Location:2008 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
In June 2004, a field trial was initiated in Fort Pierce, FL to assess the dwarfing effects of citrus viroids IIIa and IIIb infection on Valencia sweet orange. Analysis of each tree is also being evaluated semi-annually for growth rate and general appearance, and we will start to collect yield data in 2009. Viroid infection of citrus is well-known to result in dwarfing, but the underlying physiological mechanism(s) are unclear. To test the possibility that dwarfing involves reduced root growth, we determined the effect of citrus viroid III infection on root development under greenhouse conditions using three rootstock/scion combinations. Three months after slash inoculation, infected Etrog citrons were significantly shorter than uninoculated control plants; after 6 months, the inhibitory effect of viroid infection on root growth had also become statistically significant, and these effects continued to intensify between 6 and 12 months. Graft inoculation of either trifoliate orange seedlings or Valencia scions growing on trifoliate orange rootstocks resulted in a similar (though not statistically significant) inhibition of root dry weight accumulation over an 18 month period. These results provide an opportunity to rapidly identify viroid variants with enhanced dwarfing potential, thereby contributing to possible future efforts to use tree size control as part of a broad response to the threat posed by citrus greening. Viroid replication and, thus, the ability of these small RNAs to cause disease is completely dependent upon proteins encoded by the genomes of their plant hosts. Key details of this process remain obscure, and the ease of manipulation and genetic tractability of Saccharomyces cerevisiae (baker’s yeast) make this a promising model system in which to study these interactions between viroid and host. We are using brome mosaic virus RNA3 as a gene vector to launch potato spindle tuber viroid (PSTVd) replication in yeast. A variety of plasmid constructs capable of producing potentially infectious PSTVd RNAs in different subcellular compartments have been prepared, and studies of the role(s) of RNA ligase and RNase III in viroid replication are currently underway. Using the model system developed last year, we have carried out comprehensive studies in greenhouse tomato plants inoculated with PSTVd. We are examining possible coordination between mRNA responses and changes in the metabolites in the leaf apoplast. The effect of leaf developmental age is being considered also. We have succeeded in developing a highly sensitive technique to accurately monitor small changes in bacterial numbers. We used this in plant suspension cell model systems to quantify and verify the exact time that pathogens bind to their host plant cells. In addition, this technique allows us to monitor changes in the bacterial physiological status. These studies advance National Program 303 (Plant Diseases) component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors
1. Developed a method to study the movement of viroid RNAs from the cytoplasm into the chloroplast of infected plant cells. Many chloroplast proteins are synthesized in the cytoplasm and then imported into the chloroplast, but host RNAs encoded by the nuclear genome appear unable to move across the chloroplast membrane. Identification of the sequence or structural motifs that mediate viroid transport into the chloroplast offers a promising strategy to target transgene mRNAs to the chloroplast, thereby increasing the level of foreign protein expression and possibly modifying photosynthetic efficiency. These studies advance National Program 303 (Plant Diseases) component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors; namely, understanding the cellular and molecular interactions that result in susceptibility, resistance, and/or disease development.
2. Identified 20 previously undescribed variants of potato spindle tuber viroid (PSTVd) among 49 isolates collected from locations throughout Russia. The sale of untested seed potatoes is responsible for the present wide distribution of PSTVd throughout Russia, and the prevalence of this pathogen in seed potatoes and potato plants pose a serious threat to both seed production and potato breeding. Epidemiological data of this sort provide insight into pathways of viroid spread, information that is essential for the ultimate success of current efforts to detect and eliminate viroid and phytoplasma diseases from Russian seed potato production. These studies advance National Program 303 (Plant Diseases) component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors; namely, understanding the cellular and molecular interactions that result in susceptibility, resistance, and/or disease development.
3. Viroid disease is characterized by marked changes in host gene expression and activation of an RNA-based defense system known as “RNA silencing”. To better understand the relationship between RNA silencing and the appearance of visible disease symptoms, time-course analyses of viroid-specific small RNA accumulation using several viroid-host combinations were carried out. These analyses revealed the presence of two size classes of viroid-specific small RNAs in infected plants, and sequence analysis subsequently demonstrated the presence of a previously undescribed cluster of small RNAs derived primarily from (-)strand PSTVd RNA. The process by which viroid-specific small RNAs are generated appears to be more complicated than previously believed, possibly involving multiple Dicer-like activities, viroid RNA substrates, and subcellular compartments. These studies advance National Program 303 (Plant Diseases) component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors; namely, understanding the cellular and molecular interactions that result in susceptibility, resistance, and/or disease development.
4. Developed a highly sensitive technique to quantitate bacterial populations in real-time. Until now, the amount of work and materials required to determine bacterial numbers by serial dilution has made it virtually impossible to do bacterial counts for multiple treatments collected over short time periods. Our technique uses flow cytometry and allows researchers to follow previously unsuspected aspects of the initial binding phase characteristic of many pathogen bacterial interactions. These studies advance National Program 303 (Plant Diseases) component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors; namely, understanding the cellular and molecular interactions that result in susceptibility, resistance, and/or disease development.
5. Significant Activities that Support Special Target Populations
Machida, S., Yamahata, N., Watanuki, H., Owens, R.A., Sano, T. 2007. Successive accumulation of two size classes of viroid-specific small RNA in potato spindle tuber viroid-infected tomato plants. Journal of General Virology. 88:3452-3457.