past three years, we have published two papers with several others submitted or in the final stages of preparation. In addition, we have presented this work to the scientific community at conferences (International Research Conference on Huanglongbing 2017, 2019; American Phytopathological Society 2018) and for smaller working groups (Cornell Virology and Vector Biology interest group, lab symposia, etc.). These opportunities to disseminate information also brought forth discussions and valuable feedback, helping guide our efforts towards understanding and managing the pathogen. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The citrus industry in the US and worldwide has been devastated by the spread of Huanglongbing (HLB). With funding support from USDA NIFA, we have taken a multi-pronged approach to not only unravel the interactions between the causal bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and the Asian citrus psyllid (ACP), the insect that transmits the disease, but also to develop improved tools for detection of the pathogen. Over the course of this grant, we have identified lines of ACP with diverging ability to acquire and transmit CLas. This finding has opened up new areas of research to elucidate the genetic underpinnings of vector competence and might lead to novel insect and disease control strategies. We also progressed towards understanding the role of diaphorin, a toxin produced by the ACP endosymbiont 'Candidatus Profftella armatura', by solving the crystal structure and characterizing analogs that are responsive to CLas infection. Our work on developing a yeast biosensor for disease diagnosis has run into a roadblock, as we were unable to find reliable biomarkers, but complementary efforts by bright undergraduates has led to the development of a promising alternative biosensor. Finally, we have delivered proof of concept that interfering with the expression of specific ACP genes can affect CLas titer in psyllids, and is a promising strategy for the control of HLB. Progress is further detailed below. Objective 1. Discover genetic populations of ACP that segregate for CLas acquisition and transmission competency. In an effort to understand the genetic basis of vector competency, we have established and tested the ability of 15 isofemale lines of ACP to acquire and transmit CLas. These lines originated from psyllids collected from different locations to maximize genetic diversity, and there was a significant difference found in mean acquisition rates among the lines. Further examination demonstrated that acquisition and transmission rates are positively correlated, and that CLas titer in the psyllid is positively correlated with acquisition and transmission rates. We have previously found that hemocyanin, a copper binding protein, is upregulated in CLas-exposed insects. We hypothesized that this protein may affect vectoring ability due to it's responsiveness to CLas infection and known role in the immune response. To test this, we quantified the mRNA levels of hemocyanin in Laurel 8 (good vectoring ability) and Laurel 16 (poor vectoring ability), and found that the expression was significantly higher in Laurel 16. There was no difference in size or endosymbiont populations of Laurel 8 and Laurel 16, suggesting that these factors do not affect vector competency. Objective 2. Functionally characterize the ACP endosymbiont toxin diaphorin and establish whether the relationship between the ACP endosymbiont Profftella and the ACP is a viable target for ACP control. The psyllid endosymbiont 'Candidatus Profftella armatura' produces abundant quantities of the cytotoxin diaphorin, but the function is unknown. To better understand this toxin, we solved the crystal structure of diaphorin-methanol monosolvate (Szebenyi et al. 2018). We identified the minor diaphorin analogue and confirmed that it occurs at a higher proportion relative to diaphorin in CLas (-) ACP than CLas (+) ACP. This finding is consistent with previously published evidence of alterations in diaphorin biosynthesis and degradation in CLas (+) ACP (Ramsey et al. 2015). We developed an HPLC-MS method that is sensitive to diaphorin at concentrations as low as 20 picomolar. This method enables the analysis of diaphorin in ACP honeydew, bacteriomes, citrus leaves, etc., which will enhance our understanding of the distribution of diaphorin in psyllids and its function in plant-insect-pathogen interactions. Objective 3. Functionally characterize ACP proteins involved in CLas transmission. We have pursued two approaches to characterize proteins likely to impact CLas transmission: delivery of silencing constructs via artificial diets and microinjection, and development of citrus plants infected with Citrus tristeza virus (CTV) vectors producing dsRNA and siRNA designed to silence target genes. Using the first approach, we delivered dsRNA targeting four ACP proteins, and successfully silenced two of the targets. Furthermore, silencing of the hemocyanin 1 resulted in 2-fold reduction of the titer of CLas. Towards the second approach, we have received citrus plants infected with CTV silencing constructs targeting five ACP genes predicted to be involved in ACP immunity and metabolism. Infected plant material has been propagated by grafting onto healthy citrus with a subset being co-infected with CLas. CTV infection and insert stability was verified. Using the newest iteration of the ACP assembled genome, we have validated the genetic organization of the targets and developed a qPCR assay to monitor their expression in psyllids. Assays testing the effects of silencing of these ACP targets on psyllid mortality and CLas acquisition/transmission are currently on going. Objective 4. Develop an ACP-CLas yeast biosensor that can be used by growers We employed validated directed evolution strategies to engineer a yeast biosensor that specifically recognizes small protein (peptide) indicators of infection in ACP, which we hypothesize would accumulate long before the bacteria have exceeded the threshold for detection with current methods. Using optimized methods for the extraction of peptides, we identified >1000 candidate biomarkers. Attempts at validation using more sensitive methods detected the peptides in healthy insects, and we were unable to proceed as outlined originally. However, the psyllid peptidome is a rich dataset that yielded a number of candidate antimicrobial peptides and neuropeptides, and a manuscript is in preparation describing these results. In addition, this work has led to a new collaboration between the lab of Michelle (Cilia) Heck and Ronald Nachman (USDA-ARS College Station, TX) and funding via the HLB MAC initiative to study ACP neuropeptides identified as part of this work. Objective 5. Enhance cross-disciplinary undergraduate education and research at Cornell through participation in the International Genetically Engineered Machines (iGEM) Competition. The undergraduates that participated in the International Genetically Engineered Machines (iGEM) Competition sought to develop a cell-free immunoassay that could be cheaply produced, widely distributed as a freeze-dried product, and implemented directly by non-experts in the field and inspection centers at international borders. Their results successfully demonstrated that the readouts are rapid, reliable, and can be made with little to no specialized equipment. We anticipate that this new pathogen detection strategy will have a direct and profound impact on some of the objectives of this proposal. In addition, we used the iGEM framework to develop new modes of recruitment that we anticipate will bring a fresh approach to opening the diversity "pipeline". We have begun to build an active network that connects undergraduates to graduate students who will particularly resonate with them and, thereby, expose undergraduates to sustained research and faculty mentoring experiences at an R1 research institution. We are also devising professional development programs to train our graduate students to become effective mentors and engaging both Cornell faculty and visiting faculty mentors from minority-rich undergraduate institutions to form a long-lasting mentoring network of inspiring role models.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ammar, E.-D., D. G. Hall, S. Hosseinzadeh,M. (Cilia) Heck. 2018. The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen Candidatus Liberibacter asiaticus by Asian citrus psyllid isofemale lines. PLoS ONE 13(4):e0195804.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Hosseinzadeh, S., Ramsey, J., Mann, M., Bennett, L., Hunter, W. B., Shams-Bakhsh, M., Hall, D. G., M. (Cilia) Heck. 2018. Color morphology of Diaphorina citri influences interactions with its bacterial endosymbionts and 'Candidatus Liberibacter asiaticus'. PLoS ONE 14(4) :e0216599
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hosseinzadeh, S., Shams-Bakhsh, M., Mann, M., Fattah-Hosseini, S., Bagheri, A., Mehrabadi, M., M. (Cilia) Heck. 2019. Distribution and variation of bacterial endosymbiont and 'Candidatus Liberibacter asiaticus' titer in the Huanglongbing insect vector, Diaphorina citri Kuwayama. Microbial Ecol. 78(1):206-222


Progress 02/01/17 to 01/31/18

Outputs
Target Audience:The target audience for work completed this fiscal year is citrus growers and producers in areas where Huanglongbing is endemic (Florida) as well as areas at risk of infection (Texas, California). In addition, most of the personnel involved in this work have presented their research at the International Research Conference on Huanglongbing in March, 2017, which is well attended by researchers, stakeholders, and regulatory agencies. We have also focused efforts on engaging undergraduates and underrepresented minorities in experiential learning opportunities through the iGEM program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This past summer, we welcomed our first cohort of undergraduate students to our developing program designed to open the diversity "pipeline" to R1 research institutions (described in research plans for FY 2018, objective 5). One of them, Mariela A. Rivera De Jesús, a junior from the University of Puerto Rico-Mayaguez majoring in biotechnology, joined our iGEM team and spent the summer working alongside her iGEM teammates to develop the plant pathogen biosensor described above. Mariela enjoyed her experience at Cornell so much that she applied and was subsequently admitted to the PhD program in Biomedical Engineering. She recently accepted this offer and will be starting the program this coming fall. Our plan is to further grow the "Keeping the Ezra Promise" Research Experience for Undergraduates (REU) program this summer, using the USDA iGEM project as a key recruiting tool for students interested in bioengineering/synthetic biology. How have the results been disseminated to communities of interest?Many abstracts on different aspects of the grant were presented at the International Research Conference on Huanglongbing in March, 2017. This meeting, held every two years, draws a large audience of growers and researchers alike coming from many countries including the US, China and Brazil, the top three citrus producers worldwide. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. During the past few months we established several new ACP sublines and we are now in the process of testing them for both CLas acquisition and transmission. Furthermore, we are testing the effects of some other variables on vector efficiency of ACP, including gender, color morphs and the rearing host plant: orange jasmine (Murraya paniculata) vs. citron (Citrus medica). M. paniculata is a preferred host of ACP but is more resistant to CLas than citrus genotypes, and we have some preliminary results suggesting that it might affect ACP vector efficiency negatively. We also plan to study transovarial transmission of CLas to determine its possible impact on the epidemiology of this serious citrus disease. Objective 2. We will implement a modified experimental design for the use of phenyl borate cellulose columns for identification of diaphorin-interacting proteins. Diaphorin is present at such abundance in the ACP that we propose to use this system to pull down diaphorin-protein complexes using a syringe pump to flow ACP homogenate through the phenyl borate column. After identification of diaphorin-interacting proteins by mass spectrometry, we will use surface plasmon resonance to validate candidate proteins. We will continue analysis of diaphorin in ACP honeydew, bacteriomes, saliva, and citrus leaf samples which have been fed on by ACP, to enhance our understanding of the distribution of diaphorin in the insect and its function in plant-insect-pathogen interactions. Objective 3. A primary focus of our effort on Objective 3 in 2018 will be to determine whether feeding on citrus plants infected with CTV silencing constructs effectively reduces target gene expression in ACP and influences the ability of the insects to acquire or transmit CLas. The effect of ACP gene silencing on CLas transmission will be evaluated by infesting control and CTV construct plants with CLas(+) ACP and determining whether the presence of the silencing construct effects either 1) transmission of CLas to the plant by ACP, 2) acquisition of CLas by the next generation of nymphs developing on the infected plants following a defined oviposition period with CLas(+) insects, and 3) insect survivorship/mortality. Additional research into delivery of dsRNA to ACP nymphs by droplet feeding will be conducted. This is a novel strategy for delivery of molecules of interest to ACP developed by Dr. Nabil Killiny (University of Florida) in which nymph insects are placed on their backs and a droplet of liquid is placed directly on its mouthparts and is taken up. While this is a more laborious delivery system compared to artificial diet feeding, it is technically difficult to feed nymphs on artificial diet, and silencing and CLas transmission experiments may be more informative when conducted on nymphs as the bacterium must be acquired during this developmental stage for effective transmission as adults. Objective 4. Towards the development of an ACP-CLas yeast biosensor, we plan (1) validate and quantify new candidate biomarkers from our most recent dataset, (2) continue experiments examining the feasibility of targeting full-length proteins and (3) to target trypsinized and endogenous peptide biomarkers identified by the Cilia lab using our validated directed evolution approach. Preliminary experiments have shown that our biosensor receptor is capable of recognizing its cognate ligand even when the ligand is fused to a much larger protein. Subsequent experiments aim to determine if the receptor can recognize its cognate peptide ligand when the ligand is incorporated within a full-length protein. This will inform us on the ability of the receptor to detect epitopes within proteins rather than at the termini. We plan to perform these experiments using cholera toxin B subunit, a protein with well characterized structure and epitope regions. If the receptor can recognize cholera toxin in which the native epitope has been replaced by our receptor's cognate ligand, we can then apply our same directed evolution approach to march the epitope back towards its native residues, evolving a receptor that binds the epitope in the process. If we can validate this approach, we can apply it to target a protein associated with ACP-CLas infection, which have been identified in previous studies. Concurrently, we will continue to evolve receptors towards both trypsinized and endogenous peptide biomarkers identified by the Cilia lab. Once we have a receptor that binds an ACP-CLas biomarker, we can incorporate it into our biosensor platform and begin testing with growers. Objective 5. Beyond the science and engineering, we used the iGEM project framework to develop new modes of student recruitment that we anticipate will bring a fresh approach to opening the diversity "pipeline" by starting to coach promising students early in their college careers. Our program is motivated by Cornell founders Ezra Cornell and Andrew Dickson White who founded a university where "any person" could become educated in "any study". Women, students of color, rural farmhands, and first-generation immigrant children could attend Cornell University to educate themselves in any subject they desired. At the heart of our new program is a plan to build an active network that connects URM undergraduates to graduate students who will particularly resonate with them and, thereby, exposes undergraduates to sustained research and faculty mentoring experiences at an R1 research institution. Key supporting elements include professional development programs to train our graduate students to become effective mentors and engaging both Cornell faculty and visiting faculty mentors from minority-rich undergraduate institutions to form a long-lasting mentoring network of inspiring role models. Our primary goals are to increase retention of URM undergraduates to completion of degree, including women, who are underrepresented in higher career ranks, to increase the number of URM and female students applying to graduate programs, and to support URM and female graduate students towards successful career paths. We will keep the program's graduates as life-long protégées and encourage them to consider becoming faculty. Success creating more URM faculty would further reinforce the pipeline by encouraging their own future undergraduate students to "Keep the Ezra Promise."

Impacts
What was accomplished under these goals? The citrus industry in the US and worldwide has been devastated by the spread of Huanglongbing (HLB), widely considered to be the worst disease of citrus. We are taking a multi-pronged approach to not only unravel the interactions between the causal bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and the Asian citrus psyllid (ACP), the insect that transmits the disease, but also develop improved tools for detection of the pathogen. Over the past year, we have refined the isofemale lines of ACP with enhanced or diminished ability to acquire and transmit CLas, solved the crystal structure of diaphorin, a toxin produced by a bacterial endosymbiont of ACP, pursued multiple approaches to silence genes in ACP and citrus that may affect CLas, and established large set of candidate biomarkers of ACP exposure to CLas. In addition, we have engaged a team of undergraduates in a project that parallels this work, bringing a fresh approach to some of the objectives of this proposal. Progress is further detailed below. Objective 1. In our previous work, we collected and tested vector competency of 15 isofemale lines of ACP, the vector of CLas. We identified three lines as good acquirers as well as good transmitters of CLas, and three lines that were both poor acquirers and poor transmitters. CLas acquisition and transmission rates were positively correlated, and were both positively correlated with CLas titer in ACP (Ammar et al., submitted). However, further testing of these lines in 2017 showed that vector competency was not stable/consistent in some lines whereas it was still consistent with others. Thus, we have established sublines from four of these variable lines to get more homogenous ACP lines in which vector efficiency would be more stable/consistent. A manuscript is under review describing this work. Objective 2. We investigated the degradation pathways of diaphorin (1) to better understand how to handle it for use in biological studies, and (2) to characterize the degradation products to determine whether they might have biological activity relevant to the HLB pathosystem. Diaphorin is moderately stable at 25°C, slowly degrading into two products. The major product was isolated and its structure confirmed as 6-O-desmethyl diaphorin by mass spectrometry and NMR. This finding indicates that exposure to low pH in vivo can degrade diaphorin. The protocol for purifying diaphorin from ACP was optimized, and highly pure diaphorin samples are now routinely obtained in crystalline form. The crystal structure of a diaphorin-methanol monosolvate was solved and a manuscript is published. We structurally characterized a minor diaphorin analog, which occurs at a higher proportion relative to diaphorin in CLas(-) ACP than in CLas(+) ACP. NMR data were used to solve the planar structure of this compound as 17-dehydro-diaphorin. This compound represents an analog produced when the ketoreductase that adds the final ketide group in diaphorin fails to operate. This suggests that CLas infection may have an effect on the efficiency of diaphorin biosynthesis by Profftella. We developed an HPLC-MS method that is sensitive to diaphorin at concentrations as low as 20 picomolar. Using this method, we readily detected diaphorin and related compounds in ACP saliva collected via artificial diet, and found the concentration of diaphorin in dissected adult bacteriomes was elevated relative to whole insect samples. Objective 3. The focus of this objective is to characterize the function of ACP proteins which may play a role in the transmission of CLas. In our previous work we identified ACP proteins that interact with CLas proteins and which are found at higher abundance in CLas(+) compared to CLas(-) ACP, and we have pursued two independent approaches to silence the expression of a subset of these targets: delivery of synthetic dsRNA by artificial diet feeding and microinjection, and development of citrus plants infected with Citrus tristeza virus (CTV) vectors producing dsRNA designed to silence target genes. We have delivered dsRNA targeting four ACP proteins: myosin, annexin, integrin β, and hemocyanin 1. Using RT-PCR and digital drop PCR to evaluate the efficiency of target gene silencing, we observed a 70% reduction in expression of the myosin and hemocyanin 1 constructs. No significant reduction in annexin and integrin β expression was observed. Silencing was significantly more effective when dsRNA was delivered by microinjection compared to artificial diet feeding. Silencing of hemocyanin 1 by dsRNA microinjection resulted in a moderate reduction in CLas titer in exposed insects. We have received citrus plants infected with CTV silencing constructs targeting five ACP genes. In this experimental design, target ACP genes will be silenced when the insect feeds on the CTV infected plants. We have grafted budwood from the mother plants infected with each CTV construct to healthy citrus to propagate the infected plant material, and confirmed CTV transmission with ELISA. Using primers targeting the region of the CTV construct flanking the insert sequence, we confirmed that each construct contained an insert of the anticipated size. Objective 4. In the development of an ACP-CLas yeast biosensor, we optimized methods for the extraction of endogenous peptides from ACP, resulting in the identification >1000 peptides unique to infected insects. Several reduced extraction methods were evaluated, and we found that adding 50% methanol to ground psyllids and removing the debris resulted in the most enriched peptide extract of all workflows evaluated. This holds promise for the feasibility of deploying the biosensor in fields. Building on work from last period in which we developed a receptor to bind a peptide with 67% identity to one of these targets, we further evolved the receptor to bind the exact target. Although subsequent analyses showed the target was not in fact a biomarker, this accomplishment is important because it validates our directed evolution approach. CLas's demonstrated effect on the ACP proteome has provided information on trypsinized peptide and protein targets. We assessed our biosensor's viability in trypsinization conditions, finding it compatible with trypsin but sensitive to high concentrations of denaturants. We also designed and purified fusion proteins in which the biosensor receptor's cognate ligand was fused to the N- or C-terminus of a full-length protein. We found that the receptor was able to detect its cognate ligand when tethered to a much larger protein, suggesting that the biosensor is compatible with larger proteins. Objective 5. For the undergraduate synthetic biology project this past summer, we attempted to engineer a robust, easy-to-use biosensor for the detection of a variety of plant pathogens. The objective was to engineer a reporter organism that generates a visible phenotype in response to specific pathogen signatures. The signature that we focused on was a carbohydrate moiety, β-(1→6)-linked poly-N-acetyl-D-glucosamine, which is a surface capsular polysaccharide that is found on a surprisingly wide array of microbial pathogens. Importantly, rather than feed the students with the solution that our team has proposed, we challenged a team of four young, unbiased researchers to invent their own solution to this difficult problem. Their solution involved the creation of a cell-free immunoassay that could be cheaply produced, widely distributed as a freeze-dried product, and implemented directly by non-experts (e.g., growers, border inspectors, etc.) in the field and inspection centers at international borders. Their results over the summer demonstrated that the readouts are rapid, reliable, and can be made with little to no specialized equipment. We anticipate that this new pathogen detection strategy will have a direct and profound impact on some of the objectives of this proposal.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Szebenyi, D. M., I. Kriksunov, K. J. Howe, J. S. Ramsey, D. G. Hall, M. Heck and S. Krasnoff. 2018. Crystal structure of diaphorin methanol monosolvate isolated from Diaphorina citri Kuwayama, the insect vector of citrus greening disease. Acta Crystallographica E74:445-449.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Ammar, E.-D., D. G. Hall, S. Hosseinzadeh,M. (Cilia) Heck. 2018. The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen Candidatus Liberibacter asiaticus by Asian citrus psyllid isofemale lines. Revised and resubmitted to PlosOne (under revision).


Progress 02/01/16 to 01/31/17

Outputs
Target Audience:The target audience for work completed this fiscal year is citrus growers and producers in areas where Huanglongbing is endemic (Florida) as well as areas at risk of infection (Texas, California). Changes/Problems:One modification that we have made to our experimental workflow is that a magnetic bead based solution hybridization has been selected to replace a solid support resin affinity chromatography approach for identification of proteins in the ACP which interact with diaphorin. This change was made because whereas the chemical strategies to connect diaphorin to a solid support resin relied on linkage to a particular site on diaphorin, the coating on the magnetic bead allows diaphorin to bind in a wide range of orientations, ensuring that no specific epitopes are blocked, which may be critical for protein-protein interactions. What opportunities for training and professional development has the project provided?Most of the personnel involved in this work will be presenting their research at the International Research Conference on Huanglongbing in March, 2017. Work on this grant has also supported the training of a post-doctoral researcher and a visiting graduate student in the Cilia lab. In addition to scientific training, the post-doc is significantly involved in managing the grant and the team. How have the results been disseminated to communities of interest?We are planning a virtual stakeholder meeting in early 2017 where we will give updates on our progress and have time scheduled in for questions and suggestions. The invitees include participants from a variety of backgrounds and locations, including citrus growers and researchers from Brazil, California, and Florida. Engaging the stakeholders in this way will provide essential feedback, and enhance the quality of the research. Many abstracts on different aspects of the grant were submitted for presentation at the International Research Conference on Huanglongbing in March, 2017. This meeting, held every two years, draws a large audience of growers and researchers alike coming from many countries including the US, China and Brazil, the top three citrus producers worldwide. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. The Hall lab is planning to test some of the best and poorest 'acquirer' and 'transmitter' lines for at least 10 generations to ascertain the stability of the acquisition and transmission phenotypes in these lines, before crossing them for further genetic research. The Cilia lab will continue genomic, proteomic and other analyses of the good and poor vector lines. The titer of the three major endosymbionts in Laurel 8 (good transmitter) and Laurel 16 (poor transmitter) will be reexamined with a larger number of biological replicates. We have also developed an experimental plan to use a metagenomics approach to characterize differences between the strains of each endosymbiont species between the two ACP lines. Objective 2. During the next reporting period, we will use the purified diaphorin for magnetic bead conjugation and identification of interacting proteins from the ACP. We will use mass spectrometry and gel analysis to identify proteins interacting with diaphorin, and we will optimize conjugation and hybridization conditions. Once diaphorin-protein interactions have been identified, we will work to validate the putative interactions using surface plasmon resonance. Objective 3. In the coming fiscal year, we plan to develop the assays for the delivery of silencing constructs to ACP. We are also scaling up the supply of young citrus plants for the propagation of budwood infected with CTV constructs, and will design and implement assays to verify CTV infection and stability of the constructs. Objective 4. The Cornish lab is planning to (1) to continue exploring sample preparation methods, (2) to continue directed evolution efforts to obtain an initial prototype biosensor, (3) to optimize our biosensor for use in the field, and (4) to develop biosensors sensitive to other ACP-CLas peptides. Thus far, we have tested ACP samples in a modified form of Laemmli buffer; we will test other possibilities such as PBS and water as these media types may be more easily used by growers. Currently, we have developed a biosensor receptor that can bind a peptide 67% identical to the target; we will continue with additional rounds of directed evolution and alternate library generation methods to reach our final target. Once we have obtained a prototype biosensor, we will optimize readout production for use in the field by growers, focusing on minimizing time to visible redness. Finally, subsequent mass spectrometric analyses performed by the Cilia Lab may provide us with additional peptide targets specific to CLas (-) or (+) ACPs. We can use directed evolution to generate additional biosensors against these targets, which could be used collectively to create a more sensitive, specific diagnostic tool. Objective 5. Dr. DeLisa will begin distributing the iGEM job description in relevant departments at Cornell (chemistry, microbiology, bioengineering, etc.), and a group of 4-6 undergraduates will be selected from the pool of applicants. Brainstorming and new member training will take place in the spring, hands-on project work in the summer, and in the fall the team will compete at the iGEM jamboree.

Impacts
What was accomplished under these goals? The citrus industry in the US and worldwide has been devastated by the spread of Huanglongbing (HLB), a bacterial disease widely considered to be the worst disease of citrus. The causal agent, 'Candidatus Liberibacter asiaticus' (CLas), is vectored by the Asian citrus psyllid (ACP), a hemipteran insect that forms an intimate association with CLas and several bacterial endosymbionts. Our work takes a multipronged approach to unravel interactions between ACP and associated bacteria, and has direct applications for early detection and control of HLB. During this reporting period, we have accomplished several of the tasks outlined in our proposal: Objective 1. In an effort to understand the genetic basis of vector competency, the Hall lab is establishing individual ACP lines collected from different locations and host plants and screening them for differences in CLas acquisition and transmission efficiency. Factors identified that impact ACP vector competency may be exploited as targets for HLB disease control. We have established 20 isofemale lines from different locations in Florida, accomplishing the first task under this objective. Three successive generations of 14 lines have been tested for CLas acquisition efficiency and six lines for transmission efficiency. The titer of CLas in 14 lines has also been examined. We found variation in all three parameters in the lines tested. So far, we have identified two lines that are good candidates for further study. Laurel 16, the poorest vector, has the lowest acquisition rate (6.7-9.9%) and transmission rate (0-1.7%). Laurel 8, the best vector, has the highest acquisition rate (37.1-40.9%) and transmission rate (18.3-21.7%). Bacterial titer was not significantly different in these two lines; however, significant differences in titer was observed for some of the other lines tested. Linear regression analysis indicated a positive correlation between inoculation and acquisition rates in the lines tested to date. The Cilia lab received insects from the two ACP strains on either end of the transmission competency spectrum: Laurel 8 (good transmitter) and Laurel 16 (poor transmitter). We used qPCR to compare the titer of the three major ACP endosymbionts (Profftella, Carsonella, and Wolbachia) between the two lines. No significant differences were identified between the two ACP strains for any of the three endosymbionts. This experiment is being repeated with larger number of biological replicates. We have developed an experimental plan to use a metagenomics approach to characterize differences between the strains of each endosymbiont species between the two ACP strains. We have used confocal microscopy with fluorescent probes specific to endosymbiont genomic DNA sequences to visualize the distribution of the 3 endosymbionts in the Laurel 8 and Laurel 16 ACP strains. Objective 2. The ubiquitous psyllid endosymbiont 'Candidatus Profftella armatura' produces abundant quantities of the polyketide cytotoxin diaphorin. Diaphorin is found in three-fold higher concentrations in CLas + psyllids; however, the function of this toxin is unknown. The first task under this goal, optimization of a preparative HPLC protocol for the purification of the diaphorin polyketide from the ACP, has been completed. Starting from ~6000 ACP, we performed metabolite extraction in methanol and used an HPLC protocol to purify diaphorin to ~95% purity, with a final yield of ~6 grams. We used NMR to confirm the predicted structure of diaphorin, and to evaluate the room temperature stability of diaphorin. Diaphorin was found to be moderately stable: after 60 days at room temperature, the composition of the sample had changed from ~95% diaphorin to ~50% diaphorin, with increasing production of an unidentified related compound over the two month analysis period. The second task under this goal, identification of diaphorin-interacting proteins in the ACP, is in progress. We have initiated experiments to conjugate diaphorin to 1 μM magnetic beads, which will be used in solution hybridization to identify ACP proteins interacting with diaphorin. Currently we are optimizing conditions for coupling diaphorin to magnetic beads, and for extraction of proteins in their native conformation from the ACP. Objective 3. To begin the functional characterization of proteins that may play a role in CLas transmission, we have designed a series of Citrus tristeza virus (CTV) vectors producing dsRNA designed to silence target genes. These constructs were synthesized in a plasmid vector and sent to collaborators with expertise in molecular manipulation of the virus to move into CTV. In a parallel, but independent approach, we have designed dsRNAs for target gene silencing in ACP. These dsRNA sequences will be delivered to ACP via microinjection and by artificial diet feeding. Objective 4. Our team is also employing validated directed evolution strategies to engineer a yeast biosensor that specifically recognizes small protein (peptide) indicators of infection in ACP, which we hypothesize would accumulate long before the bacteria have exceeded the threshold for detection with the current methods. In the development of an ACP-CLas yeast biosensor, the Cornish lab has (1) developed an ACP sample preparation strategy, (2) assessed the compatibility of our yeast biosensor model with ACP samples, and (3) used directed evolution to modify our biosensor receptor for target binding. The Cilia Lab has developed a protocol for the extraction of peptides for mass spectrometric analysis. Similarly, we extracted peptides from ACP samples by grinding up ACPs, resuspending in a modified form of Laemmli buffer, then sonicating. We then tested the viability of one of our unrelated fungal biosensors in media containing this ACP suspension, confirming the compatibility of our biosensor model with ACP samples and resuspension buffer constituents. In a mass spectrometric analysis of CLas-negative (-) and -positive (+) ACPs, the Cilia Lab identified peptides specific to healthy ACPs. We have selected a peptide to target through directed evolution based on peptide sequence similarity with currently functioning biosensors. Thus far, we have used directed evolution to develop a fungal receptor that can bind a peptide with 67% identity to the target, representing significant progress towards the target. Along with the work being performed in parallel at the Cornish lab, the Cilia lab has continued to work on improving the psyllid peptide extraction protocol and assessing the repeatability of preliminary results. A second a set of peptide extracts from CLas + and - psyllids has been prepared and sent for LC-MS/MS analysis. Objective 5. A draft job description for the iGEM team has been written by Dr. DeLisa and the Cornish lab.

Publications