Characterizing RNA translocation in the parasitic weed Cuscuta pentagona

LeBlanc, Megan Leanne

Characterizing RNA translocation in the parasitic weed Cuscuta pentagona

dc.contributor.author	LeBlanc, Megan Leanne	en
dc.contributor.committeechair	Westwood, James H.	en
dc.contributor.committeemember	McDowell, John M.	en
dc.contributor.committeemember	Okumoto, Sakiko	en
dc.contributor.committeemember	Khodaparast, Giti A.	en
dc.contributor.department	Plant Pathology, Physiology, and Weed Science	en
dc.date.accessioned	2015-05-30T06:01:11Z	en
dc.date.available	2015-05-30T06:01:11Z	en
dc.date.issued	2013-06-03	en
dc.description.abstract	The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications.	en
dc.description.degree	Ph. D.	en
dc.format.medium	ETD	en
dc.identifier.other	vt_gsexam:863	en
dc.identifier.uri	http://hdl.handle.net/10919/52865	en
dc.publisher	Virginia Tech	en
dc.rights	In Copyright	en
dc.rights.uri	http://rightsstatements.org/vocab/InC/1.0/	en
dc.subject	plant parasitism	en
dc.subject	Cuscuta pentagona	en
dc.subject	mRNA trafficking	en
dc.subject	optoperforation	en
dc.subject	laser-mediated plant cell disruption	en
dc.subject	host-parasite relations	en
dc.title	Characterizing RNA translocation in the parasitic weed Cuscuta pentagona	en
dc.type	Dissertation	en
thesis.degree.discipline	Plant Pathology, Physiology, and Weed Science	en
thesis.degree.grantor	Virginia Polytechnic Institute and State University	en
thesis.degree.level	doctoral	en
thesis.degree.name	Ph. D.	en