Jose Alonso

Overview. Changes in gene expression are at the core of many biological processes, from forming a multicellular organism from a fertilized egg to surviving pathogen attacks or coping with environmental pressures. Although transcription regulation plays a critical role in modulating gene expression, growing lines of evidence indicate that gene-specific regulation at the translational level is also critical for many of these important biological processes. Unfortunately, the existing technologies to quantify changes in translation, both at genome-wide and single-gene levels, are technically demanding and costly, thus hindering the widespread investigation of this type of regulation. The development of technologies that make the quantification of translation efficiency routine has the potential to transform the field of gene regulation, allowing for the discovery of many more processes and genes regulated at the translational level. This, in turn, will open new opportunities to manipulate gene expression for both basic and applied purposes. Currently, the most widely used approach to determine the translation level of a gene is the expensive and technically demanding ribosome profiling (aka Ribo-seq) which involves quantifying the levels of each transcript and the corresponding number of associated ribosomes. We argue that this information could also be obtained by a much simpler process of determining the position of just the first or last ribosome (most 5��� or most 3���) in each transcript and then comparing the distribution of these first or last positions between two different experimental conditions. Although in principle, there is no conceptual reason to think that this Ribosome Position Inference (RiboPI) approach would not work, critical technical unknowns make this a high-risk, high-reward proposal. Intellectual merit. The main objective of this proposal is to develop an efficient, simple, and scalable RiboPI technology to quantify translation rates at both genome-wide and single-gene levels. If successful, RiboPI will make translation regulation information as accessible as RNA-seq did for transcriptomics, reducing the cost and time requirements, the complexity of the experimental procedures, and the amount of biological material needed. Not only will this make translation analysis a routine technique in many labs, but it could also bypass some of the limitations of the current technologies, such as the difficulty of mapping the very short ribosome footprints to specific splice variants, alleles, or even homeologs in polyploid species, or enable targeted studies for a group of genes. To achieve this goal, we propose to develop RiboPI, an experimentally simple approach to capture the position of the first or last ribosome in each transcript and computational methods to compare the distribution of these ribosome positions between different experimental conditions to estimate translational levels from this information. The proposed experimental pipeline involves testing novel combinations of in vivo and in vitro molecular biology procedures to efficiently and specifically map the first/last ribosome in a transcript. Some of the unknowns that make this proposal high-risk are (1) the uncertainty of whether suitable experimental conditions can be found (e.g., that preserve ribosome binding and promote reverse transcriptase activity but melt the secondary structure of mRNA) and (2) the ability to infer the efficiency of translation from the distributions of first/last ribosomes on transcripts. By comparing results obtained with classical Ribo-seq to those obtained with RiboPI, we will be able to determine the reliability of the new approach. Broader impacts. In addition to the clear benefits of developing a new experimental approach to quantify gene-specific translation efficiency and popularizing this type of analysis, this project will provide an ideal training platform for undergraduate students to experience first-hand the translation of basic biological knowledge into potentially transformative new technologies.

Title: Transcriptional and translational regulatory networks of hormone signal integration in tomato and Arabidopsis. PI: Jose M. Alonso (Plant Biology, NCSU), Co-PIs:Anna Stepanova (Plant Biology, NCSU), Steffen Heber (Computer Science, NCSU), Cranos Williams (Electric Engineering, NCSU). Overview: Plants, as sessile organisms, need to constantly adjust their intrinsic growth and developmental programs to the environmental conditions. These environmentally triggered ����������������adjustments���������������� often involve changes in the developmentally predefined patterns of one or more hormone activities. In turn, these hormonal changes result in alterations at the gene expression level and the concurrent alterations of the cellular activities. In general, these hormone-mediated regulatory functions are achieved, at least in part, by modulating the transcriptional activity of hundreds of genes. The study of these transcriptional regulatory networks not only provides a conceptual framework to understand the fundamental biology behind these hormone-mediated processes, but also the molecular tools needed to accelerate the progress of modern agriculture. Although often overlooked, understanding of the translational regulatory networks behind complex biological processes has the potential to empower similar advances in both basic and applied plant biology arenas. By taking advantage of the recently developed ribosome footprinting technology, genome-wide changes in translation activity in response to ethylene were quantified at codon resolution, and new translational regulatory elements have been identified in Arabidopsis. Importantly, the detailed characterization of one of the regulatory elements identified indicates that this regulation is NOT miRNA dependent, and that the identified regulatory element is also responsive to the plant hormone auxin, suggesting a role in the interaction between these two plant hormones. These findings not only confirm the basic biological importance of translational regulation and its potential as a signal integration mechanism, but also open new avenues to identifying, characterizing and utilizing additional regulatory modules in plants species of economic importance. Towards that general goal, a plant-optimized ribosome footprinting methodology will be deployed to examine the translation landscape of two plant species, tomato and Arabidopsis, in response to two plant hormones, ethylene and auxin. A time-course experiment will be performed to maximize the detection sensitivity (strong vs. weak) and diversity (early vs. late activation) of additional translational regulatory elements. The large amount and dynamic nature of the generated data will be also utilized to generate hierarchical transcriptional and translational interaction networks between these two hormones and to explore the possible use of these types of diverse information to identify key regulatory nodes. Finally, the comparison between two plant species will provide critical information on the conservation of the regulatory elements identified and, thus, inform research on future practical applications. Intellectual merit: The identification and characterization of signal integration hubs and cis-regulatory elements of translation will allow not only to better understand how information from different origins (environment and developmental programs) are integrated, but also to devise new strategies to control this flow for the advance of agriculture. Broader Impacts: A new outreach program to promote interest among middle and high school kids in combining biology, computers, and engineering. We will use our current NSF-supported Plants4kids platform (ref) with a web-based bilingual divulgation tools, monthly demos at the science museum and local schools to implement this new outreach program. Examples of demonstration modules will include comparison between simple electronic and genetic circuits.

In the last two decades, biological research has had an emphasis on deciphering the sequence of whole genomes and on starting to identify the genetic variants responsible for the phenotypic diversity in plant and animal species. This information, together with the development and constant improvement of genome editing techniques, is making a profound impact not only on the way researchers approach fundamental biological questions, but also on how this basic knowledge can be translated into agricultural and medical practical applications. At the foundation of the current genome editing approaches is the ability of a cell to precisely replace/repair a given sequence in the genome with a repair template sequence that shares some, but often not all, of the same sequence by means of a process called homologous recombination (HR). In some organisms, such as S. cerevisiae, the high innate rates of HR can be harnessed by researchers to introduce precise changes in the genome sequence. In most organisms, however, the natural rates of HR are too low to be of practical use in genome editing. To bypass these limitations, several methods to enhance the rates of HR have been developed expanding genome editing to a wide range of organisms. One such way to enhance the rates of HR is by means of introducing double-stranded (ds) DNA breaks in the proximity of the sequence to be modified in the genome. With the recent development of easy-to-program nucleases such as Zinc finger and TALE nucleases and the CRISPR-Cas based systems, these types of approaches have gained popularity among researchers. There are, however, other strategies to enhance HR that do not rely on introducing dsDNA breaks in the genome. Among these approaches, one of the most widely used methods is the Lambda-Red system based on the expression of a set of proteins from the bacteriophage Lambda. Although this system has proven to be extremely efficient, so far, its application has been restricted to bacterial systems.

The main goal of this project is to generate a series of optimized synthetic transcriptional reporters to simultaneously monitor the activity of up to nine major plant hormones (auxin, ethylene, ABA, cytokinins, gibberellins, brassinosteroids, salicylic acid, jasmonate, and strigolactones) in a single plant. Single-hormone synthetic reporters (e.g., DR5 and EBS) have been shown to work across a broad range of plant species, making the proposed new tool useful for many plant species, from Arabidopsis to tomato and maize. By applying the synthetic biology principles of standardization and reusability to all ����������������genetic parts��������������� (e.g., synthetic minimal promoters, CDSs, or terminators) and ����������������modules��������������� (whole transcriptional units, or TUs) generated, these new tools will be highly customizable and upgradable whenever new fluorescent proteins or promoters become available. Thus, in addition to producing a single-locus multi-hormone reporter, this project will also: A) popularize synthetic biology tools, such as the GoldenBraid1 (GB) gene assembly technology, among plant biologists; B) streamline rapid and quantitative pipeline to evaluate the function of individual genetic parts and modules; C) test the limit on the number of genes that can be routinely monitored simultaneously by ����������������generic��������������� labs not specialized in imaging techniques; D) explore new approaches that combine CRISPR-Cas9 and recombineering to stack multiple genes (up to 150 Kb) in a single TAC clone.

Auxin is an essential plant hormone that participates in the regulation of nearly every aspect of plant life cycle, from embryo development to meristem maintenance, and from defense against pathogens to shade avoidance. Despite the key role of auxin, the biosynthetic pathways that plants utilize to produce this hormone are largely unknown. In fact, none of the several proposed routes of auxin biosynthesis have been elucidated in their entirety and, in most cases, just a single gene of a multistep pathway has been identified. Thus, for example, the IAM pathway is defined by the AtAMI1 gene, the IPyA pathway is defined by the TAA1 gene family (and now also the YUC family), etc. Despite major gaps in our understanding of auxin synthesis in plants, it is firmly established that indole-3-acetic acid (IAA), the prevalent form of auxin in plants, can be produced from the amino acid tryptophan (Trp) or from the Trp biosynthetic intermediate indole-3-glycerol phosphate. Trp, on the other hand, in addition to serving as a precursor in the IAA biosynthesis, is also an essential building block for proteins, as well as the precursor for a number of defense compounds such as indole glucosinolates and camalexins. Trp itself is synthesized via the shikimate pathway along with two other aromatic amino acids, phenylalanine (Phe) and tyrosine (Tyr). Phe and Tyr, like Trp, serve as the precursors to a large array of secondary metabolites including anthocyanins, flavonols, lignins, etc. Thus, one can imagine the auxin biosynthetic pathway as one of the many final branches of the large metabolic tree of the shikimate pathway. This raises another important question about auxin biosynthesis, i.e. how do plants coordinate the activity of all these different metabolic routes that feed on common precursors? Supposedly, this is achieved by a refined (and yet unknown) mechanism that coordinates the activity of the different metabolic branches that comprise the shikimate pathway and that operates at the cellular level. The current proposal will focus on addressing two open questions in auxin biology. What are the genes that compose the different auxin biosynthetic routes? And, how are the auxin biosynthetic pathways coordinated within the large metabolic network in which they are embedded? To address these key questions, we will focus on the following two objectives. (1) We will systematically examine the proposed indole-3-pyruvic acid (IPA) independent routes of auxin biosynthesis. Several genes previously implicated in key steps of these routes will be characterized using a combination of genetic and biochemical approaches to re-evaluate their role in auxin biosynthesis. Furthermore, to shed light on the unknown components of the IPA-independent routes of auxin production, novel genetic and chemical screens will be conducted. (2) We will also define, at cellular resolution, the regulatory network responsible for the coordinated activity of the different branches of the shikimate pathway. This will be achieved by monitoring the spatial and temporal expression of a set of 84 genes from selected branches of the shikimate pathway. Perturbation of the system using pharmacological means will be used to identify the interconnection between the different components of the network.