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United States Department of Agriculture

Agricultural Research Service

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2010 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. Objective 4: Determine mechanisms that enhance plant defenses against diseases caused by bacteria and viroids.

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 large-scale 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. Objective 4 will use physiological and molecular approaches developed in the previous objectives to study plant defense mechanisms against both viroid and bacterial pathogens in a common host.

3. Progress Report
Obj. 1. We have been able to identify a new phenomenon that appears to be unique to pathogenic bacteria in our model system. The event appears to involve the binding of bacteria to gather and possibly to plant cells. We are exploring if this is signaled by redox status of the cells and if it is a critical point for pathogenesis. Obj. 2. A series of chimeric viroid RNAs was constructed in which one or more copies of a short RNA 'hairpin' binding site for MS2 bacteriophage coat protein were inserted at key points in eggplant latent viroid. By fusing the MS2 coat protein to different fluorescent reporter proteins it should be possible to use fluorescence microscopy to monitor viroid movement into the chloroplast of living cells. We have constructed a series of yeast expression plasmids that launch potato spindle tuber viroid (PSTVd) RNAs by nuclear transcription and established an RT-PCR system to detect the presence of linear versus circular molecules in total RNA from transformed yeast. Processing of several different PSTVd constructs in vivo was compared with cleavage/ligation in vitro using host plant and yeast nuclear extracts, and the formation of circular, potentially infectious molecules of PSTVd in yeast was demonstrated. Mutational analysis of citrus dwarfing viroid has yielded a variant containing a single non-lethal sequence change in the so-called "loop E motif". Bioassay on Etrog citron revealed a dramatic reduction in foliar symptoms, and the effects of this change on root growth (and, potentially, dwarfing under field conditions) are currently under study. Obj. 3. We completed the quantification of tomato metabolites that change during infection by the PSTV viroid. This was a complex project involving numerous metabolites in multiple leaves of multiple treatments over time. We have collected mass spectral data on all metabolites which allowed identification of several. The determination of redox potentials of the individual metabolites is currently underway. Obj. 4 Recently-completed microarray and large-scale sequencing analyses carried out in tomato plants infected with Potato spindle tuber viroid have provided the first genome-wide picture of viroid disease, revealing changes in the expression levels of 20-25% of all host mRNAs (tomato) as well as several microRNAs responsible for coordinating entire developmental pathways.

4. Accomplishments
1. Identified a previously undescribed event in plant bacterial pathology. Bacterial pathogens must make contact with host cells in order to transfer various factors that block plant resistance and allow disease to progress. Using cytometeric techniques developed earlier in conjunction with plant cell suspension model systems, the initiation of this event was determined by observing a decrease in the number of free bacteria. Using fluorescent and confocal microscopy it was apparent that the bacterial cells were coming together and forming aggregates, in many cases the aggregates included plant cells. This aggregation event did not occur with non-pathogenic bacteria. This aggregation event appears to be a critical step for bacteria to cause disease in plants. It appears to be subject to plant secondary metabolites and antioxidants which may lead to a means to disrupt the event and reduce disease.

2. First genome-wide picture of viroid disease. Recently completed microarray analyses and large-scale sequencing of small regulatory RNAs isolated from tomato plants infected with potato spindle tuber viroid (PSTVd) provide the first genome-wide picture of viroid disease. Changes in host gene expression following infection are extensive and highly cultivar-dependent, affecting both biosynthesis of gibberellins, brassinosteroids, and abscissic acid and downstream hormone signaling. Together with studies of a protein that appears to regulate the cell-to-cell movement of PSTVd, these results identify potential control points at which it may be possible to disrupt infection and block the disease process.

3. New insight into the epidemiology of a disease that remains a serious problem in Russia and many other portions of the former Soviet Union. Characterization of a large number of novel, naturally-occurring variants of potato spindle tuber viroid (PSTVd) collected over a 25+ year period from different locations in the former Soviet Union has provided new insight into the epidemiology of a disease that remains a serious problem in Russia and many other portions of the former Soviet Union. Over the last several years outbreaks of PSTVd and other related viroids affecting greenhouse-grown tomato and various ornamental species have been reported from Australia/New Zealand, Europe, and North America (including the U.S.). Controlling such outbreaks requires that the initial source of infection be identified and eradicated. Knowledge of sequence variation among naturally-occurring isolates of PSTVd (and other viroids) is critical to these disease control efforts.

Review Publications
Baker, C.J., Owens, R.A., Whitaker, B.D., Mock, N.M., Deahl, K.L., Roberts, D.P., Averyanov, A.A. 2010. Effect of viroid infection on the dynamics of phenolic metabolites in the apoplast of tomato. Physiological and Molecular Plant Pathology. 74:214-220.

Baker, C.J., Whitaker, B.D., Mock, N.M., Rice, C., Roberts, D.P., Deahl, K.L., Ueng, P.P., Aver'Yanov, A.A. 2009. Differential induction of redox sensitive extracellular phenolic amides in potato. Physiological and Molecular Plant Pathology. 73:109-115.

Last Modified: 2/23/2016
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