1a.Objectives (from AD-416):
Objective 1: Identify changes in host gene expression and small RNA-mediated regulation associated with viroid and bacterial infection and disease development as potential targets for disease management. Objective 2: Identify key metabolites that are involved in the early stages of pathogenesis and may have global effects on disease resistance through either their bioactive nature or effect on redox-status. Sub-objective 2.A. Identify and quantify secondary metabolites induced upon infection of tomato with either P. syringae or potato spindle tuber viroid (PSTVd). Sub-objective 2.B. Determine bioactivity of secondary metabolites induced upon infection of tomato with either P. syringae or PSTVd. Sub-objective 2.C. Determine the effect on redox status of secondary metabolites induced upon infection of tomato with either P. syringae or PSTVd. Objective 3: Identify the molecular signals and pathways used by viroids to move through the cytoplasm, enter the nucleus or chloroplast of the host cell, and begin replication. Sub-objective 3.A. Determine the role of host protein 4/1 (and other proteins interacting with 4/1) in the intra- and intercellular movement of PSTVd. Sub-objective 3.B. Use sequence motifs derived from Eggplant latent viroid (ELVd) to redirect mRNAs encoding enzymes involved in terpenoid biosynthesis into the chloroplast.
1b.Approach (from AD-416):
This project seeks to elucidate specific signaling mechanisms that are involved in plant disease resistance. Plants respond to biotic stress by the integration of responses located in two different cellular compartments, the symplast and the apoplast. Improvement of existing resistance to plant disease requires a more comprehensive understanding of key apoplastic and symplastic responses to pathogen invasion and how they are integrated. In the apoplast, we will examine the complex interplay between secondary metabolites and redox signaling that controls early events in bacterial and viroid pathogenesis as well as other responses triggered by long distance signaling. In the symplast, we will examine the molecular interactions between viroids and their hosts to determine how these small RNA molecules replicate and move between organelles within a single cell or over long distances between cells. The long-term objective of this multidisciplinary project is to understand the role of plant-pathogen signaling in disease resistance in sufficient detail that novel strategies can be developed to render plants resistant/immune to pathogen infection. In the intermediate term, we will test the ability of certain viroid-derived targeting signals to redirect mRNAs to the chloroplast, thereby adding novel biosynthetic capabilities to chloroplast metabolism with the need for chloroplast genome transformation.
This is a new project and we have started working on the first year milestones. The initial microarray analysis and large scale sequencing have been nearly completed and initial gene constructs made to help determine whether viroid infection affect hormone signaling and signaling in tomato via RNA silencing. Some of this work will be done in collaboration with another lab using materials that we supplied. Preliminary results to monitor the redox potential in these plants have indicated that electrodes placed in the plant vascular tissue are able to detect tissue responses. The chemistry of the redox changes can be related to specific metabolites which have been identified and are being tested in metabolic studies. Two in particular appear to help determine the timing of the oxidative burst which is often noted in plant pathogen interactions.