Linda Hanley-Bowdoin
Professor
William Neal Reynolds Distinguished Professor
Partners Building III 360C
Bio
Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genomes that replicate through double-stranded intermediates. Because of their limited coding capacity, geminiviruses supply only the factors required to initiate viral replication and depend on host DNA polymerases to amplify their genomes. Many geminiviruses replicate in differentiated plant cells that no longer contain detectable levels of host DNA polymerases and associated factors.
Geminiviruses are major impediments to food production in sub-Saharan Africa and south Asia, which together account for more than 60% of the 820M undernourished people worldwide. We are working on peptide aptamers and trans-dominant negative mutants that interfere with viral replication to confer stable, general resistance against these important plant pathogens and collaborating with researchers in Africa and Asia to move the technology into key crops.
In eukaryotes, DNA replication begins at specific sites in their genomes designated as origins of replication. Even though origins have been mapped to specific sequences in yeast, it has proven more difficult to define origins in higher eukaryotes. We are collaborating with other scientists to characterize plant origins of replication and to determine their relationships to matrix attachment regions, DNA methylation sites, recruitment of modified histones and transcriptional activity.
Courses Taught:
- BCH703 (Macromolecular Synthesis)
- BCH/GN761 (Advanced Molecular Biology of the Cell)
Area(s) of Expertise
DNA Replication and Transcription, Geminivirus Infection
Publications
- Cassava begomovirus species diversity changes during plant vegetative cycles , Frontiers in Microbiology (2023)
- Genome segment ratios change during whitefly transmission of two bipartite cassava mosaic begomoviruses , Scientific Reports (2023)
- SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana , FRONTIERS IN PLANT SCIENCE (2023)
- An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing , JOURNAL OF VIROLOGICAL METHODS (2022)
- Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning , SCIENTIFIC REPORTS (2022)
- Vector acquisition and co-inoculation of two plant viruses influences transmission, infection, and replication in new hosts , SCIENTIFIC REPORTS (2022)
- A New Type of Satellite Associated with Cassava Mosaic Begomoviruses , JOURNAL OF VIROLOGY (2021)
- A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants , CELL HOST & MICROBE (2021)
- A protocol for genome-wide analysis of DNA replication timing in intact root tips , Methods in Molecular Biology series (2021)
- Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation , CURRENT BIOLOGY (2021)
Grants
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.
Geminiviruses represent a serious threat to food security in Africa. This proposal is a subcontract of a larger proposal entitled "Disease Diagnostics for Sustainable Cassava Productivity in Africa" submitted by Dr. Joseph Ndunguru of Tanzania. The proposed research will identify a resistance gene for geminiviruses that loses effectiveness in the presence of DNA satellites isolated from cassava. Standard QTL mapping of a segregating population will be used to fine map the gene, followed by targeted disruptions of candidate loci. No resistance gene has ever been identified at the molecular level for a plant DNA virus. This information is expected to help determine the mechanism of action for resistance-breaking DNA satellites and may uncover new ways to safeguard cassava against viruses.
Cassava is an important staple crop in Africa and Asia, where it is eaten by over 700 million people every day. It is grown by subsistence farmers in the poorest villages and is often the only food source when other crops fail or are destroyed by conflict. Cassava can grow under drought, high temperature and poor soil conditions, but its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is caused by a DNA virus complex that includes seven geminivirus species. Two satellites (SatII and SatIII) associated with the complex cause increased viral levels and symptoms. Cassava brown streak disease (CBSD) is caused by a potyvirus that can act synergistically with cassava geminiviruses to en??A?hance disease, but it is not known if SatII and SatIII also interact with CBSD. There has been consider??A?able effort to develop CMD resistant cassava, but the utility of these cultivars has been reduced due to resistance breaking by SatII and their high suscepti??A?bility to CBSD. SatII and SatIII do not resemble other geminivirus satellites and their mechanisms of action are not known. This project will determine how SatII and SatIII are propagated during infec??A?tion and interact with their helper viruses and host, and possibly with CBSV, to enhance disease proc??A?esses. Aim 1 will charac??A?terize the transcription products of SatII and SatIII and ask if they are nec??A?essary for enhanced symptoms and resistance breaking. This analysis will lay the groundwork for Aim 2 that asks if SatII and SatIII act through a protein or a small RNA and how they function during infection. The two satellite show only limited homology, cause different phenotypes, and may act via different mechanisms that will require dif??A?ferent approaches to overcome their activities. Aim 3 asks if SatII and SatIII are encapsidated and trans??A?mitted by whiteflies like their helper viruses and/or are released from the cassava genome during infec??A?tion. Aim 4 asks if the satellites enhance syner??A?gism between CMD and CBSD. CMD has constrained cassava production in Africa for more than a century, but changes in the nature of the disease have led to losses on an unprecedented scale in the last 20 years. Sev??A?eral factors have con??A?tributed to the pandemic, including recombination between cassava geminiviruses resulting in en??A?hanced virulence and the emergence of resistance breaking satellites. The increased incidence of CBSD and its synergy with CMD have compounded the disease losses. In 2005, the total crop losses due to CMD were 4M metric tons/year in Tanzania, Uganda, Rwanda and Burundi, while the estimated losses due to CBSD were between 35-70M US$ in just Tanzania. The impact of SatII and SatIII on cas??A?sava yields has not yet been docu??A?mented, but it is very likely that they will significantly increase losses and lead to food insecu??A?rity in Africa if measures are not taken to contain their effects. To do this, we must first understand how SatII and SatIII enhance disease processes and are transmitted and/or released in in??A?fected cassava fields. The basic research proposed here will increase our knowledge of the mechanism(s) of action and trans??A?mis??A?sion of SatII and SatIII and will enable the rational development of CMD resistance in the context of the sat??A?ellites and CBSD. The research will provide unique training opportunities for a postdoctoral re??A?searcher in the U.S. and a graduate student in Tanzania that include international collaboration as well as bench and field experi??A?ence. The project will prepare the trainees to become globally engaged, independent sci??A?entists who can effectively address future disease problems caused by rapidly evolving plant viruses. The studies will also transfer expertise on geminivirus replication and host interac??A?tions to facilitate future studies on cassava geminiviruses and their satellites in Africa, and increase resources for these types of studies.
Intellectual merit: The TCPs constitute a large family of plant-specific transcription factors that are defined by a conserved basic helix-loop-helix (bHLH) domain. They fall into two classes with Class I TCPs promoting cell proliferation, and Class II members promoting leaf and floral differentiation. TCPs are sequence-specific DNA binding proteins that bind to shared or overlapping motifs as homo- or heterodimers. Given the complexity of the TCP family and its potential to function combinatorially, it is likely to be governed by an intricate array of regulatory mechanisms. The PIs recently showed that a subset of the 24 TCPs encoded by the Arabidopsis genome is phosphorylated in vitro by the GRIK-SnRK1 kinase cascade (Geminivirus Rep Interacting Kinase ?SNF1-Related Kinase). SnRK1 is best known for controlling and coor??A?dinat??A?ing carbon metabo??A?lism and energy balance in plants, but there is increasing evidence that it also plays an essential role in plant development. GRIK, which acti??A?vates SnRK1, only accumu??A?lates in young plant tissues, cultured cells and gemi??A?nivirus-infected cells, all of which replicate plant chro??A?mosomal DNA. The related kinases, SNF1, AMPK and their upstream kinases, have been implicated in the cell cycle in yeast and animals. It has been proposed that SnRK1 controls plant gene expression by phosphorylating transcription factors and altering their activities. The PIs found that Class I TCP bind??A?ing motifs are enriched in SnRK1-down regulated genes, strongly supporting a role for SnRK1 phosphorylation in modulating TCP function in plants. The goal of this project is to characterize the GRIK-SnRK1 target residues of selected TCPs and to examine the consequence of altering their phosphorylation status on TCP function. Aim 1 will use a reconstituted heterotrimeric SnRK1 kinase complex to characterize putative TCP targets and to map the in vitro phosphoryla??A?tion sites by liquid chromatog??A?raphy tandem mass spectrometry (LC/MS/MS) and site-directed mutagene??A?sis. Aim 2 will use a combination of molecular and analytical approaches to determine the phosphorylation status of endogenous and recombinant epitope-tagged TCP pro??A?teins in Arabidopsis suspension cells and plants. Aim 3 will examine the role of TCP phosphorylation by assessing the capacities of TCP phosphomutants to complement TCP mutant phenotypes in plants as well the impact of phosphorylation status on TCP stability, intracellular partitioning and genome-wide occupancy profiles in cultured Arabidopsis cells. Broader impact: The proposed experiments represent an important first step in under??A?standing how TCPs are regulated by phosphorylation and the role that post-translational mechanisms play in modulat??A?ing this unique transcription factor family in plants. They will also iden??A?tify genes that are direct targets of a TCP family member at the whole genome level and lay the groundwork for future studies that compare genome-wide occupancy and protein partners across TCP family members, which are essential for under??A?standing the combinatorial potential of the TCP family. The studies may also uncover new roles for GRIK and SnRK1 in the cell cycle and define very early steps in the integration of energy balance and nutrient availability with cell divi??A?sion, de??A?velopment and geminivirus infection. A better under??A?stand??A?ing of TCP regulatory mecha??A?nisms and the role of the GRIK-SnRK1 cascade in TCP control could lead to the devel??A?opment of new strategies for modulating plant growth, especially under metabolic stress conditions. The breadth of technical approaches incorporated in the proposed research provides a unique training opportunity for undergraduate, graduate and senior researchers. The PIs will work together to foster intellectual and collaborative interactions between their labs to ensure that participants are broadly trained and develop the skills necessary to apply inte??A?grated approaches to complex biological problems. The PIs and participants will also expand upon a successful collaboration with local edu??A?cators and the NC Museum of Li
Emerging plant diseases and pests plague African food crops. The risk of introduction of pests and pathogens with trade between countries requires monitoring and improved diagnostic capabilities for important foods crops in Africa and deployment of resistant varieties. In Phase One we will hold a conference on ?Ecosystem Services and Emerging Infectious Plant Diseases of Africa?. At the conference, we will ask: ?Can the spread of emerging plant pathogens and pests be reduced using ecosystem service impact modeling and transformational technologies?? Plant diseases can impact ecosystem services directly through their effect on plant productivity or indirectly via the management practices imposed. We will focus on the understudied but high economic impact relationships. We will dissect the ecology of the emerging pathogens and their vectors that plague African crops such as cassava mosaic virus, wheat rust (UG99), potato late blight, bacterial, viral and fungal diseases of rice, corn, banana, sweet potato, taro, and tomato and use an ecosystem services model to direct research to mitigate their impacts. We will coordinate an outreach network of US plant scientists including pathologists, entomologists, breeders, ecologists and soil scientists that will provide PhD level training to a cadre of promising African women. Funded assistantships from six land grant universities (NC State, UC Davis, Cornell, Colorado State, Kansas State and Ohio State University) will be sought to kick start the effort with the ultimate goal of 20 deployed scientists in east African countries of Uganda, Kenya, Tanzania, and Malawi by 2020.
ARI-R2: Renovation of the North Carolina State University Phytotron for Improved Environmental Control and BSL-3 Containment. North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art controlled, environmental facility (Phytotron) with containment for investigation of high-risk plant pathogens and other microbes. The four-story Phytotron was built in 1968 with most of the construction (~$1.5M) and first eight years of operating funds provided by the NSF, and 33 years of continued maintenance and staffing provided by NCSU. After 41 years however, the NCSU Phytotron is in dire need of upgrades to meet present and growing demands for controlled environmental research facilities for plants, insects and small mammals. This application proposes critical infrastructural upgrades to (1) renovate the core environmental system by replacing chillers and pumps, connecting to university chilled water lines, re-insulating chilled water and glycol lines, and applying epoxy coating to chamber and greenhouse floors; (2) replace controllers that modulate the environmental conditions for individual environmental chambers, and modernize and increase the precision of CO2 controls; and (3) convert a plant dark room and greenhouse into a BLS-3 facility for investigations with highest risk viral, bacterial, fungal and nematode plant pathogens and select agents. Intellectual Merit. Biologists in the post genome era face a major challenge in determining the function of the thousands of identified genes, many of which are under environmental control. Often, the phenotype of a mutant and the expression pattern of responsible gene(s) can be properly defined only in precise environmental conditions. Renovation of the Phytotron will provide researchers with the large-scale controlled environment capacity to take full advantage of publically accessible genetic tools not only to accelerate information-rich phenotyping in model plants but to translate the same high-throughput screens to crops, wild relatives and non-native species. Control over CO2 concentration will open new opportunities for NCSU researchers involved in global change research and predicting the responses of individual organisms and ecosystems to environmental change. These demands, as well as the growing need for studies of phenotypic plasticity needing reproducible combinations of climatic factors and ecological studies that require the capacity to simulate a variety of environments, make this facility an essential resource for future, competitively funded investigations. Similarly, upgrading a designated area of the facility for BSL-3 containment will provide a research environment and foster international collaborations to better understand the fundamental biology of plant pathogens that already cause severe disease problems in subtropical/tropical regions and pose an increasing threat to the U. S. and global food supply chain. Broader Impacts. The NCSU Phytotron is an outstanding testament to the value of investing in research infrastructure. This NSF-funded legacy is a valuable University-wide facility and focal point for training and research at all levels. The infrastructure renovations proposed here will allow this unprecedented resource to continue to fulfill this mission by becoming a high-throughput public facility for phenotypic analyses of many different plants. Graduate and undergraduate students from NCSU, summer REU programs involving students from non-Research I universities and other outreach programs for underrepresented groups use the Phytotron for independent research projects under the direction of a diverse representation of faculty who serve as role models and mentors. The renovations will have immediate scientific impacts as NCSU researchers can acquire pathogens and microbes from collaborators around the world by having a centralized facility inspected and known to meet or exceed containment guidelines. Unemployment in the State of North Carolina is over 11% and above the national average.