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United States Department of Agriculture

Agricultural Research Service

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2012 Annual Report

1a. Objectives (from AD-416):
This project merges two historically successful projects with distinct origins but a common goal, elucidation of critical signals in plant/pathogen interaction and exploitation of the signaling process to improve plant disease resistance. Previously, one project was focused on the biochemical and physiological signals of host/bacterial interactions in the apoplast, while the other examined the molecular signals mediating viroid/host interaction in the symplast. The knowledge base and technical expertise associated with these two projects complement each other very well, and combining them provides a broader and much stronger base for interdisciplinary studies in three specific areas: Objective 1 - Determine the role of plant apoplastic redox metabolism in signaling early events in host resistance responses. Objective 2 - Identify structural features of viroid genomes that serve as signals for symplastic replication, movement and pathogenicity. Objective 3 - Evaluate the relative roles of redox and RNA-based signaling in long distance coordination of host resistance responses.

1b. Approach (from AD-416):
Objective 1 will use physiological assays previously developed in a cell suspension model system to 1) identify a limited number of plant bacterial interactions that display different physiological states of basal resistance or suppression and 2) characterize the corresponding changes in all detectable apoplastic phenolics and changes in redox status. The same bacterial isolates will also be used in whole plant experiments to 3) characterize the corresponding changes in apoplastic phenolics and redox status. Finally, we will 4) test whether addition of identified phenolics to suspension cells or whole plants interferes with basal resistance elicitation and suppression. Objective 2 will also involve multiple experimental approaches. A combination of Agroinfiltration and in situ hybridization techniques will be used to 1) identify the structural feature(s) of Eggplant latent viroid (ELVd) responsible for its ability to enter the chloroplast. A field study currently underway in Fort Pierce, FL will 2) test the ability of variants of citrus viroid III (CVd-III) to dwarf citrus growing under subtropical conditions. Finally, we will use a Saccharomyces cereviseae (baker’s yeast) experimental system to 3) study viroid transport from the cytoplasm to the nucleus and other fundamental features of viroid-host interaction. Objective 3 will examine the role of redox and viroid-induced RNA silencing in regulating host responses to infection. Using 454 DNA sequencing technology, we will compare small RNA profiles from four sets of tomato plants; i.e., uninfected control plants; plants infected with either a mild or severe strain of PSTVd; and transgenic plants that constitutively express a noninfectious hairpin RNA derived from PSTVd. Effects on host gene expression will be monitored by microarray analysis with special emphasis on down-regulated genes potentially involved in redox metabolism.

3. Progress Report:
We improved our technique for sampling leaflets for the metabolites that they produce. We found a way to reliably sample smaller leaf samples and incorporated an internal standard that was identified last year. We demonstrated that similar leaflets of the same leaf have almost identical metabolites and serve as replicates. We continued and finalized analysis of changes in gene expression and small RNA synthesis accompanying viroid infection in tomato. Differences between the responses of sensitive and tolerant tomato varieties indicate a key role for ‘RNA silencing’ in the disease process. We demonstrated that a cellular protein known as ‘4/1’ plays a key role in viroid movement from cell to cell. Disrupting this interaction between 4/1 and the incoming viroid RNA may render potential host plants resistant/immune to viroid infection.

4. Accomplishments
1. Demonstrated that a new compound could be used as the first internal standard for apoplast metabolite analysis. Identified and demonstrated that dinotefuran, which is not found in plants, can mimic apoplast phenolic behavior and therefore be used to improve apoplast analysis. The improved precision of the technique will allow secondary metabolites to be analyzed over a period of time to determine the sequence of metabolic events as well as their temporal placement.

2. Demonstrated additional cultivar-specific differences in tomato gene expression and small RNA levels associated with potato spindle tuber viroid infection. These differences indicate that hormone signaling pathways (especially gibberellin and brassinosteroid signaling) and ‘RNA silencing’ play important roles in disease symptom development.

Review Publications
Deahl, K.L., Perez, F.G., Jones, R.W., Baker, C.J., Mcgrath, M. 2010. Natural occurrence of Phytophthora infestans on woody nightshade (Solanum dulcamara) in New York. Plant Disease. 94:1063.

Owens, R.A., Sano, T., Duran-Vila, N. 2012. Plant viroids: Isolation, Characterization/Detection, and Analysis. Methods in Molecular Biology. 894:253-271.

Last Modified: 2/23/2016
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