Zachary Lippman - US grants

PI: Zachary B. Lippman (Cold Spring Harbor Laboratory)
Collaborator: Dani Zamir (Hebrew University)

The agricultural yields of the world's major crop plants have increased significantly in the last century, thus providing food for an ever-growing population. This was made possible primarily through the century-old discovery that crossing different poor-yielding inbred varieties of corn causes a remarkable increase in yield among the inter-crossed, or hybrid, plants. Such hybrid vigor, termed "heterosis", has since become a foundation for crop breeding and a focus of intense research. While studies have revealed important generalizations about what controls heterosis, the individual genes and molecular networks responsible have still not been identified. This project will test a new hypothesis, namely that a mutation in a single gene, which is seemingly detrimental to plant growth, can cause heterosis when hybridized back to a normal non-mutant plant. The resulting partial gene activity in the "mutant hybrid" results in a new state of growth that is beneficial to yield through a genetic phenomenon called "dosage". Preliminary research in tomato has already identified multiple mutant hybrids that increase fruit production. Based on these findings, the goals of this project will be to: i) search for additional tomato mutants that show heterosis when hybridized with normal plants, ii) characterize the changes in growth (e.g. branch number, flower production) that are the basis for heterosis in two previously identified mutant hybrids, and iii) study novel changes in the activity of all tomato genes in mutant hybrids. Unlike previous studies, this project will be able to link heterotic effects of a single gene with specific changes in growth and relevant changes in gene activity. This new knowledge can then be harnessed to identify genes causing heterosis in other crop species.

To promote educational outreach about heterosis and the principles of this project to the general public, a teaching program about plant breeding and food production has been developed. Specifically, a three-part lesson explaining the processes by which plants make flowers, fruits, and seeds has been developed for an elementary school with an underrepresented student population in Queens, New York. Live plants from multiple crops will be used to illustrate the dramatic changes in growth that occur as plants switch from making leaves to making flowers. Discussion sections will emphasize how the environment interacts with plants to stimulate flower and fruit production, and how plant breeders leverage this knowledge to develop new crop varieties. It is anticipated that this outreach project will serve as a model for future programs that will target additional schools in New York City. Finally, all data generated in this project will be made publicly available through the Solanaceae Genomics Network (http://sgn.cornell.edu/). In addition, seeds from mutant hybrids can be requested from Cold Spring Harbor Laboratory (http://www.cshl.edu/public/SCIENCE/lippman.html) and from the Tomato Genetics Resource Center (http://tgrc.ucdavis.edu/).

A Research Experience for Undergraduates (REU) Sites award has been made to Cold Spring Harbor Laboratory (CSHL) that will provide research training for 8 students, for 10 weeks during the summers of 2012- 2014. The program trains participants on the present and growing need to integrate biological research with sophisticated computational tools and techniques. CSHL has over 40 faculty members, including members of a newly established Quantitative Biology Department, who will serve as bioinformatics and computational biology mentors in fields ranging from plant biology to machine learning for biology. Through this NSF-REU support, students are afforded the opportunity to conduct full-time research in an appropriately matched lab based on mutual interests and goals. CSHL REU participants have access to individual and shared laboratory facilities such as flow cytometry, high throughput sequencing and analysis, imaging, and proteomics facilities. Participants attend multiple seminars and workshops, such as the responsible conduct in research, professional communication skills, the graduate school application process, and introduction to science careers. REU participants also are invited to attend the CSHL summer courses or meetings, which cover a range of topics such as Computational Neuroscience and Single Cell Analysis. All students are housed on campus within walking distance of their laboratories and the CSHL cafeteria, where they receive the majority of their meals. The multilayer recruitment effort consists of both traditional and digital mailings to potential students and their professors, as well as recruitment visits to universities throughout the country. Students are selected based on academic record, motivation for the proposed program of study, and potential as future researchers. Alumni successes are monitored to determine their continued interest in their academic field of study, their career paths, and the long-term impact of their research experience. Information about the program will be assessed using faculty and student evaluations, as well as the use of an REU common assessment tool. More information is available by visiting http://www.cshl.edu/education/urp/nsf-sponsored-reu-in-bioinformatics-and-computational-biology, or by contacting the PI (Dr. Zachary Lippman at lippman@cshl.edu) or the co-PI (Dr. Doreen Ware at ware@cshl.edu).

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Funds are provided for Cold Spring Harbor Laboratory (CSHL) to renovate and upgrade existing plant growth facilities at our Uplands Farm Research Field Station. CSHL has an extensive greenhouse complex offsite where seminal work in Arabidopsis and crop plants has been conducted. The research focus on crop yield and adaptation of crops to substandard conditions has global impact and this will increase with these improved facilities. The greenhouses and growth rooms are used to support the research programs and also support the plant genetics teaching programs of the CSHL Dolan DNA Learning Center. These aging and outdated facilities are inadequate to meet the demands of current genome driven plant biology research. The infrastructural improvements will provide appropriate growing conditions for a greater diversity of plant species and will increase the energy efficiency of the facilities. In the proposed renovations, CSHL intends to:
1 Replace boilers in two smaller greenhouses, and two boiler burner units in the largest of the three greenhouses.
2 Install updated evaporative cooling units in each of the three greenhouses.
3 Install automated ventilation, sunshade, and irrigation systems in all three greenhouses.
4 Replace the aging, hazed acrylic sheathing on the largest greenhouse.
5 Renovate and improve the head house of the largest greenhouse.
6 Replace the cooling unit in the Field Station Laboratory with a modern and more efficient unit with an economizer ventilation unit.
7 Replace outdated lighting fixtures in the Arabidopsis growth facilities with improved, high-efficiency units and install a dedicated heating loop.

PI: Zachary B. Lippman (Cold Spring Harbor Laboratory)

Co-PIs: Michael C. Schatz (Cold Spring Harbor Laboratory) and Joyce Van Eck (Boyce Thompson Institute for Plant Research)

Key Collaborators: Molly Hammell and Jesse Gillis (Cold Spring Harbor Laboratory)

Plants show remarkable variation in the number of flowers they produce during their lifetime. This widespread variation traces back to differences in how, when, and where plants switch from making leaves to making flowers - the flowering transition. Although vitally important to crop yields, the transition to flowering and the subsequent effects on shoot growth and flower production remain poorly understood in many types of plants. For example, it is still not known why one plant will form just a single flower each time there is a flowering transition, as in pepper, and yet another plant will grow dozens of branches bearing hundreds of flowers, as in some types of tomato. To address this fundamental question in plant biology, this project is uniting a unique set of genetic, genomic, and natural variation tools in tomato and related Solanaceae plants, such as pepper, potato, and petunia, to reveal the genes and networks controlling how, when, and where plants undergo flowering transitions throughout development to continuously generate new branches and flowers. By analyzing a wide range of tomato mutants and wild Solanaceae species reflecting a wide range of flower production, this research will identify and characterize the differences in gene expression and DNA sequences that underlie variation in flowering transitions and flower production. This multi-dimensional project will provide the most detailed information yet on the key genetic regulators that drive the initiation and production of flowers in both agricultural and wild plants, which will enable the application of novel strategies to improve crop yields. The Solanaceae comprise the most valuable family for vegetable crop production, and we will deliver to both the public and scientific community broad genetic and genomic data in tomato, pepper, and edible wild Solanaceae species that have the potential to become agriculturally important crops.

This project will train high school and college students in interdisciplinary plant research, and a unique outreach program has been developed with an elementary school in Queens, New York to excite young students about plant biology and to explain the importance of integrating multiple research disciplines to create the knowledge and tools that will ensure food security. Students will meet scientists, experience plant genetic research in their own school, experiment in a "Virtual Greenhouse" with kid-friendly genetics games, and practice science writing. Each year, several students will be awarded a daylong visit to CSHL to experience firsthand, modern plant biology research. All data from this project, including gene expression, genetic mapping, network analyses, and computational tools for analyzing DNA sequences will be made publically available immediately after passing quality control. All DNA sequence data will be deposited in Genbank (http://www.ncbi.nlm.nih.gov/Genbank/), the SOL Genomics Network (SGN) website (http://www.sgn.cornell.edu/), and a project web site that will be developed.

In both nature and agriculture plant productivity depends on flowers, which are the foundation for fruits and seeds. Depending on the plant, the number of flowers that form on reproductive branches, known as inflorescences, can vary substantially. Discovering the genes responsible for this species-specific diversity, and understanding how these genes work together to control flower production, is a major focus in plant biology with direct relevance for crop improvement. In tomato and its close relatives in the nightshade (Solanaceae) family, such as eggplant, pepper, and potato, flower production on each inflorescence varies dramatically, from a solitary flower on a single branch, as in pepper, to dozens of flowers on many branches, as in several wild tomatoes. This project will take advantage of variation in flower production found in tomato to study a group of genes that are required for generating multi-flowered inflorescences, and thus the familiar "tomatoes-on-the-vine" architecture characteristic of all tomato varieties. By mutating these genes using new gene-editing technology, it will be possible to dissect how the proteins encoded by these genes control precisely when, where, and how many flowers and fruits are produced on each tomato plant. Results from this project should reveal new flower-production genes and their modes of action, which can then be targeted for modification using both classical and modern genetic tools to improve yields in tomato and many related crops. Additionally, an outreach program at an inner-city middle school will educate young students on the process of genetic engineering to help shed popular misconceptions about genetically modified food.

All above ground plant growth originates from shoot meristems, small populations of stem cells that give rise to vast architectural diversity, particularly in inflorescences. At the heart of this diversity lies a critical, yet poorly understood, process of meristem maturation. A major question in plant development is how timing of meristem maturation, and thus inflorescence and flower production, is controlled in different plants. Compared to knowledge on meristem maturation in other model plants, much less is known in tomato and related Solanaceae, despite representing the widespread sympodial growth habit. Recent work in tomato has exposed a new maturation program, defined by a novel transcriptional regulator encoded by the TMF gene. This project integrates genetics, genomics and biochemistry to study the mechanisms by which TMF and its interacting protein partners regulate meristem maturation. In Aim 1, TMF transcriptional co-factors will be studied genetically, molecularly, and developmentally. In Aim 2, transcriptional targets of TMF and its expression network will be explored by integrating RNA-seq and ChIP-seq. In Aim 3, TMF family members will be characterized using CRISPR/Cas9. This project comprises a first molecular mechanistic study to understand how meristem maturation is fine-tuned to quantitatively control flower production in sympodial plants. The findings should reveal new principles of meristem maturation that can enable modulation of inflorescence architecture and flower production to benefit agriculture.

Plant genome research over the past 20 years has provided a deep understanding of genetic pathways that underlie economically important processes in crop plants. However, as in most organisms, many plant genes have "backup" copies, or duplicates representing genetic redundancy. Very little is known about the effect of such redundancy on plant improvement efforts. This lack of knowledge complicates the efficient use of genetic resources. This project will focus on a known group of signaling genes to understand the basic principles that underlie genetic redundancy in plants. It will therefore advance knowledge in a fundamental area of plant genome biology. Outcomes from this project will have the potential to bring improvements to US agriculture by providing new knowledge and tools to develop high yielding crops. The project will also train a number of young scientists at various levels, as well as promote outreach and education in plant genomics. Project personnel will develop new teaching modules to highlight the importance of plant genomics in crop domestication, and will present these in schools and workshops that target female students and underrepresented minorities. Outreach activities will also target rural farming communities in the New York, Massachusetts and North Carolina areas, where open house displays and lab visits will be used to educate these groups about the importance of plant genomics research in agriculture.

This project asks how redundancy in signaling pathways has evolved across the plant kingdom. It will develop a genome-level understanding to link genes and pathways to complex phenotypes, by testing the hypothesis that genetic redundancy in plants is controlled by Responsive Backup Circuits (RBCs). A second hypothesis to be tested is that signaling network outputs can be modulated and exploited using weak promoter alleles. Three species will be used, the model system Arabidopsis, to rapidly test hypotheses, and tomato and maize, divergent and economically important crop species. Genetic redundancy is a major limitation to the ability to link genes to phenotypes in plants, and this project will use a subset of Leucine Rich Repeat Receptor Like Kinases and their predicted ligands as a model network. Signaling genes selected by phylogenetic analysis will be targeted for knockouts using genome editing technologies (CRISPR/Cas9). Genome-wide transcript profiling will then be used to deduce redundancy mechanisms and reiteratively design new knockouts to address the effect of disrupting redundant paralogs. At each stage, careful phenotyping will be used to understand the effect of multiple gene knockouts at different developmental stages relevant to crop productivity. Redundancy in gene regulatory sequences (promoters) will also be addressed by developing a generalizable CRISPR/Cas9 multiplex knockout strategy to make semi-random mutations across gene regulatory sequence regions. These lines will be screened en masse, and represent a new approach to mutagenesis in plants, with a potential to generate new genetic diversity, and to recover weak alleles with enhanced yield traits.

Genome DNA sequences for many crops have been determined in the last two decades, providing the blueprints to discover genes that underlie key agricultural traits. However, a great challenge is identifying the differences in DNA between related varieties of the same crop, which are responsible for the subtle trait variation that plant breeders exploit to improve productivity. A major contributor to this trait variation is 'genome structural variation' where pieces of DNA are deleted, inserted, or rearranged resulting in changes in gene expression. This project will focus on how structural variation contributed to domestication and breeding of tomatoes. A related goal is to expand and develop new molecular tools to create structural variation for crop improvement. This project will improve US agriculture by providing new knowledge and tools to efficiently and predictably enhance crop productivity. A major part of the project will also include training of young scientists in fundamental principles of plant genome research that can be applied to agriculture. This knowledge will also be shared through outreach programs in inner city New York schools that do not have access to research opportunities. Project personnel will develop hands-on teaching activities that will highlight the importance of plant genomics and new genome editing technologies to improve crops and meet the agricultural needs of the 21st century.


Limited knowledge on the extent and diversity of structural variation in plant genomes is hindering the ability to link genes to important crop phenotypes. This project will unite new long-read sequencing technologies, computational biology, developmental and quantitative genetics, and genome editing to elucidate and manipulate structural variation (SV) at a scale never before achieved for a major crop. Tomato provides a powerful system due to its relatively small and high quality reference genome and availability of resequenced genomes. By applying SV-detection algorithms to existing short-read Illumina sequencing data from hundreds of accessions, more than 40 genomes will be selected, capturing the majority of predicted SV diversity, to establish new reference genomes using the latest long-read sequencing technology (PacBio and 10X Genomics). From these data, a compendium of validated SVs will be generated and integrated with ongoing genome-wide association studies. Significant gene-associated SVs, including those affecting gene activity measured by genome-wide transcript profiling, will be characterized using CRISPR/Cas9 gene editing and quantitative phenotypic analyses, focusing on reproductive traits that drive crop productivity. In parallel, CRISPR/Cas9 gene editing will be used to generate a collection of SV mutations in known yield and fruit quality genes in two related wild Solanaceae with agricultural potential, with the goal of achieving major steps towards domestication and for comparative developmental genetics studies. This project will greatly expand our knowledge of genomic diversity in tomato, and provide a road map for dissecting SVs in other crops, where such knowledge can be exploited to improve productivity.

Crop plants provide food, feed for livestock, and other essential materials. Breeders are continuously improving our crops; however, there is an urgent need to accelerate crop improvement in the face of climate change and limited resources. Natural genetic variation in the form of DNA mutations is widespread in crops, and is the starting material for their improvement, but such variation is often not useful or is unpredictable in its effect on plant growth. Genes that control important yield traits are expressed at specific levels, locations and times during plant growth, and tuning these expression programs may enhance crop productivity. Gene expression is controlled by regions of DNA surrounding genes known as cis-regulatory elements. Despite their fundamental biological significance, the identification of such elements and their use in agriculture has been challenging. This research project, a collaborative effort between scientists at Cold Spring Harbor Laboratory, the University of Massachusetts-Amherst, and the Hebrew University of Jerusalem, will predict regulatory elements using a newly developed computational algorithm, Conservatory, combined with existing genome sequences from many plant families. These elements will then be modified using CRISPR genome editing tools. These new variants will be tested for changes in phenotype that lead to improvements in yield and other important agronomic traits. The project will train young scientists at various levels, as well as promote outreach and education in plant genomics in partnership with Genspace, a Community Biology lab in Brooklyn, NY. The project will develop a new curriculum for high school students from under-resourced Title I schools and demographic groups historically excluded from the life sciences to explore applications of CRISPR in agriculture, including hands-on labs in plant transformation and CRISPR editing.

This project will test the hypothesis that genes with conserved functions are regulated by deeply conserved cis-regulatory elements (CREs) across angiosperms, and that characterizing these CREs will provide a new level of understanding in linking genotype to phenotype. The project will exploit the recent explosion in high-quality sequenced genomes to identify conserved regulatory elements across angiosperm diversity using the Conservatory algorithm. The functions of the elements identified by Conservatory will be tested by precise genome editing, with a focus on developmental regulators and architectural traits. Functional dissections will be performed in two species in each of three diverse plant families, spanning eudicots and monocots, which will allow the assessment of CRE functional evolution over shallow and deep timescales. The catalog of conserved regulatory elements identified, and the editing strategies developed to test their functions, will reveal fundamental principles governing gene expression control and will accelerate innovative approaches to fine-tune crop productivity traits. Critically, the tools, techniques and fundamental principles emerging from this multi-disciplinary project will comprise a valuable community resource, enabling the engineering of diverse systems and phenotypes, such as biotic and abiotic stress tolerance, nutritional quality, and symbiosis. All project outcomes will be widely accessible through long-term public data and genetic repositories.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The growing population and climate extremes are threatening food security. Agriculture is largely based on a few major crops, and revolutionary technologies in genome sequencing and CRISPR genome engineering are accelerating their improvement. These technologies can also improve “orphan” crops, which are not widely cultivated or studied but have the potential to increase the diversity and resilience of food production. Orphan crops are related to major crops, allowing translation of knowledge between them. However, orphan crops lack research tools, and an even greater challenge is determining whether specific genetic mutations that benefitted major crops can be engineered to improve traits similarly in orphan crops. This is because gene sequence and function change as species evolve, especially among genes that become duplicated, which is common in plants. This project will take advantage of the nightshade family – a source of many major and orphan crops, such as eggplant, pepino, and tomato – to study how duplicated genes evolve and affect agricultural traits in related species. Combining genome sequencing and CRISPR will reveal sequence diversity among thousands of duplicated genes and enable improved predictability in engineering genes and traits across species. This project will train young scientists with a focus on diversity and inclusion, as well as promote public understanding of genome engineering in plant biology through a community science program on orphan crops. Finally, new curricula and research opportunities for undergraduate students at a small liberal arts college will broaden participation and training of underrepresented groups in the plant sciences.<br/><br/>This project will exploit advances in large-scale reference genome sequencing, gene co-expression analyses, and CRISPR genome editing to dissect how paralog diversification impacts species-specific phenotypes in a genus of both fundamental and applied importance. Fifty Solanum species, including 16 orphan crops, will be sequenced to establish a Solanum Pan-Genome with telomere-to-telomere reference assemblies, providing a foundation for genus-wide comparative genomics and functional genetics. Computational approaches based on genomics data will be developed for precise assembly and comparison of complex genomes, and identification and classification of paralogs and their relationships based on their variants and expression patterns. Simultaneously, transformation protocols and genome editing will be developed and deployed for an array of Solanum to test how paralogs impact genotype-to-phenotype relationships within and between species. By focusing on major domestication gene families and the adaptation and productivity traits they control, this synergistic work will provide both a new understanding of paralog diversification in evolution and a more robust translation of agriculturally relevant genotype-to-phenotype relationships to orphan crops. Beyond a valuable community resource of Solanum reference genomes, expression data, and CRISPR lines for plant researchers and breeders, this multidisciplinary project will result in new tools, resources, and principles that will enable the study and engineering of other taxa and traits of significance to both plant biology and crop improvement.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.