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Nicotiana sylvestris is a diploid tobacco plant that is amenable to laboratory manipulation including facile transformation of nuclear and plastid (chloroplast) genomes. In three separate studies, I used this model organism to observe biological processes with evolutionary and biotechnological implications. The first addresses the mechanisms of horizontal gene transfer by demonstrating cell-to-cell movement of plastids. We grafted Nicotiana sylvestris plants with selectable transgenic plastid genomes to Nicotiana tabacum plants with selectable transgenic nuclear markers. Grafting triggers formation of new cell-tocell contacts, creating an opportunity for organelle movement between the plant cells. I present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. My second objective was to determine whether exceptional pollen transmissionof plastids is accompanied by paternal mitochondria transmission in Nicotiana sylvestris. Plastids and mitochondria in Nicotiana are normally both inherited from the maternal parent. We observed that plastids from the N. sylvestris father were transmitted at a low (~0.002%) frequency via pollen. The plants that inherited paternal plastids did not carry paternal mitochondrial DNA, indicating that leakage of plastids via pollen can produce plant lines with unrelated plastids and mitochondria. My third objective was to observe the behavior of an individual high-copy retrotransposon in N. sylvestris, its native host. Long terminal repeat (LTR) retrotransposons are major components of the nuclear genomes of plants, animals and fungi. The “copy-and-paste” life cycle of retrotransposons accounts for their accumulation in host genomes and permits the assumption that LTRs are identical at the time of insertion. Our objective was to experimentally determine if an introduced synthetic element would interact with native high-copy elements during retrotransposition. I present evidence that S-TNT1 co-packaged with native TNT1 elements to produce hybrid insertions with swapped LTRs and multiple recombinations within the gag-pol gene. We can best explain our observations by dimerization and co-packaging of TNT1 gRNAs in the cytoplasm, followed by template-switching during minus-strand DNA synthesis, which we term the “mixand- paste” pseudodiploid mating system for LTR-retroelements.

ETD_3974

Ph.D.

Includes bibliographical references

Includes vita

by Gregory N. Thyssen

http://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000065276

doi:10.7282/T33F4NKH

ETD doctoral

The author owns the copyright to this work.

22377984

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