Linda Hanley-Bowdoin

DNA replication is a highly choreographed process that integrates many aspects of genome structure and function, including transcriptional activity, chromatin structure, epigenetic states, and 3-D structure. However, almost all of our knowledge about DNA replication in higher eukaryotes comes from studies in metazoans. Evolutionary variation in DNA replication programs and their genetic control have not been studied in any plant system. The project will provide summer research and professional development experiences to undergraduates from underrepresented groups. The students will be actively recruited through established connections with HBCUs. The project will support the continuation of an annual summer workshop for under-represented highschool students, exposing them to maize genetics and modern plant research techniques. It will also support the Maize-10-Maze demonstration project during Year 3, and the production of a book combining the artistry and science of maize mutants to illustrate the genetic potential of important crops to the general public.

Cassava mosaic disease (CMD) is one of the most important diseases of cassava and a serious constraint on production across Africa. We identified and characterized two DNA sequences designated as SEGS-1 and SEGS-2 that enhance CMD symptoms and break resistance. A better understanding of the origins of the SEGS and how they alter disease etiology is necessary to develop sustainable CMD control measures for cassava. In this project, we will use cassava and Arabidopsis to address the following questions with the ultimate goal of translating the information to cassava. (1) How are the SEGS activated/transmitted in cassava? (2) What infection/defense processes are altered by the SEGS to enhance symptoms and break resistance? (3) What is the nature of the begomovirus resistance in Arabidopsis? (4) How our findings be translated to cassava to improve resistance to CMD?

This proposal establishes a research and training partnership between scientists in the U.S. and East Africa to study the evolution of plant DNA viruses, which have emerged as leading pathogens and now threaten crops worldwide. Africa������������������s future depends on increasing food production to feed its growing population. There has been dramatic growth in the investments by governments, nongovernmental organizations, international donors and the private sector to develop the scientific expertise and infrastructure necessary to find solutions to the problems that limit African agriculture. The Biosciences Eastern and Central Africa-International Livestock Research Institute (BecA-ILRI) Hub in Kenya and the Mikocheni Agricultural Research Institute (MARI) in Tanzania were created to solve problems facing African farmers and limiting food security. A U.S.-East Africa partnership represents an excellent international opportunity for research synergy and training of U.S. students and early career scientists. Key features include the establishment of a research exchange program between laboratories in the U.S. and East Africa. Postdoctoral researchers, graduate students and undergraduates will be mentored by a strong international research team, which includes experts on viral population genetics, insect vector transmission and population dynamics, virus/vector/plant interactions, and STEM education. The multidisciplinary nature of the research will provide trainees experience in laboratory and field-based research as well as bioinformatics. This will prepare them to become globally engaged, independent scientists with a solid foundation in a range of research methodologies and environments and first-hand experience in international and multidisciplinary collaborations.

This proposal will develop a new approach using viral vectors to express targeted nucleases to introduce site-specific modifications into the cassava genome. The system will use a viral vector based on a cassava geminivirus to express a meganuclease designed to introduce mutations into the cassava gene encoding phytoene desaturase (PDS), the first enzyme in the carotenoid biosynthetic pathway. The bleached phenotype of a PDS mutant will be used to screen for gene knockouts caused by the transient gene editing system and establish its efficacy. The impact of several parameters, e.g. the inoculation method, inoculation site, virus movement and cassava sequences that enhance infection, on the efficiency of the system will be tested. Experiments will also characterize progeny plants for the presence of mutant PDS sequences as well as for viral and meganuclease sequences. These studies will indicate if vegetative propagation of the inocu���������lated cassava increases the efficiency of detecting a site-specific mutation and if progeny plants do not retain viral or meganuclease DNA.

Intellectual Merit In spite of continued strong interest in epigenetic aspects of genome function, we do not yet know very much about the process by which plant and animal cells transmit epigenetic marks through multiple rounds of DNA replication and cell division. Because cell-to-cell inheritance is crucial to the function of epigenetic marks, understanding their biology requires an intimate knowledge of DNA replication as well as tools to study events occurring in S phase. In the proposed project, we will use the tools and knowledge of DNA replication that we developed in our previous PGRP project to characterize epigenome dynamics during S phase in two important model systems, Arabidopsis and maize. We will use combinations of flow cytometry and in vivo labeling to dissect multiple stages within S phase of cells in suspension culture and in planta. Using a genome-wide approach and making extensive use of deep sequencing technology, we will then investigate questions such as, Are post-translational modifications made immediately after replication of a given stretch of DNA? Are there differences in the timing of different types of modifications, or of modification events in different parts of the genome? To what extent does siRNA contribute to heterochromatin replication and inheritance? Do matrix attachment sites define domains with similar replication and modification kinetics? Do the attachment sites change during S phase? In addition, we will carry out experiments with mutants and artificial micro RNA knockdowns to explore the functional consequences of inhibiting selected modification pathways. Broader Impacts Knowledge of epigenome dynamics and the underlying mechanisms of epigenetic inheritance will enhance our understanding of fundamental processes in development and evolution, and will have practical impacts on plant tissue culture and micropropagation, plant breeding, and biotechnology. The effort will bring together investigators with expertise in biochemistry, molecular biology, genetics, genomics, and bioinformatics, and support a productive collaboration between two major research institutions. An excellent training environment for graduate and postdoctoral students will be provided, and selected undergraduates will be given the opportunity to participate in various aspects of the research. We plan two principal outreach efforts, one at each institution. At NCSU, we will continue and expand our successful collaboration with two Granville County Middle School teachers and the North Carolina Museum of Life and Science. We will update the ?Science in a Suitcase? unit on Genetics that we created in our present project, and continue to hold workshops for teaching training. At CSHL, we will inaugurate a program in epigenetics at the Dolan DNA Learning Center. This program will target advanced high school and faculty at two year and agricultural colleges. It will seek to update faculty and provide resources for teaching about epigenetics, and will provide a combination of web materials and podcasts as well as resources for experiments on imprinting and epigenetic inheritance. We will also host a regional workshop at a location to be determined, perhaps in conjuction with a plant biology professional meeting. The program at CSHL is expected to involve staff and PIs from NCSU as well as CSHL personnel.