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United States Department of Agriculture

Agricultural Research Service

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2011 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 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 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 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
Potato spindle tuber viroid (PSTVd) infects potatoes, tomatoes, and a variety of ornamental species where it can cause devastating crop losses. PSTVd is a very small, circular molecule of naked nucleic acid that spreads throughout the host plant causing a systemic infection. We are studying the effects of PSTVd on gene expression in tomato, measuring the associated biochemical changes as the viroid spreads from leaf to leaf throughout the plant. In FY 2011, we developed a technique to reliably analyze smaller samples of leaf tissue for the metabolites that they contain. We identified a compound that can serve as an internal standard for analyzing these metabolites, thereby allowing more precise quantitization of each metabolite. Metabolites that alter redox status in the host could be critical for PSTVd multiplication and may provide a means to control the disease by interfering with the viroid multiplication. The information will be of greatest use to plant scientists working to improve resistance of crop plants to diseases caused by viroids and viruses. Potato spindle tuber viroid (PSTVd) is able to move from cell-to-cell in infected plants and cause disease without assistance from any pathogen-encoded proteins. By characterizing these processes at the molecular level we hope to i) better understand the operation of certain key regulatory systems controlling growth and development in healthy plants and ii) identify possible strategies to protect such plants against viroid infection. Last year, we completed an analysis of changes in tomato gene expression and small RNA synthesis accompanying PSTVd infection of tomato. Differences between the responses of sensitive and tolerant tomato varieties point to a key role for a regulatory system known as ‘RNA silencing’ in the disease process. A cellular protein known as ‘4/1’ was also shown to play a key role in viroid movement from cell to cell. Disrupting the ability of 4/1 to interact with incoming viroid RNA may render potential host plants resistant/immune to viroid infection.

4. Accomplishments
1. A new technique to examine the interaction between plants and pathogens. Many, if not most, leaf pathogens gain entry to the plant through the stomata and occupy the air chamber inside. The first few hours of this occupation involve an exchange of chemical and physiological signals between the plant and pathogen. We developed a technique to allow a more detailed examination of this exchange. The improved precision of the technique in both quantification and timing will allow the role of secondary metabolites to be determined and a sequence of events to be determined. This type of information will allow plant scientists to improve the disease resistance of crop plants and reduce our reliance on chemical additives.

2. Potato spindle tuber (and other) viroids affect plant hormones and RNA silencing. Viroids are able to move from cell to cell in infected plants and cause disease without assistance from any pathogen-encoded proteins. By characterizing these processes at the molecular level, we hope to better understand the operation of certain key regulatory systems that control growth and development in healthy plants, and identify possible strategies to protect such plants against viroid infection. Recently completed studies have demonstrated cultivar-specific differences in gene expression and small RNA levels in PSTVd-infected tomato plants. These differences indicate that hormone signaling pathways (especially gibberellin and brassinosteroid signaling) and ‘RNA silencing’ play important roles in controlling disease development.

Review Publications
Zakharenkova, T.S., Averyanov, A.A., Pasechnik, T.D., Lapikova, V.P., Baker, C.J. 2010. Release of elicitors from rice blast spores under the action of reactive oxygen species. Russian Journal of Plant Physiology. 57:615-619.

Kim, E., Xiao, Y., Baker, C.J., Owens, R.A., Bentley, W.E., Payne, G.F. 2011. Fabricated catecholic films are capable of redox-cycling and H2O2-generation in the absence of enzymes. Biomacromolecules. 12:880-888.

Baker, C.J., Owens, R.A., Whitaker, B.D., Mock, N.M., Deahl, K.L., Roberts, D.P., Orlandi, E., Averyanov, A.A. 2011. Detection of bacterial aggregation in cell suspensions treated with pathogenic bacteria. Physiological and Molecular Plant Pathology. 75:170-175.

Baker, C.J., Romanova, T.S., Aver'Yanov, A.A., Pasechnik, T.D., Lapikova, V.P. 2009. Rice resistance to blast caused by leaf surface moistening prior to inoculation. Russian Journal of Plant Physiology. 56:(3):389-393.

Verhoeven, J.J., Roenhorst, J.W., Owens, R.A. 2011. Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid. Archives of Virology. 156:1433-1437.

Wang, I., Shibuya, M., Taneda, A., Tasuku, K., Senda, M., Owens, R.A., Sano, T. 2011. Accumulation of Potato spindle tuber viroid-specific small RNAs is accompanied by specific changes in tomato gene expression. Virology. 413-72-83.

Last Modified: 2/23/2016
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