Cellular signaling is a system of communication between cells and their surroundings which governs basic cell functions, detection of pathogens and other environmental stressors. In my lab, we focus on the study of cell signaling in plants. Signaling relays on pathways composed of proteins with enzymatic functions able to assemble in complexes and transmit signals over long distances within the cell. We aim to discover stress-triggered signaling pathways in several plant experimental models, to understand their functions and mechanisms of regulation. For this, we employ large-scale approaches to obtain information on proteins, such as interactions or chemical modifications, and assemble this information in signaling networks. Individual network components are selected and studied using classical, low-throughput molecular biology and biochemistry tools. This hybrid strategy helps us understand the overall architecture of cellular signaling pathways and how discrete errors in this system impact signal transmission and cellular responses. Our work has tremendous practical potential, as it may lead to engineering synthetic signaling pathways in plants, for higher tolerance to diseases and environmental stressors.
Area: Stress-activated signaling pathways: mechanisms and regulation
Receptor-mediated immune signaling is modulated by reticulons which facilitate receptor transport to the cell surface. Receptors associated with the plasma membrane (PM) are the primary elements in signal transduction pathways that activate the immune response after recognition of diverse pathogen-associated molecular patterns (PAMPs). The mechanism for receptor transport at the PM is a critical yet understudied aspect of plant immune receptors in particular, and of the PM-associated proteins in general. It has been largely unknown how receptors reach their active sites and to what degree the transport process impinges on a key receptor feature: competency in activating forward signaling pathways. In a recent study, we have used several known immune receptors as probes for protein microarrays (PMAs) to gain insights into their function. We discovered that FLS2 interacts with two reticulons (RTNLBs) and that FLS2-mediated PAMP-triggered immunity is under the regulation of reticulon-mediated transport pathways. These results led to a model whereby intracellular transport of receptors to the cell membrane is a key aspect in receptor biogenesis and a determinant of the receptors’ optimal functionality at the cell surface. Although the functions of the endoplasmic reticulum (ER) in receptor maturation were known at the time, we were the first to identify molecular components that mediate receptor transport out of the ER as part of their maturation, and to show that receptor exit from the ER is a critical factor in the trafficking and signaling activity of immune receptors (Lee et al., 2011; Popescu, 2012).
A novel signaling pathway, activated by biotic or abiotic stimuli, modulates early cellular stress responses. We continue to be interested in the early aspects of immune signaling and its connections with the PM- and/or ER-associated proteins. Our previous microarray screen for calmodulin targets identified a member of the integrin-linked kinase family (ILK1) as a candidate interactor of two calmodulins known to function in plant response to multiple types of stress (Popescu et al., 2007b). These results positioned ILK1 gene as a possible component of a shared biotic/abiotic stress response pathway. ILK1, a component with roles in response to multiple stressors, is an ideal molecular target for engineering stress tolerance in crops. A provisional patent on ILK1was filed through BTI (Popescu and Brauer, 2012).
Mechanisms for the manipulation of kinase-mediated signaling by phytopathogens. We are applying the knowledge and reagents we have accumulated using Arabidopsis as an experimental model, to study signaling in plants with economic importance. We have begun work in this direction as part of a large collaborative endeavor (NSF-Plant Genomes). Understanding kinases’ functions in tomato is important from a basic research perspective; however, it also has wider implications as it will provide a key to deciphering signal transduction networks in Solanaceae. From the perspective of my lab’s research interests and our aims within the project, the systematic study of Solanaceae kinases has been limited by the lack of a solid base of appropriate reagents. To address this, we have developed a set of resources (Singh et al., 2014) including (i) a high-confidence tomato kinome, (ii) an ORFeome (ToKn) containing over 300 tomato kinase cDNAs cloned in a recombination-based vector, and (iii) a large-scale quantitative method for detecting protein-pathogen molecular interactions in live tomato cells. Using these resources, we are moving towards identifying in vivo kinase targets of pathogen effectors and performing mechanistic studies to understand the function and regulation of kinase-effector interactions in tomato. Our resources facilitate functional studies in Solanaceae by my team and other laboratories in the field.
Cellular proteolytic pathways and salicylic acid-mediated signaling. As part of a large collaborative project (NSF 2010) we identified proteins that are part of immune signaling pathways regulated by salicylic acid (SA). Classical methods for isolating SA binding proteins (SABPs) routinely miss possible candidates due to low abundance and transient or weak binding of SA. To circumvent these difficulties, my team has developed a PMA-based method to detect SABPs (Moreau et al., 2013). We have screened ~5,000 Arabidopsis proteins and identified a set of chloroplast-localized SABPs with proteolytic functions, including the TOP1/TOP2 thimet metallo-endopeptidases and a meprin-like MATH domain protein (MAT1). Even though enzymes in SA biosynthetic pathways are known to be chloroplast localized and SA was shown to accumulate first in chloroplasts early after pathogen infection or other stress treatments, there were no characterized chloroplast SA targets previous to our work. Overall, our results suggest that SA might exercise its functions by binding to more targets than previously considered and, importantly, that SA regulates proteolytic pathways. Currently, we focus on key questions such as the cellular roles of TOP-generated peptides, their contributions to stress-activated signaling.
Area: Systems biology of stress and predictive modeling of stress-activated signaling networks
TOP-mediated regulation of cellular stress response. Signaling networks are complex systems containing large numbers of components that establish diverse relationships and feedback connections. As a consequence, signaling networks are resilient to perturbations and non-linear in response (Stelling et al., 2004; Tsuda et al., 2009; Keurentjes et al., 2011; Diercks and Aderem, 2013). Thus, from the perspective of an experimenter, it is challenging to directly connect the individual elements of a signaling network to the downstream cellular responses and outputs using only biological research tools. By analyzing TOPs functions from a systems’ perspective, we seek to establish a platform for analytical and quantitative approaches to the study of plant stress response and create a starting point for future real-world predictions of plant cellular dynamics after stress exposure.
Popescu SC, Brauer EK (2012) U.S. Provisional Patent Application No. 61/670,839: Compositions and methods comprising use of ILK1 modulating agents for controlling the cellular stress response.
ATPROTEINCHIP1: Expression collection of 5000 Arabidopsis thaliana ORFs printed on a single array at http://abrc.osu.edu/protein-chip (Stock number: 5529827344);
TOKN, a tomato kinase ORFeome at ftp://ftp.solgenomics.net/
The tomato kinome at ftp://ftp.solgenomics.net/
A comprehensive understanding of living systems lays in observing and deciphering the behavior of proteins in cells. How can we identify all these molecular interactions that occur in the cells? What are the tools that will help us achieve this goal? …these are some of questions that scientists are trying to answer.
In our lab we study how plant proteins assemble in complexes and signaling pathways to carry out various biological processes. We are interested specifically in signal pathways initiated by the interactions between plants and their environment. In our research, in addition to classical molecular biology and biochemistry techniques, we use a novel technology we developed, the functional protein microarrays for the model plant system Arabidopsis thaliana.
Protein microarrays represent a novel technology for the unbiased, large scale characterization of molecular interactions. They have been used for a wide variety of applications such as the study of enzyme-substrate, protein-protein, and protein-nucleic acid interactions, profiling antibody specificity, and searching for protein posttranslational modifications. In our lab we produce protein microarrays containing thousands of proteins by using the model plant, Nicotiana benthamiana, as a “protein factory” for the synthesis of large amounts of proteins. Purified proteins which are then deposited on microscope slides using a process very similar to the one used for making DNA microarrays. Once the microarrays have been printed they can be used in various biochemical assays and applications.
Another important focus of our lab is to understand signaling in plants from a system perspective. We plan to use our protein profiling data gathered on protein microarrays to build qualitative and quantitative models of signaling pathways. Such models should provide a way to uncover higher-order properties of signaling pathways and a better understanding of how protein networks operate to generate complex cellular functions.
For more information about the Popescu Research Group, please visit the lab’s webpage at the Boyce Thompson Institute.