Location:2009 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 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. 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
A visit to the Fort Pierce field site earlier this year (May, 2009) revealed that the trees in our trial have been severely affected by the uncontrolled spread of both citrus canker and citrus greening. Fruit collection is scheduled to begin this fall, and serious consideration is being given to terminating the trial after this first collection. Mutational analysis of the loop E motifs of CVd-IIIa and CVd-IIIb is continuing. A longer-than-full-length PSTVd cDNA whose RNA transcript is expected be processed in vivo into full-length, infectious PSTVd by Dicer (a family of host-encoded RNase III-like enzymes) has been constructed. Plasmids containing this PSTVd cDNA construct have been expressed in both yeast and Cryphonectria parasitica (the chestnut blight fungus), and total RNA preparations from the fungi are currently being examined for the presence of the expected processing/cleavage products. Microarray analysis involving over 9200 individual gene transcripts has shown that expression levels of more tomato genes are down-regulated by PSTVd infection than are up-regulated in the cultivar Rutgers; i.e., 292 vs. 244. Additional studies with PSTVd-infected Moneymaker seedlings as well as transformed Moneymaker plants constitutively expressing small PSTVd-related RNAs in the absence of viroid infection are currently underway. HPLC analysis revealed significant differences in the profiles of apoplastic phenolics recovered from the three types of plants. Techniques for observing changes in apoplastic phenolics of both whole plants and cell suspensions were tested and finalized. Using these techniques we have isolated specific phenolics that appear to be involved in the early stages of plant stress caused by either bacteria or viroids. The identification of these phenolics has been facilitated by new mass-spec UPLC techniques. Studies using fluorescent stains have been initiated to allow a more visual approach plant-microbe early interactions. New data handling strategies have been introduced to help interpret the electrophysiological data of both whole plant and cell suspension studies.
1. ARS scientists in Beltsville, MD in collaboration with scientists at the Russian Research Institute of Phytopathology (VNIIF) have identified more than 30 previously undescribed variants of potato spindle tuber viroid (PSTVd) collected from locations throughout Russia. The widespread distribution of this pathogen in Russia poses a serious threat to both seed production and potato breeding; elsewhere, increasing globalization of the ornamentals industry has been accompanied by a troubling increase in the number of plants infected by PSTVd or related viroids. Regulatory agencies like APHIS have begun to demand increased testing in order to reduce (and ultimately eliminate) viroid spread, and epidemiological data of this sort provide important insights into pathways of viroid spread - information that is essential for the ultimate success of current control efforts.
2. Translation on chloroplast rather than cytoplasmic ribosomes offers a promising strategy to increase expression of valuable foreign proteins in plant cells. Many proteins synthesized in the cytoplasm of a plant cell are later imported into the chloroplast, but the messenger RNAs which encode these proteins are unable to cross the chloroplast membrane. Viroids like eggplant latent viroid (ELVd) replicate in the chloroplast; thus, these RNA molecules must contain molecular signals that allow them to enter the chloroplast. Using Agrobacterium to introduce a gene encoding ELVd into the nuclear genome of both host (eggplant) and non-host (tobacco) plants, we have shown that newly-synthesized ELVd is able to leave the nucleus of a host cell, enter the chloroplast, and begin replication. In non-host cells, ELVd was transported to the cytoplasm but failed to enter the chloroplast. In addition to increasing the level of foreign protein expression, it may also be possible to use this strategy to modify photosynthetic efficiency.
3. Developed a technique that demonstrates when bacterial pathogens bind to plant cells. The ability of bacterial pathogens to cause disease in plants is dependent on their ability to exchange molecular signals with the plant. Likewise the ability of plants to actively resist pathogens depends on this molecular exchange with the pathogen. Therefore knowledge of precisely when this binding occurs is important in order to know when to find and identify the molecular signals and processes critical to susceptibility and resistance. By using fluorescent dyes to label bacteria and by counting them with a laser cytometer (cell counter), the binding of bacteria could be followed. This knowledge has allowed us to focus on other processes and metabolites that are present at this time and will hopefully lead to better a understanding of how to improve disease resistance.
Owens, R.A., Girsova, N.V., Kromina, K.A., Lee, I., Mozhaeva, K.A., Kastalyeva, T.B. 2009. Russian isolates of Potato spindle tuber viroid exhibit low population diversity. Plant Disease. 93:752-759.