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. / Ph. D.

Cuscuta pentagona is an obligate parasitic plant that hinders production of crops throughout the world. Parasitic plants have unique morphological and physiological features, the most prominent being the haustorium, a specialized organ that functions to connect them with their host's vascular system. The Cuscuta haustorium is remarkable in that it enables mRNA movement to occur between hosts and parasite, but little is known about the mechanisms regulating cross-species mRNA transfer or its biological significance to the parasite. These questions were addressed with genomics approaches that used high throughput sequencing to assess the presence of host mRNAs in the parasite as well as parasite mRNAs in the host. For the main experiment Cuscuta was grown on stems of Arabidopsis thaliana and tomato (Solanum lycopersicon) hosts because the completely sequenced genomes of these plants facilitates identification of host and parasite transcripts in mixed mRNA samples. Tissues sequenced included the Cuscuta stem alone, the region of Cuscuta-host attachment, and the host stem adjacent to the attachment site. The sequences generated from each tissue were mapped to host reference genes to distinguish host sequences, and the remaining sequences were used in a de novo assembly of a Cuscuta transcriptome. This analysis revealed that thousands of different Arabidopsis transcripts, representing nearly half of the expressed transcriptome of Arabidopsis, were represented in the attached Cuscuta. RNA movement was also found to be bidirectional, with a substantial proportion of expressed Cuscuta transcripts found in host tissue. The mechanism underlying the exchange remains unknown, as well as the function of mobile RNAs in either the parasite or host. An approach was developed to assay potential translation of host mRNAs by detecting them in the Cuscuta translatome as revealed by sequencing polysomal RNA and ribosome-protected RNA. This work highlights RNA trafficking as a potentially important new form of interaction between hosts and Cuscuta. / Ph. D.