Linda L. Walling - US grants

The interactions between the tomato plant (Lycopersicon esculentum) and its pathogen, Pseudomonas syringae pv. tomato, provide an excellent system for the study of plant gene expression during host-pathogen interactions. Genes which are expressed in a disease-resistant plant ("defense" genes) during bacterial pathogen infection will be isolated, and their structure and gene expression programs will be determined. The sequences which are important in the regulation of "defense" gene expression will be identified by deletion mutant and gene fusion analysis. Intact and modified genes will be introduced into the disease-sensitive tomato genome; transformed plants will be regenerated and analyzed for the structure, organization, and expression of the "defense" gene. In addition, transformed plants will be tested for the expression of the disease-resistance phenotype. These experiments will provide new and basic information about how plant gene expression is regulated during a biological stress in a higher eukaryotic.

This is a Research Opportunities for Women Career Advancement Award, to enable her to carry out important studies on cytoplasmic (chloroplast-mediated) inheritance in soybeans. Dr. Walling is at a critical stage in her career, and the research she will do with this award is anticipated to boost her career substantially.

Substrate adhesion molecules (SAMs) belong to a family of adhesive glycoproteins which occur in the extracellular matrix (ECM) of a variety of organisms. Vitronectin (VN) is a SAM that has been implicated in cell adhesion and migration. The PI's have established that a homolog to VN occurs in the primary cell wall of plants, and have evidence for its role in facilitating pollen tube extension in the style. The proposed research is a multi- faceted approach to understanding the expression, localization, and function of vitronectin-like molecules during plant development. The experiments proposed will specifically test the role of VN- like molecules during pollination. Immunocytochemical and fluorescence staining techniques will be used to examine the relationship between components of the cytoskeleton, VN-like molecules, and VN receptors (integrins) during pollen tube elongation and during shoot ontogeny. Synthetic peptides that are known to mediate adhesion of VN to its integrin will be used to determine their effect on pollen tube elongation in vivo and in vitro. cDNA clones that encode VN-like proteins will be isolated and characterized. The levels of VN RNA during plant development will be examined by RNA blot and in situ hybridizations. The role of VN-like proteins during plant development will be tested by overexpressing and underexpressing VN RNAs using chimeric gene constructs in transgenic plants.

9318260 Walling Aminopeptidases are exopeptidases that hydrolyze the N-terminal amino acid from peptides and proteins. In animals, aminopeptidases have an important role in nutrition and modulation of the activities of gut and brain peptide hormones by converting them to active or inactive forms. In addition, these exopeptidases may have a role in modulating half-lives of proteins in vivo. Surprisingly; little is known about aminopeptidase activity during plant development. Although aminopeptidases are known to be induced after germination and during senescence, the expression of these genes during the plant life cycle or in response to biotic or abiotic stress has not been characterized. Dr. Walling has isolated and characterized a leucine aminopeptidase (lap) cDNA clone from Lycopersicon exculentum (tomato). Leucine aminopeptidase mRNAs are induced to high levels after mechanical injury, insect feeding, and Pseudomonas syringae pv. tomato infection. LAP proteins and activity are also induced after wounding. Two wound-induced lap genes are present in the tomato. The proposed research is a multi-faceted approach to understanding the expression, localization, and function of LAPs during the plant-defense response. Furthermore, these studies will be exteded to gain a comprehensive understanding of lap gene expression throughout plant development and in response to abiotic stress. Only recently, have plant leucine aminopeptidase genes been isolated, and few directed efforts have been made to understand their expression and impact on the plant responses to the environment and to developmental cues. Elucidation of the role of LAPs during the plant defense reaction and during development will enhance our understanding of the mechanisms that favor optimal growth and development. These factors will directly impact on optimal crop productivity and potentially yield insights for strategies to enhance a plant's endogenous protection mechanisms. ***

Little is known about the degradation of proteins during plant development and in response to stress. In particular, there is a gap in the understanding of the class of proteases called aminopeptidases. Two classes of leucine aminopeptidase genes (LapA and LapN) from tomato have been characterized. LapA genes are expressed in response to wounding, caterpillar feeding, and some bacterial and fungal pathogens. In contrast, LapN genes are expressed at all times and in all tissues. The analysis of genetically engineered tomato plants that express a LapA antisense gene (35S:asLap) showed that down-regulation of Lap genes impairs the ability of tomatoes to mount a wound response. This proposal builds on a strong foundation of knowledge about the tomato LAPs built during the previous granting period. This proposed integrated research effort is focused on understanding the location of LAPs and their role in responses wounding, pathogens, water deficit, and salinity.
Cell fractionation studies show that LAPs reside in two cellular compartments: the cytoplasm and the chloroplast. Obviously, the substrates available for LAP action are determined by the location of LAPs within the tomato cell. Therefore, LAP-A- and LAP-N-specific antibodies will be used to determine the location of these two classes of aminopeptidases within the cell. The mechanisms that control the accumulation of LAP-A and LAP-N in two cellular locations will be tested.
The LapA antisense plants (35S:asLap) do not effectively induce wound-response genes. These data suggest that LAP-A may be one of the first peptidases to regulate a plant-signaling pathway. Since this conclusion is novel for the plant community, these data should be confirmed using additional strategies. Three sets of transgenic tomato plants that have altered patterns of Lap gene expression will be evaluated. First, plants expressing two Lap antisense genes will be made. These plants express the LapA antisense RNA from both the wound-induced LapA promoter (LapA:asLap) and the constitutive 35S promoter (35S:asLap). These plants should afford the tightest regulation of the wound response. Second, co-suppressed plants will be characterized. Unlike the antisense plants that down regulate both LapA and LapN, the co-suppressed plants only down regulate LapA. Third, transgenic tomatoes that express high levels of LAP-A constitutively will be characterized. A tight correlation of LapA and/or LapN down regulation with the suppression of the wound response will implicate LAPs as protease that regulates plant stress responses. If LAPs are shown to modulate the wound response, the site of LAP action will be determined by feeding Lap down-regulated plants systemin, abscisic acid, jasmonic acid and intermediates of the octadecanoid pathway. If systemin activation is implicated, the activity of LAP-A and LAP-N on putative systemin precursors will be characterized.
Transgenic tomatoes that are up- and down-regulated for Lap gene expression will be used to determine if LAPs are important for controlling bacterial, viral, and fungal infections, as well as insect infestations and tolerance to water-deficit and salinity.
The understanding of the expression and impact of LAPs in plant development and in responses to stress has increased greatly. However, continued efforts are essential to elucidate the exact roles of LAPs in stress responses. These studies will enhance our understanding of the mechanisms that favor optimal growth and development. These factors will directly impact on increased crop productivity and may yield significant insights for the development of cogent strategies to enhance a plant's endogenous protection mechanisms to an array of stresses.

This 2010 project will determine the specificity and mechanisms of N-terminal modifications of the Arabidopsis proteome. The N-terminal modification enzymes (NTMEs), which include 28 peptidases and 11 transferases, will be studied. NTMEs control important fundamental aspects of protein maturation, activation or regulation, degradation, and potentially of plant gene regulation. State-of-the-art methods from plant genetics, cell biology and both basic and high throughput biochemistry are integrated in this program to transition the NTME field from "activity by homology" to one grounded in solid biochemistry. The project will address the following questions: (1) Do Arabidopsis NTME proteins have enzymatic activities? (2) What are the specificities of NTME enzymes as determined by combinatorial peptide libraries? (3) When and where do NTME RNAs and proteins accumulate? (4) Do NTME proteins act co- or post-translationally? (5) What are the impacts of NTME T-DNA knock-out mutants? Collectively, these data will establish the specificity and location of each NTME and thereby establish a set of rules for the proteome, which will predict the fate of a protein by its subcellular localization and the primary sequence of its N-terminus.

Broader Impacts: This project will: (1) produce and distribute antisera to 39 NTME proteins, (2) develop methods to express and assay plant NTMEs, (3) synthesize novel combinatorial peptide libraries for determining the extended specificities of NTMEs, and (4) produce a collection of homozygous single and double mutants for NTMEs. The interactive NTME web site (http://www.cepceb.ucr.edu/members/walling/2010_NTMEs) will provide a list of the Arabidopsis NTME genes, NTME assays, links to the peptidase and transferase research communities, and mechanisms to access the resources developed by this project. The Arabidopsis Biological Resource Center (ABRC) will distribute mutants generated in this project. In addition to establishing the first biochemical rules to predict the fate of a protein in a subcellular compartment, this program provides a unique opportunity to train undergraduates, graduate students and postdoctoral fellows in a newly emerging field of N-terminal modifying enzymes in plants.

This proposal focuses on understanding the tomato leucyl aminopeptidase A (LapA). LAP controls tomato defenses after wounding and insect feeding. When LAP protein is reduced in transgenic LapA-SI plants, the proteins and chemicals that antagonize insect growth are produced at lower levels. LapA-SI plants incur more damage and tobacco hornworms are larger relative to insects from wild-type plants. In addition, on plants that make excessive amounts of LAP-A protein (LapA-OX), caterpillars consume less foliage and larval masses are reduced. The LapA-OX plants provide an important and new mechanism of insect resistance, since plant stature and fertility are not impaired. Understanding how LAP-A controls tomato defenses will be an important addition to our general knowledge of gene regulation in plants.
Goal 1 is to determine the susceptibility or resistance of LapA-SI and LapA-OX plants to insects that use different modes of feeding. If LapA-OX plants provide resistance to many insect species, an important and new mechanism to protect plants from insect pests will have been discovered. Goal 2 is to use genetic, biochemical and plant transformation technologies to determine if the peptidase activity of LAP-A is essential for its role in controlling defenses against insects. This is important since LAPs from other organisms have multiple functions. Finally, Goal 3 is to determine the cellular process that is regulated by LAP-A. A new model that links the many signals that influence wounding in tomato and LAP-A is proposed. This model will be rigorously tested using molecular, biochemical and transgenic plant strategies. By supporting or refuting this model, these experiments will aide in defining the complex and largely unexplored defense network that plants employ against insects.
These studies should result in new strategies for engineering an environmentally safe resistance to insects into crops and horticultural plants, benefiting agriculture and society at large. The proposed experiments range from whole organism studies to molecular responses and spans the fields of plant biology and entomology. This research program will provide a unique, interdisciplinary training environment for a graduate and undergraduate students and a postdoctoral fellow.

The field of vector-borne diseases spans the biology and genomics of the arthropod vectors of mammalian and plant disease agents, the microbes and viruses transmitted by insects and ticks, and host responses to vectors and vector-borne diseases. Advances in molecular biology and genomics have created an unprecedented opportunity for deciphering common evolutionary adaptations of vectors and pathogens they transmit. This award will support participation of early-career scientists in the "Facing the Challenges of Vector-Borne Diseases in the 21st Century" symposium at the University of California at Riverside, March 27-28, 2010. The UCR symposium is the first to bring together researchers in the area of vector-borne disease of plants and animals. Seven scientific sessions and two forumswill provide opportunities to discuss cross-disciplinary topics. This meeting will synergize experts from the animal and plant communities, who often do not interact, in order to bring about a greater understanding of the current themes and challenges for the future, providing a new paradigm for meetings in this research area.

Broader Impacts: It is anticipated that by bringing together scientists from the plant and animal disease-vector communities using a range of approaches from molecular genetics, genomics and social sciences, this symposium will enable synergistic interactions and stimulate research innovations and collaborations in these fields. With only 104 participants, the symposium affords researchers, including graduate students and postdoctoral associates, an opportunity for one-on-one discussions and exchanges to an extent that is not usually possible at large professional society meetings. The NSF funds will be used to support the travel of early career investigators including Assistant Professors, postdoctoral fellows and graduate students. Women, underrepresented minority participants, and postdocs and students from the labs of Assistant Professors will be encouraged to participate.

Over 40% of the world's food supply is lost to damage caused by pathogens, pests and weeds. Therefore, novel strategies to enhance plant productivity and resistance to pests will significantly increase the security of the world's food supply. To achieve this goal, a deep understanding the mechanisms that control resistance to insects is needed. This project is at the cutting edge of plant-insect interactions and will reveal how chloroplasts communicate with nuclei to coordinate a robust defense after injury and insect attack; this knowledge will enable new strategies for insect resistance in the future. This project is a fertile training ground for a graduate student. Graduate student leadership skills will be developed through guiding undergraduate's in this project. UCR is a minority-serving institution and this project will recruit and engage students with diverse backgrounds. Results from the project will be disseminated by publications in scientific journals, lay talks to Riverside K-12 schools and the public, and talks at meetings and universities.

This project will study the importance of a tomato chloroplast-localized protein called leucine aminopeptidase (LAP-A) and its role in generating a signal that controls nuclear gene expression after insect feeding and wounding. This project will identify the LAP-A-dependent signal that mediates this chloroplast-to-nucleus communication. The protein/peptide (target) that LAP-A acts on will be identified and the target may be the signal or may create the signal. LAP-A has two activities. LAP-A is an aminopeptidase and may produce the signal by modifying the N-terminus of a target protein/peptide that resides in the chloroplast. LAP-A is also a molecular chaperone and may help targets stay in an active folded state. These activities should influence the target's structure, activity and/or stability and thereby signal production. This project will use cutting-edge proteomics technologies to measure proteins/peptides in three tomato lines that express different levels of LAP-A (LapA-silenced, LapA-overexpressing, and wild type). Aim 1 will identify chloroplast proteins that are (1) cut by LAP-A and (2) have altered stability that is strictly correlated with the presence/absence of LAP-A. Aim 2 will determine the LAP-A-dependent changes in a small peptide called glutathione (GSH). Preliminary data link LAP-A and GSH turnover. GSH is an excellent candidate for the signal as it regulates two defense signals (reactive oxygen species and redox status) in other systems. The identity of LAP-A's targets will reveal the nature of the LAP-A-generated signal that mediates chloroplast-nucleus communication.