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INVESTIGATOR: Beckmann, J. F.

PERFORMING INSTITUTION:
YALE UNIVERSITY
105 WALL ST
NEW HAVEN, CONNECTICUT 06511-6614

MOLECULAR MECHANISM OF CYTOPLASMIC INCOMPATIBILITY AND INSECT CONTROL

NON-TECHNICAL SUMMARY: Insects cause massive amounts of agricultural losses with respect to food, time, and money, both in the United States and all over the world. Furthermore, many of these pests are developing resistance to pesticides. Alternative approaches to insect pest control aside from/supplementing pesticide use are of great importance. One such approach is SIT, which involves the release of sterile insects and has been previously utilized to drive down populations of insect pests due to sexual competition with infertile males. Certain bacteria are capable of sterilizing insect sperm. Understanding how these bacteria sterilize insect sperm will allow us to use this as a tool to manipulate insect pest populations.I will use biochemistry based research approaches to understand how one particular bacterium, Wolbachia, induces reproductive sterility in insects and seek to develop this knowledge into a new tool for insect population reduction. If I am successfully able to determine the function of this reproductive sterility it might be continually used in place of or supplemental to pesticides. Furthermore this research may reduce the amounts of money and food lost each year as a result of insects increasing the productivity and competitiveness of agriculture in the United States.

OBJECTIVES: The research proposed here seeks to develop approaches to investigate the molecular functions of cytoplasmic incompatibility with respect to two previously identified Wolbachia proteins. These genes may have future applications in pest control via CI induced SIT. In order to evaluate the biochemistry of these two specific genes I will: 1. Clone and characterize the expression of WPIP0282 and WPIP0283; 2. Determine the substrate specificity of the Wolbachia Ulp1-like SUMO Protease (WPIP0283); 3. Find binding partners of the wPip 0282-0283 operon proteins; 4. Express the proteins within insect cells and eukaryotic yeast cells and investigate phenotypes; 5. Evaluate the potential of the wPip 0282-0283 operon to induce CI in arthropods by expression in Drosophila melanogaster.

APPROACH: Methods include PCR, molecular cloning, site-directed mutagenesis, pull-down assays, western blots, and SDS PAGE, and DNA sequencing with respect to the proteins WPIP0282 and WPIP0283 as well as their orthologs and paralogs in Drosophila. I am also considering developing quantitative proteomics based approaches utilizing iTRAQ techniques in order to quantitatively assess substrate recognition of the uknown wolbachia protease WPIP0283. Furthermore, methods will include expression and microscopic analysis within yeast, Drosophila S2 cells, and Drosophila melanogaster flies. Our methods and experimental results will be evaluated by sequentially reaching various research milestones including: 1. Expression of recombinant proteins within E.coli and yeast vectors. 2. Identifying positive interacting partners. 3. Generation of positive evidence suggesting a link of the wPip 0282-0283 operon to CI induction/rescue. 4. Expressing the operon within Drosophila melanogaster flies.

PROGRESS: 2014/09 TO 2016/12
Target Audience:Students: 2 Graduate student at Yale University were mentored during their rotation in the Hochstrasser lab. The second student, Mr. Hongli Chen, joined our lab and has been mentored for the last year of the project. Yale Entrepreneurial Institute: Contact and mentorship was provided during the award period by Susan Froshauer and Param Sreekanth. Public: I have maintained a twitter account and tweeted tweets with respect to my project for the public and agricultural community to observe as well as connected with other Agricultural researchers funded by NIFA and the USDA on twitter. My research has been published in the journal Nature Microbiology and was featured in media articles including but not limited to: Nature News and Views, WIRED, The Scientist, CT post, New Haven Register, Yale Daily News, VOA news, and the Faribault Daily News. My work was Presented during invited talks at the Entomological Society of America Pacific Branch, the University of Minnesota, the University of Kentucky, Hope College, the Wolbachia conference, the American Society for Rickettsiology, and Vanderbilt University Changes/Problems:There were no major changes. I accomplished literally everything I proposed to do within the award period. What opportunities for training and professional development has the project provided?1. I have been able to study genetic techniques with respect to yeast and E.coli model organisms. Examples of this include: optimization of protein cloning and expression within these organisms, extensive utilization of diverse vectors, plasmids, selective systems, and growth conditions. 2. Cooperative efforts with other researchers within Yale's department of molecular biophysics and biochemistry have provided me the opportunity to familiarize myself with protein crystallography and have expanded this projects scope through new avenues of research directly pertinent to the aims of the grant. 3. I have been able to access training and mentorship with respect to biotech entrepreneurship by reaching out and interacting with Yale's Office of Cooperative Research. 4. The project has provided me with the opportunity to attend the PD training program directed by the USDA in Washington DC. 5. The project has provided me the opportunity to report my research results and network at multiple research conferences including the American Society for Rickettsiology and the Wolbachia 2016 meeting. 6. The project has provided me the opportunity to train in advanced insect genetic technologies through a course at the University of Maryland as part of the Insect Genetic Technologies Research Coordination Network. How have the results been disseminated to communities of interest?Results have been communicated via a publication in Nature Microbiology and through social media venues such as twitter and blogs as well as interviews with the popular press. I have also presented my research at multiple scientific meetings (see above). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2014/09 TO 2016/12
What was accomplished under these goals? Goal: 1. Clone and characterize the expression of WPIP0282 and WPIP0283 This goal has been achieved and was reported to the public. WPIP0283 was chacacterized as a CI Inducing Deubiquitylase. Goal 2. Determine the substrate specificity of the Wolbachia Ulp1-like SUMO Protease (WPIP0283); This goal has been achieved. I have determined that the enzyme was a deubuiquitylase. It's substrate is Ubiquitin. Goal 3: Find binding partners of the wPip 0282-0283 operon proteins; This goal has been achieved. I have found that the two genes interact with each other and have also found binding partners for each gene* as well as potental pathway interactions within yeast cells*. These results will be published at a later date. Goal 4: Express the proteins within insect cells and eukaryotic yeast cells and investigate phenotypes; This goal has been achieved. I have expressed the proteins within eukaryotic cells and demonstrated phenotypic effects from these experiments and have also begun expression in insect cells*. Goal 5: Evaluate the potential of the wPip 0282-0283 operon to induce CI in arthropods by expression in Drosophila melanogaster. This goal was achieved. I was able to induce Cytoplasmic Incompatibility independently of the bacterium within a transgenic Drosophila. These results were reported in our publication.

PUBLICATIONS (not previously reported): 2014/09 TO 2016/12
Type: Journal Articles Status: Published Year Published: 2017 Citation: Beckmann JF, Ronau JA, Hochstrasser M. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nature Microbiology. 2017;2:17007. doi: 10.1038/nmicrobiol.2017.7 http://www.nature.com/articles/nmicrobiol20177#supplementary-information.

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:Students: 1 Graduate student at Yale University was mentored during her rotation in the Hochstrasser lab. Biotec Companies/VC companies: I participated in Yale's biotec bootcamp which allowed me to network and discuss my ideas with potental investors and seek advice from experienced biotec entrepreneurs. Public: I have maintained a twitter account and tweeted tweets with respect to my project for the public and agricultural community to observe as well as connected with other Agricultural researchers funded by NIFA and the USDA on twitter. Lab Members: Because the expermental work is unpublished most of the communication for this first reporting was kept within our laboratory until we are ready to completely reveal the full research data. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. I have been able to study genetic techniques with respect to yeast and E.coli model organisms. Examples of this include: optimization of protein cloning and expression within these organisms, extensive utilization of diverse vectors, plasmids, selective systems, and growth conditions. 2. Cooperative efforts with other researchers within Yale's department of molecular biophysics and biochemistry have provided me the opportunity to familiarize myself with protein crystallography and have expanded this projects scope through new avenues of research directly pertinent to the aims of the grant. 3. I have been able to access training and mentorship with respect to biotech entrepreneurship, intellectual property, and company formation by reaching out and interacting with Yale's Office of Cooperative Research. 4. The project has provided me with the opportunity to attend the PD training program directed by the USDA in Washington DC. How have the results been disseminated to communities of interest?The results acquired with respect to the aims of this grant have, in our opinion, been extremely significant and therefore cannot be revealed until publication and patent filing. What do you plan to do during the next reporting period to accomplish the goals?I will continue my work with respect to transgenic drosophila and expect to have a significant publication by the termination of the grant period.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? Goal: 1. Clone and characterize the expression of WPIP0282 and WPIP0283 This goal has been achieved. I have successfully cloned both the above genes and developed 343 new constructs addressing the physiology of these proteins. I now know at least one function of both proteins which will be reported in later publications*. Goal 2. Determine the substrate specificity of the Wolbachia Ulp1-like SUMO Protease (WPIP0283); This goal has been achieved. I have determined at least one specific substrate for the protease*. Goal 3: Find binding partners of the wPip 0282-0283 operon proteins; This goal has been achieved. I have found binding partners for each gene* as well as potental pathway interactions within yeast cells. Goal 4: Express the proteins within insect cells and eukaryotic yeast cells and investigate phenotypes; This goal has been achieved. I have expressed the proteins within eukaryotic cells* and demonstrated phenotypic effects from these experiments*. Goal 5: Evaluate the potential of the wPip 0282-0283 operon to induce CI in arthropods by expression in Drosophila melanogaster. This goal is in progress and on track to be completed by the completion of the grant period. I have built the constructs for transgenesis* and inserted them into Drosophila melanogaster. The research with respect to these organisms will be conducted in throughout the rest of the grant period. * asterisks represent sensitive data that at this point remains private because it contains unpublished results and potential intellectual property.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
No publications reported this period.